Frédéric Checler
email: checler@ipmc.cnrs.fr

Elissa Afram

Raphaëlle Pardossi-Piquard

Inger Lauritzen

Alexandre Bourgeois

Wejdane El Manaa

Eric Duplan

Mounia Chami

Audrey Valverde

Bauer Charlotte1

Frederic Checler
email: checler@ipmc.cnrs.fr.

Bauer Charlotte

Frederic Checler

Frédéric Checler

"The η-secretase-derived APP fragment ηCTF is localized in Golgi, endosomes and extracellular vesicles and contributes to Aβ production."

Keywords: Alzheimer's Disease, ηCTF, Aβ-Production, Extracellular Vesicles, Endosomes, Autophagic Degradation Publications et communications APLP Intracellular Alzheimer's disease, ηCTF, endosomes, extracellular vesicles, autophagic degradation, Aproduction

The amyloid-β (Aβ) peptides, which accumulate in amyloid plaques, are formed by canonical cleavages of the amyloid precursor protein (APP) by β-and γ-secretases. Under physiological conditions, APP is cleaved by α, and γ-secretases, precluding the production of Aβ. Recently, the matrix metalloproteinase MT5-MMP, referred to as η-secretase, has been identified as a novel APP cleaving enzyme. The η-secretase targets the APP protein in its amino-terminal part and produces a soluble fragment, the sAPPη, and a transmembrane fragment, the ηCTF, which can then be a substrate of the αor β-secretase activity generating the soluble fragments Aηα or Aηβ, respectively. Although Aηα and Aηβ fragments have recently been described as being able to affect synaptic activity, the pathophysiological function of the ηCTF fragment has not yet been studied. My PhD project has consisted in studying by different approaches the fate, biology and cellular localization of this fragment and in evaluating its contribution in the pathology associated with Alzheimer's disease (AD).

Using pharmacological approaches, we first showed that ηCTF undergoes degradation in the autophagic and proteasomal pathways. We established that ηCTF is a precursor for APP-CTFs (C99 and C83), and we also revealed for the first time that overexpressing ηCTF in fibroblasts lacking APP promotes the secretion of Aηα as well as the production of Aβ peptides, demonstrating that ηCTF operates as a novel genuine precursor of Aβ. Remarkably, we designed a novel immunological probe referred to as ηCTF-NTer that specifically interacts with the N-terminal part of η-CTF, thus targeting η-CTF, Aη, Aη but not C99, C83 nor A. Immunostaining experiments, using this novel antibody, in vitro, as well as ex vivo, revealed a major Golgi localization of ηCTF and a lower proportion in endosomes. Interestingly, and in agreement with this endosomal localization, we demonstrated the presence of ηCTF in exosomes purified from culture medium of SH-SY5Y cells expressing wild-type APP as well as in exosomes obtained from wild type mice expressing ηCTF after intracerebroventricular injection of AAV-ηCTF. Additionally, ηCTF fragments were found surrounding amyloid plaques by immunostaining performed on brain slices of 20 months old 3xTgAD mice. Finally, preliminary long-term potentiation (LTP) experiments performed on brain sections from wildtype mice showed impaired synaptic activity in the presence of recombinant Aηα and Aηβ peptides.

Aβ is certainly not the only pathological actor in AD, as other APP catabolites may also be involved. Indeed, independently of Aβ, the C99 fragment has been shown to contribute to the molecular and cellular dysfunctions in AD. In a review in which I contributed, we proposed that γ-secretase should be considered as a beneficial C99-inactivating enzyme and highlighted how the toxicity of C99 may be one of the reasons why inhibiting γ-secretase failed in clinical trials.

Much research is undoubtedly still needed in order to fully understand the implication of ηCTF in AD; however, as a precursor of C99 and of A, ηCTF displays a theoretical toxic potential. The accumulation of ηCTF, its detection around amyloid plaques, as well as its presence in exosomes, which may be responsible for the spread of neuropathology, may all support its potential contribution to AD pathology. Conclusively, this demonstrates the complexity of AD, the multifactorial disease with various toxic factors that are still being investigated, which likely explains the challenges in discovering a curative and effective treatment.

Résumé

Le peptide amyloïde-β (Aβ) qui est le constituant principal des plaques séniles est formé suite aux clivages canoniques de la protéine amyloïde précurseur amyloïde (APP) par les activités βet γ-sécrétases. Dans les conditions physiologiques, l'APP est clivée par les activités α, et γsécrétases, limitant ainsi la production d'Aβ. Récemment, la métalloprotéinase matricielle MT5-MMP, appelée η-secrétase, a été identifiée comme une nouvelle enzyme de clivage de l'APP. L'η-secrétase clive la protéine APP dans sa partie amino-terminale et produit un fragment soluble, le sAPPη, et un fragment transmembranaire, l'ηCTF qui est aussi substrat de l'activité αou β-secrétase générant respectivement les fragments solubles Aηα ou Aηβ. Ces fragments Aηα et Aηβ ont récemment été décrits comme pouvant altérer l'activité synaptique mais la fonction physiopathologique du fragment ηCTF n'a pas encore été décrite. Mon projet de thèse a consisté à étudier par différentes approches le catabolisme, la biologie et la localisation cellulaire de ce fragment pour évaluer sa contribution dans la pathologie associée à la maladie d'Alzheimer (MA).

En utilisant des approches pharmacologiques, nous avons d'abord montré que l'ηCTF est dégradé par les voies du protéasome et de l'autophagie. Nous avons aussi établi pour la première foisque l'ηCTF est un précurseur des APP-CTFs (C99 et C83), et du peptide Aβ. En effet, sa surexpression dans des fibroblastes dépourvus d'APP induit non seulement comme attendu la sécrétion d'Aηα mais aussi la sécrétion du peptide Aβ40, démontrant que l'ηCTF se comporte comme un nouveau précurseur véritable d'Aβ. De manière importante, nous avons également développé un nouvel anticorps nommé ηCTF-NTer qui interagit spécifiquement avec la partie N-terminale de l'η-CTF en ciblant l'η-CTF, l'Aηβ, l'Aηβ mais pas le C99, le C83 ni l'Aβ. Des expériences d'immunomarquage, utilisant ce nouvel anticorps, in vitro, ainsi qu'ex vivo, ont révélé une localisation majeure de l'ηCTF dans le Golgi et une proportion plus faible dans les endosomes. De manière très intéressante et en accord avec cette localisation endosomale, nous avons démontré la présence de l'ηCTF dans les exosomes purifiés à partir du milieu de culture de cellules SH-SY5Y exprimant l'APP sauvage ainsi que dans les exosomes obtenus à partir de souris sauvages exprimant l'ηCTF suite à l'injection intracérébroventriculaire de virus exprimant l'ηCTF (AAV-ηCTF). De plus, un marquage spécifique de l'ηCTF a été détecté autour des plaques amyloïdes par immunomarquage réalisé sur des coupes de cerveau de souris 3xTgAD âgées de 20 mois. Enfin, des expériences préliminaires de potentialisation à long terme (LTP) réalisées sur des coupes de cerveau de souris sauvages ont montré une activité synaptique altérée en présence des peptides recombinants Aηα et Aηβ.

Aβ n'est certainement pas le seul acteur pathologique de la MA, puisque d'autres catabolites de l'APP peuvent également être impliqués. En effet, indépendamment de l'Aβ, notre laboratoire a établi que le fragment C99 pouvait contribuer aux dysfonctionnements moléculaires et cellulaires observés dans la MA. Dans une revue dont je suis co-auteur, nous avons proposé que la γ-secrétase soit considérée comme une enzyme bénéfique inactivant le C99 et avons expliqué comment la toxicité du C99 peut être l'une des raisons pour lesquelles l'inhibition de la γ-secrétase a pu échouer dans les essais cliniques. Secondly, I want to convey my appreciation to Dr. Frederic Checler for placing his trust in me with a project that is so important to him. Without you, Dr. Frédéric, I would'nt have had the chance to earn my PhD. I can't thank you enough for this opportunity, and I hope I've reached your expectations.

I would like to deeply thank my thesis director, Dr Raphaëlle Pardossi-Piquard. I consider myself quite blessed to have had you as my thesis supervisor, Raphaëlle, as you combine maternal tenderness with research expertise. I genuinely thank your invaluable guidance during my three years of thesis, your constant availability, and your constructive remarks. All of this has helped me mature, and I will always be extremely grateful. Without forgetting to thank you for helping me with my thesis's posters, paper, manuscript, and oral defense. Thank you for everything you did! I'll miss you dearly.

I would like to thank Dr. Inger Lauritzen for teaching me how to perform the immunostaining and exosome purification experiments, as well as for always being available to answer to my never-ending questions. Inger, you have always been a bundle of positivity, and I have always enjoyed seeing your contagious and motivating smile in the office.

Dr. Julie Dunys, first, thank you for your help with the LTP experiments, and most importantly, thank you for being a delight to work with. Dr. Cristine Alves Da Costa and Dr. Mounia Chami, the two future team leaders with scientific and leadership abilities; you are excellent role models and inspirations.

Dr. Eric Duplan, first, I would like to thank you for all of your assistance with the molecular biology experiments! And, of course, thank you for our occasional scientific and non-scientific talks. I've always looked forward to our small hallway discussions and laughs.

Dr. Bruno Vincent, it was a pleasure to meet someone with such a youthful spirit.

Thank you, Charlotte, for everything you do! Especially for dealing with orders and maxipreps.

All of the projects rely on you, and you certainly handle a lot while working on your PhD! And best of luck with your thesis.

Audrey, having you next to me in the office when I first started my thesis was a blessing. You were always there for me, morally and scientifically, to support and help me. I value the knowledge you've given to me, the assistance you've offered me with various techniques, and our chats, which I've always loved.

Fanny, thank you for all the pleasant evenings. Thank you, Aurore, for always being such a sweetheart. Thank you Loan for training me for the lentivirus production, and I will never forget your calm attitude and amazing sense of humor. Arnaud, thank you for always being helpful anytime I came into your office to ask a lot of questions, which happened quite frequently! Julien, thank you for believing in my babyfoot skills despite the fact that I absolutely suck. Thank you, Paula and Ligia, for being the first to welcome me when I arrived, which made it easier for me to adapt to a new environment. Céline, thank you for being a delightful and adorable office coworker. Juliane and Gwendoline, although I have only known you for a few months, our small talks have always been pleasant and cheerful.

Thank you, Team Checler, for being my second family, for being such nice, sweet, and loving people. Thank you for providing a fantastic work atmosphere, and thank you for all of the memories that will bring a smile to my face for the rest of my life.

To all my friends in Lebanon, you've always been on my mind. The thought of having such supportive and caring friends back home motivates me to keep going. Pamela, you're more than just a best friend; you're a sister. I can't thank you enough for everything you've done for me; for being the best listener and most supportive person on the planet! Thank you for seeing me at my worst and yet being there to support and help me.

To my friends in Antibes: Joe Kh, Sacha, Elio, Wassim, George and Cynthia, I'll be forever grateful for having you as a second family. Thank you for sticking with me over the past three years.

Joe, Jouj Habibi. Even in the most difficult times, talking to you always made everything better. I appreciate how well you always handle my attitude and anxiety. Thank you for believing in me, and never giving up on me. Thank you for being the most considerate and loving partner I could ever ask for. You bring joy to my heart. I love you. Thank you for always prioritizing my happiness before yours. Thank you for going above and beyond my needs. I'll be forever grateful, and I pray that God gives you health, happiness, and everything else you desire in this life.

Mom

Last but not least, Jesus, thank you for holding my hand through every step of the way. Alzheimer's disease (AD) is an neurodegenerative disorder named by Dr. Emil Kraepelin, in reference of the German psychiatrist Alois Alzheimer, who was the first to identify the disease in 1906 [START_REF] Moller | The case described by Alois Alzheimer in 1911. Historical and conceptual perspectives based on the clinical record and neurohistological sections[END_REF]. The disease is induced by neuropathological alterations that majorly affect the cerebral cortex and the hippocampus and but also spread to different areas of the brain, at rates that vary considerably from one patient to another. The term AD is used today by specialists to represent a clinical entity that typically begins with a progressive amnestic disorder and then progresses to various cognitive, behavioral, and neuropsychiatric alterations that affect social function and daily living tasks.

Involved in 60 to 80% of demented cases in people over 65, AD is the most prevalent neurodegenerative dementia from middle age to the elderly. Dementia is a syndrome with multiple causes, characterized by deterioration in cognitive ability, behavior, social abilities and capacity for independent living. While AD is the leading cause of dementia in the elderly, other neurodegenerative disorders such as vascular dementia, Lewy body, and frontotemporal dementia (also termed frontotemporal lobar degeneration) can also be associated to dementia.

Non-neurodegenerative diseases such as tumors, vitamin deficiencies or infections for example, may also contribute to patients' dementia syndrome [START_REF] Gale | Dementia[END_REF].

In 2020, 55 million people across the world were diagnosed with dementia. Due to longer life spans, cases will almost double every 20 years. By 2050, the number of people aged over 60 years will have increased by 1.25 billion, and the number of people with dementia is expected to reach 139 million cases. It is worth noting that the global prevalence of AD is between 5 and 6% at age 65 and 30% in individuals over 85 (Figure 1).

Without effective pharmacologic treatments for Alzheimer disease to date, social and economic burden on families, society and health care systems will continue to grow due to the needs of persistent care and therapy. Total estimated cost of dementia today for health care, is 1.3 trillion dollars and is expected to rise to 2.8 trillion dollars by 2030.

(https://www.alzint.org/about/dementia-facts-figures/dementia-statistics/)

B) NEUROPATHOLOGICAL CHANGES IN AD AND BIOMARKERS

Prior to breakthroughs in technology and science in the 80s, AD was only diagnosed after the patient died, when a marked reduction in brain volume associated with microscopic histopathological hallmarks such as senile plaques (Glenner and Wong, 1984;[START_REF] Masters | Amyloid plaque core protein in Alzheimer disease and Down syndrome[END_REF] and neurofibrillary tangles [START_REF] Braak | Neuropathological stageing of Alzheimer-related changes[END_REF][START_REF] Brion | Neurofibrillary tangles of Alzheimer's disease: an immunohistochemical study[END_REF][START_REF] Grundke-Iqbal | Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology[END_REF] were observed.

Macroscopic Brain Atrophy

Typical macroscopic alteration of AD is cerebral cortical atrophy, primarily affecting the medial temporal lobes, including the entorhinal cortex, the hippocampus and the amygdala [START_REF] Dickerson | Functional abnormalities of the medial temporal lobe memory system in mild cognitive impairment and Alzheimer's disease: insights from functional MRI studies[END_REF][START_REF] Jack | Comparison of different MRI brain atrophy rate measures with clinical disease progression in AD[END_REF]. This atrophy is accompanied of widened sulcal spaces and gyri shrinking (Figure 2 middle part).

Both neuronal and synaptic losses contribute to this brain atrophy, and as a result of this cortical thinning, the frontal and temporal horns of the lateral ventricles appear prominently dilated [START_REF] Apostolova | Hippocampal atrophy and ventricular enlargement in normal aging, mild cognitive impairment (MCI), and Alzheimer Disease[END_REF].

Microscopic Features 2.1. Neurofibrillary Tangles (NFTs)

Neurofibrillary tangles (NFT) were one of the key brain abnormalities highlighted by Alois Alzheimer when he originally characterized AD in 1906. However, it took decades for Tau protein to be identified [START_REF] Brion | Neurofibrillary tangles of Alzheimer's disease: an immunohistochemical study[END_REF][START_REF] Grundke-Iqbal | Abnormal phosphorylation of the microtubule-associated protein tau (tau) in Alzheimer cytoskeletal pathology[END_REF] as the primary component of NFTs [START_REF] Naseri | The complexity of tau in Alzheimer's disease[END_REF]. Macroscopic brain atrophy in AD with the two histopathological markers: extracellular amyloid plaques and intraneuronal neurofibrillary tangles (NFTs). Created with BioRender.com free software.

The MAPT (Microtubule Associated Protein Tau) gene, which is found on chromosome 17q21, encodes tau, a microtubule-associated protein that is mostly produced in the axons of neurons.

Since microtubules are the primary components of the cytoskeleton, Tau's binding to microtubules helps to assemble and stabilize them, controlling the cytoskeleton's dynamics as well as axonal transport (Figure 2 left part) [START_REF] Barbier | Role of Tau as a Microtubule-Associated Protein: Structural and Functional Aspects[END_REF]. Mutations and posttranslational modifications, principally phosphorylation, control Tau's physiological function.

Tau's conformation is altered by hyperphosphorylation or phosphorylation on proline-rich regions, which facilitates the formation of neurotoxic fibrils and reduces its affinity for microtubules, resulting in the formation and accumulation of NFTs (Figure 2 right part) [START_REF] Wegmann | A current view on Tau protein phosphorylation in Alzheimer's disease[END_REF].

Tauopathies are neurodegenerative diseases caused by intracellular accumulations of abnormal tau filaments that form NFTs [START_REF] Takashima | Tau Biology[END_REF].

Amyloid Plaques

Unlike NFTs, extracellular amyloid plaques, also known as senile plaques, are a more specific neuropathological diagnostic criterion for AD. Amyloid plaques are composed primarily of βamyloid peptides (Aβ) (Glenner and Wong, 1984;[START_REF] Masters | Amyloid plaque core protein in Alzheimer disease and Down syndrome[END_REF], which are produced by the proteolytic processing of a larger glycoprotein known as amyloid precursor protein (APP).

APP metabolism and Aβ peptides will be discussed in greater detail later.

Amyloid plaques can range from 10 up to 120 μm in cross-sectional dimension, and the density and degree of compaction of the Aβ fibrils that constitute the extracellular core vary greatly among plaques (Figure 2 right part). Both inside of this amyloid deposit and immediately around it, dystrophic neurites can be seen. These neurites are frequently tangled and twisted, and are characterized by ultrastructural abnormalities such as enlarged lysosomes, numerous mitochondria, and paired helical filaments [START_REF] Selkoe | Alzheimer's disease: genes, proteins, and therapy[END_REF].

Biomarkers

Newer techniques for early AD diagnosis, based on the detection of the abnormal accumulation amyloid β and Tau, are currently in use [START_REF] Anoop | CSF Biomarkers for Alzheimer's Disease Diagnosis[END_REF]. For instance, methods for measuring the levels of total tau (T-tau) and neurofilament light (NFL) as biomarker of neurodegeneration, phosphorylated tau (P-Tau), and amyloid β (Aβ42 and Aβ42/40 ratio) in cerebrospinal fluid (CSF) have been established. Lumbar puncture is used to collect CSF in the search for the "Alzheimer CSF profile," which is characterized by elevated levels of T-tau and NFL (reflecting neurodegeneration) and phosphorylated tau (marker for tangles), as well as decreased levels of Aβ42 and Aβ42/40 ratio (reflecting brain amyloidosis and plaques).

Multiple studies have found a decrease in CSF Aβ42 in AD patients [START_REF] Andreasen | Cerebrospinal fluid tau and Abeta42 as predictors of development of Alzheimer's disease in patients with mild cognitive impairment[END_REF][START_REF] Fagan | Cerebrospinal fluid tau/beta-amyloid(42) ratio as a prediction of cognitive decline in nondemented older adults[END_REF]. Given that Aβ42 is a significant component of senile plaques, reduced CSF-Aβ42 levels are believed to be induced by high plaque deposition of Aβ42 in brains.

Since the 1980s, imaging techniques like magnetic resonance imaging (MRI) and fluorodeoxyglucose-positron emission tomography (FDG-PET) have been used to assist with the diagnosis of AD. Although extremely expensive, amyloid positron emission tomography (PET), which first became available in 2004 [START_REF] Klunk | Imaging brain amyloid in Alzheimer's disease with Pittsburgh Compound-B[END_REF], and tau PET, which followed a few years later [START_REF] Chien | Early clinical PET imaging results with the novel PHF-tau radioligand [F18]-T808[END_REF], are the first true disease-specific imaging biomarkers for AD. They mostly consist of the detection of AD biomarkers, which are used to assess patients' overall health and pathological state [START_REF] Khan | Recent Advancements in Pathogenesis, Diagnostics and Treatment of Alzheimer's Disease[END_REF][START_REF] Lashley | Molecular biomarkers of Alzheimer's disease: progress and prospects[END_REF].

These biomarkers appear to be valuable elements, allowing early detection of the disease, as well as monitoring the disease's evolution and tracking the efficacy of a treatment.

Dr. Dubois and his colleagues proposed the "A/T/N" system in 2016 [START_REF] Jack | A/T/N: An unbiased descriptive classification scheme for Alzheimer disease biomarkers[END_REF], which divides AD biomarkers into three categories: "A" refers to the value of β-amyloid (amyloid PET or CSF Aβ42); "T," the value of a Tau (CSF P-Tau, or Tau PET); and "N," neurodegeneration biomarkers (FDG-PET, structural MRI, or CSF T-Tau). This A/T/N approach is not influenced by an individual's cognitive condition and avoids syndromic or clinical labeling, making it an unbiased strategy suited mainly to the diagnostic of AD, but also to cognitive aging research in general. However, biomarker data interpretation without a clinical investigation can be challenging, and positive AD biomarkers can be confused with other agerelated diseases. So, while the A/T/N biomarker system allows the detection of the disease in the preclinical phase, it will require clinical information about each individual and must be used in conjunction with a complementary system for a complete diagnosis.

C) AD PROGRESSION AND SYMPTOMS

The findings of PET scanning and CSF analysis showed that the pathological alterations begin two decades before symptoms occur. In fact, AD progresses on a spectrum of three major phases (2022):

1. The preclinical asymptomatic phase in which neuropathological changes have already taken place in the brain, without any clinical symptoms leading to diagnosis. This phase is relatively long, several years or decades before any symptoms start developing [START_REF] Sperling | Toward defining the preclinical stages of Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease[END_REF].

2. The prodromal phase also referred to as mild cognitive impairment (MCI) in some references. Patients can be diagnosed with AD during this mild symptomatic stage based on their clinical presentation and biomarker-confirmed Alzheimer's pathology. Patients start losing the ability to carry out everyday activities, but still manage to be independent. Symptoms may include episodic memory loss, mild decline in semantic memory and working memory, motivation, mood and behavioral disorders [START_REF] Albert | The diagnosis of mild cognitive impairment due to Alzheimer's disease: Recommendations from the National Institute on Aging-Alzheimer's Association workgroups on diagnostic guidelines for Alzheimer's disease[END_REF].

3. The final dementia stage -the more severe form of AD -is characterized by severe impairment in talking, walking, sitting and, swallowing, and severe decline in memory and cognitive skills. Patients at this stage lose their motivation, their ability to communicate and their complete autonomy. At this point, 24-hour care should be provided, and hospitalization might also be required since patients become more vulnerable to infections.

Clinical phenotypes are extremely complex and difficult to understand, and it's crucial to remember that each person may experience symptoms differently and go through the stages in a different way (TABLE 1).

D) TREATMENTS AND ONGOING CLINICAL TRIALS

With AD being now a global health concern, finding a cure is crucial to easing the severe physical, emotional, and financial burdens placed on patients and society.

Only four medications were approved by the Food and Drug Administration (FDA) for the treatment of AD until 2021; all four are only for treating symptoms but not curative drugs.

Signs of Alzheimer's Dementia

Typical Age-Related Changes Memory loss that disrupts daily life: One of the most common signs of Alzheimer's dementia, especially in the early stage, is forgetting recently learned information. Others include asking the same questions over and over, and increasingly needing to rely on memory aids (for example, reminder notes or electronic devices) or family members for things that used to be handled on one's own. Sometimes forgetting names or appointments, but remembering them later.

Challenges in planning or solving problems: Some people experience changes in their ability to develop and follow a plan or work with numbers. They may have trouble following a familiar recipe or keeping track of monthly bills. They may have difficulty concentrating and take much longer to do things than they did before.

Making occasional errors when managing finances or household bills

Difficulty completing familiar tasks: People with Alzheimer's often find it hard to complete daily tasks. Sometimes, people have trouble driving to a familiar location, organizing a grocery list or remembering the rules of a favorite game.

Occasionally needing help to use microwave settings or record a television show. Confusion with time or place: People living with Alzheimer's can lose track of dates, seasons and the passage of time. They may have trouble understanding something if it is not happening immediately. Sometimes they forget where they are or how they got there.

Getting confused about the day of the week but figuring it out later.

Trouble understanding visual images and spatial relationships: For some people, having vision problems is a sign of Alzheimer's. They may also have problems judging distance and determining color and contrast, causing issues with driving.

Vision changes related to cataracts.

New problems with words in

They may be easily upset at home, at work, with friends or when out of their comfort zones.

Developing very specific ways of doing things and becoming irritable when a routine is disrupted.

Table 1: Signs of Alzheimer's dementia compared with typical age-related changes. Adapted from (https://www.alz.org/media/documents/alzheimers-facts-and-figures) Donepezil, Rivastigmine, and Galantamine were three of them that were acetylcholinesterase enzyme inhibitors. Acetylcholine (ACh) is crucial for cognitive function, and in AD, cholinergic synaptic loss and cholinergic neuronal degeneration were reported. So these inhibitors prevent acetylcholinesterase from degrading ACh, allowing it to be persistently preserved and activate cholinergic receptors. Memantine, an N-Methyl-D-aspartate (NMDA) receptor antagonist, is the fourth medicine to receive FDA approval. It protects against the overstimulating and toxic stimulation of these receptors that occurs in AD. Unfortunately, these medications neither cure nor prevent the disease; they simply serve to improve the symptoms [START_REF] Breijyeh | Comprehensive Review on Alzheimer's Disease: Causes and Treatment[END_REF].

In 2021, Aducanumab was the first disease-modifying therapy (DMT) to be approved for AD.

Aducanumab is a monoclonal anti-amyloid antibody that targets plaques and oligomers.

However, the medicine was approved only in United States by FDA with significant debate, and phase III clinical trials will proceed until 2023 [START_REF] Walsh | Aducanumab for Alzheimer's disease?[END_REF].

There are currently 143 other molecules in 172 ongoing trials of treatments for AD. The most common agents being studied are DMTs, and 21 of these agents have reached phase III studies.

These DMTs have a wide range of targets, including tau, synaptic plasticity, oxidative stress, metabolism, and amyloid (Aβ monomers, oligomers, protofibrils, and amyloid plaques), etc…

The majority of these DMT target amyloid, including Donanemab and Lecanemab, two monoclonal plaque-lowering antibodies that are now being evaluated by the FDA [START_REF] Cummings | Alzheimer's disease drug development pipeline: 2022[END_REF]. Eisai and Biogen made an exclusive announcement on 27 th of September 2022, on the positive results of the Phase 3 Clarity trial of Lecanemab. They claimed in this exciting statement that Lecanemab significantly slowed the progression of cognitive decline by 27% over 18 months in patients with very mild to mild AD, and that both companies intend to apply for regulatory approval in the United States, Europe, and Japan. The findings of this trial has been presented at the Clinical Trials on Alzheimer's Congress, which has been held in San Francisco in November, and will be published in a peer-reviewed journal soon (https://www.alzforum.org/news/research-news/finally-big-win-all-outcomes-lecanemabphase-3-topline-results).

E) AD ETIOLOGY

AD exists in two forms: hereditary/familial and sporadic. Only 1 to 2% of patients have the hereditary form, which is caused by one of three monogenic autosomal dominant mutations in one of three genes: APP (amyloid precursor protein), PS1 (presenilin 1), or PS2 (presenilin 2).

The remaining 98-99% of cases, however, are sporadic, in which the disease is not caused by a dominant mutation but rather by a combination of factors including age, genetic predispositions, and environmental factors.

According to the age at which symptoms first appeared, AD patients can also be divided into two groups: those with early-onset AD (EOAD), which develop symptoms before the age of 65, and those with late-onset AD (LOAD) where symptoms are detected after the age of 65.

Dementia and the two histological markers (amyloid plaques and NFTs) are common hallmarks of EAOD and LOAD, indicating that distinguishing the two AD age groups at the end-stage of disease can be challenging.

Familial Forms of AD (FAD)

Patients suffering from familial forms of AD (FAD) start developing symptoms between their 30s and 50s, consequently, FAD is characterized mostly by EOAD. Autosomal dominant mutations in APP, PSEN1 or PSEN2 genes have been generally described to induce an abnormal increase in the production of total Aβ or an aggregated form of Aβ. Interestingly, only 10% of EOAD are explained by a mutation of these genes, suggesting that the remaining 90% of the disease's early manifestations, whose origins are still undetermined, are more likely to have autosomal recessive causes [START_REF] Wingo | Autosomal Recessive Causes Likely in Early-Onset Alzheimer Disease[END_REF].

APP Mutations

Since the aggregation of the Aβ peptide into senile plaques is one of the two histopathological hallmarks of AD, mutations in the APP gene, the precursor of Aβ, are consequently directly linked to the disease.

Currently, 73 APP gene mutations are documented on the Alzforum website, with over 53 of them linked to FAD (https://www.alzforum.org/mutations/app). These mutations alter the cleavage of APP by certain secretases (see chapter APP Cleaving Enzymes), resulting in AD via various mechanisms. For instance, the first APP mutation discovered, known as the 'London' mutation (V717I), causes an increase in Aβ42 (Goate et al., 1991). However, the arctic mutation (E693G), enhances Aβ aggregation [START_REF] Kamino | Linkage and mutational analysis of familial Alzheimer disease kindreds for the APP gene region[END_REF][START_REF] Nilsberth | The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation[END_REF].

In my thesis project, I used cellular and mouse models including the triple transgenic mouse model of AD (3xTgAD) mice that overexpress the human APP with the Swedish double mutation (APPswe) (KM670/671NL). This mutation promotes APP cleavage at the β site (see chapter β-secretase), increasing total Aβ levels [START_REF] Johnston | Increased beta-amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation[END_REF][START_REF] Mullan | Familial Alzheimer's disease: second gene locus located[END_REF] , and replicating amyloid pathology with senile plaque deposits in mice brains. Not all APP mutations are pathogenic; for example, the Icelandic mutation (A673T) has been identified as protective, reducing Aβ40 and Aβ42 by 40% in HEK293 cells [START_REF] Jonsson | A mutation in APP protects against Alzheimer's disease and age-related cognitive decline[END_REF].

Furthermore, since the APP gene is located on chromosome 21 (21q21.3), a duplication of this gene results in autosomal dominant EOAD [START_REF] Rovelet-Lecrux | APP locus duplication causes autosomal dominant early-onset Alzheimer disease with cerebral amyloid angiopathy[END_REF]. Thus, the presence of an extra copy of APP in Down Syndrome patients (due to a trisomy on chromosome 21) leads to development of typical AD phenotypes by the age of 40 [START_REF] Lott | Dementia in Down syndrome: unique insights for Alzheimer disease research[END_REF].

PSEN1 and PSEN2 Mutations

PSEN1 and PSEN2 encode PS1 and PS2 respectively, which are subunits of γ-secretase, a key enzyme in APP processing and Aβ production (see chapter γ-secretase). To date, 87 mutations in PSEN2, located on chromosome 1 (1q42.13), have been discovered and described as an uncommon cause of EOAD, whereas more than 300 mutations in PSEN1, located on chromosome 14 (14q24.2), have been reported and represent the most common cause of EOAD (https://www.alzforum.org/mutations).

PSEN1 mutations mostly increase the Aβ42/40 ratio, either by decreasing Aβ40 levels or raising Aβ42 levels. For instance, the first reported PSEN1 mutation (M146V), identified in 1995 [START_REF] Alzheimer's Disease Collaborative | The structure of the presenilin 1 (S182) gene and identification of six novel mutations in early onset AD families[END_REF], results in increased levels of secreted Aβ42 and an elevated Aβ42/40 ratio. This is one of the most common mutations reported in FAD patients, and it is also present in the 3xTgAD mouse model.

Sporadic Forms of AD (SAD)

The sporadic form of AD (SAD), unlike FAD, is not caused by an autosomal dominant mutation, but it can be genetically impacted. Mainly referred to as LOAD because symptoms occur after the age of 65, it is a multifactorial disorder influenced mostly by environmental factors, genetic predispositions, patient's sex, and age, with age being the primary risk factor [START_REF] Rice | Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission[END_REF].

Genetic Predisposition

APOE

The Apolipoprotein E (APOE) is glycoprotein, important for transporting lipids and for the metabolism of cholesterol. The central nervous system (CNS) contains this glycoprotein, which is widely expressed in astrocytes, and to a lesser level in stressed neurons. By binding to cellsurface APOE receptors, this glycoprotein redistributes cholesterol and other lipids to neurons, critical for the maintenance of myelin and neuronal membranes [START_REF] Rice | Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission[END_REF][START_REF] Yamazaki | Apolipoprotein E and Alzheimer disease: pathobiology and targeting strategies[END_REF].

APOE is found on chromosome 19 and consists of three allelic variants: APOE-ε2 (Cys-112, Cys-158), . Each APOE isoform's functionality is impacted by the structural changes brought by the various amino acids at these sites [START_REF] Verghese | Apolipoprotein E in Alzheimer's disease and other neurological disorders[END_REF]. This polymorphism has been identified as the most important genetic risk factor for LOAD. The replacement of cysteine for arginine at position 112 and 158 in APOE4, raises plasma cholesterol and LDL levels and puts the person greater risk for cardiovascular and neurodegenerative diseases like AD [START_REF] Rice | Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission[END_REF]. One copy of the ε4 allele increases risk by fourfold, while two copies increase risk by fourteen [START_REF] Corder | Gene dose of apolipoprotein E type 4 allele and the risk of Alzheimer's disease in late onset families[END_REF], with each copy decreasing onset by eight years [START_REF] Verghese | Apolipoprotein E in Alzheimer's disease and other neurological disorders[END_REF].

The processes through which APOE4 is related to AD are reviewed in Yamazaki's article [START_REF] Yamazaki | Apolipoprotein E and Alzheimer disease: pathobiology and targeting strategies[END_REF]. In summary, it appears to interfere with inflammation and synaptic plasticity, as well as the phosphorylation of the Tau protein, and the clearance and aggregation of the Aβ peptide in senile plaques. In fact, APOE4 interacts with Aβ and decreases its perivascular drainage, its phagocytosis and its clearance by enzymatic degradation or through the blood-brain barrier (BBB).

Other genetic risk factors for AD

APOE was assumed to be the only genetic predisposition factor associated to AD until 2009, but today, 75 independent loci for AD and related dementias have been reported [START_REF] Bellenguez | New insights into the genetic etiology of Alzheimer's disease and related dementias[END_REF]. These genetic risk factors are mainly involved in cholesterol metabolism, endosome/lysosome trafficking/function, or innate immunity and microglial activation (Figure 3). The internal color corresponds to their understood function; where there are two internal colors, the gene has been implicated in more than one pathway. Genes circled in yellow are also thought to influence amyloid precursor protein metabolism; genes circled in red are thought to influence tau metabolism (Lane, Hardy and Schott, 2018).

Non-Genetic Factors

Age

Age continues to be the most important risk factor for AD and is a key element in the progression of the illness. However, phenotypes comparable to those reported in AD brains, such as reduced brain volume, enlarged ventricles, synapses loss, and even amyloid plaques, have been observed in normal aging and cognitively normal brains. This means that AD is not the consequence of normal aging alone, and that age without other factors, is insufficient to develop the disease (A. Armstrong, 2019).

Sex

When it comes to gender as a risk factor of AD, females are more affected than men after the age of 80. This was explained by the fact that women have a longer life expectancy than men, and it was believed that when men's lives are estimated to be as long as women's in a few years, this AD risk factor would be spread equally between the two sexes [START_REF] Rice | Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission[END_REF].

However, hormones and post-menopausal hormonal changes have been proven to have an impact on increasing AD risk in women [START_REF] Mosconi | Perimenopause and emergence of an Alzheimer's bioenergetic phenotype in brain and periphery[END_REF][START_REF] Scheyer | Female Sex and Alzheimer's Risk: The Menopause Connection[END_REF]. Similar sex difference is also observed in AD mouse models such as 3xTgAD brain.

Other environmental risk factors for AD

LOAD can also be influenced by environmental and lifestyle factors. These include mainly: diet and malnutrition, obesity, education, alcohol consumption, education, and physical exercises.

The risk of AD can also be increased by other events including traumatic brain injury, and some diseases such as diabetes and vascular diseases can also increase risk for AD. All these factors are reviewed in (A. [START_REF] Armstrong | Risk factors for Alzheimer's disease[END_REF].

The Amyloid Cascade Hypothesis

The amyloid cascade hypothesis, introduced by John Hardy and Gerald Higgins in 1992, places Aβ peptide at the root of the disease. According to this theory, Aβ accumulation is the initial cause of AD, and that the formation of neurofibrillary tangles is triggered by the aggregation of Aβ, and dementia arise later as a direct result of these deposits (Hardy and Higgins, 1992;Selkoe and Hardy, 2016).

The amyloid hypothesis dominates the AD study field since it is supported by a significant number of evidence such as genetics and histopathology (Pimplikar et al., 2010). Indeed, Aβ plaques first appear in the cortical regions and then spread to the remainder of the brain as the disease progresses, and histopathologically, they are regarded as one of the diagnostic hallmarks of AD and a key element of AD pathogenesis. Moreover, genetic research served as a foundation for the amyloid theory. Strong evidence in favor of the amyloidogenic hypothesis, for example, comes from mutations in FAD that increase the production of the Aβ peptide or modify the Aβ42/Aβ40 ratio, as well as from the fact that individuals with Down's syndrome develop typical Alzheimer neuropathology due to an increased APP copy number. Besides the APP mutations inducing the pathological phenotypes of AD, there is also the Icelandic mutation on APP, identified as protective, since it reduces β-secretase cleavage and Aβ production.

Along with the autosomal dominant mutations, genome-wide association studies (GWAS) studies have identified risk factors for SAD that are related directly or indirectly to APP metabolism and Aβ clearance.

In spite of being the most widely accepted hypothesis, it is heavily debated, and even rejected by some scientists due to some of its weaknesses. However, this hypothesis has evolved as studies and research on the disease have progressed (Selkoe and Hardy, 2016).

For instance, it has been reported that many healthy individuals have amyloid deposits, whereas many AD patients present very few amyloid deposits [START_REF] Edison | Amyloid, hypometabolism, and cognition in Alzheimer disease: an [11C]PIB and [18F]FDG PET study[END_REF], hence implying that the accumulation Aβ is related to aging rather than the development of AD. Moreover, it was claimed that NFTs correlate better than amyloid plaques with the progression of cognitive defects in AD [START_REF] Braak | Neuropathological stageing of Alzheimer-related changes[END_REF]. But then as counterarguments, Selkoe and Hardy explained that Aβ42 oligomers start to accumulate very early in the brain, inhibiting long-term potentiation (LTP), impairing memory and causing neuronal dysfunction even before the formation of plaques. Thus, intracellular Aβ oligomers instead of Aβ deposits are now considered as the initial trigger responsible for AD. Furthermore, the deposits detected in healthy individuals are frequently diffuse plaques with little Aβ oligomers and have a lower neurotoxic state.

In addition to all this hypothesis' flaws, the failure of drugs targeting amyloid plaques or β and γ-secretases in clinical trials raised doubts concerning the amyloidogenic hypothesis. Despite optimistic outcomes in mouse models and the recent but contested Aducanumab FDA approval, the large majority of trials with anti-amyloid drugs conducted in humans with AD have failed to improve cognitive impairment in spite of their abilities to remove completely the brain from Aβ deposits. Until the plaque-lowering antibody Lecanemab Clarity trial revealed encouraging results in favor of the amyloidogenic hypothesis in September 2022. Despite Aβ's neurotoxic properties, the peptide could not be the only factor triggering AD.

Without a doubt, AD is a complex pathology, and Aβ, Tau, and even multiple APP C-terminal fragments participate in the neuropathological cascade of AD; hence, a multitargeting treatment for AD is required.

2) APP PROTEIN

A) APP: GENE, STRUCTURE AND FUNCTION 1. Gene APP is a type I integral membrane protein that belongs to a family that includes APP and two mammalian homologs known as APP-like proteins, APLP-1 and APLP-2. The three have similar domain structures, with APP and APLP-2 being evolutionarily more closely related, and APLP-1 representing the APP gene family members' ancestral form [START_REF] Coulson | What the evolution of the amyloid protein precursor supergene family tells us about its function[END_REF]. The different secretases process all three proteins in a similar manner, but the Aβ sequence is unique to APP and FAD mutations were only found on APP genes but not on those of its homologs.

The APP gene, which is found on chromosome 21, has 18 exons. Multiple isoforms of APP are produced as a result of various alternative splicings of this gene, each with a different size and expression levels depending on the organ. APP770, APP751, and APP695 are those expressed in the CNS, with APP695 being the predominant neuronal form from which Aβ is preferentially produced [START_REF] Nalivaeva | The amyloid precursor protein: a biochemical enigma in brain development, function and disease[END_REF].

Structure

Structurally, as presented in (Figure 4) these proteins share a long N-terminal extracellular domain whose length varies according to the isoforms, a hydrophobic transmembrane domain, containing the Aβ sequence, and a short domain C-terminal intracellular tail. The extracellular N-terminal part contains a cysteine-rich globular E1 domain, an extension domain (ED), an acidic subdomain (AcD), a helix-rich E2 domain, and a juxtamembrane region (JMR). The E1 and E2 domains of APP contain heparin binding domains (HBD), which are extracellular matrix (ECM) components. The first HBD on E1, known as the GFLD (Growth Factor Like Domain) can form three disulfide bridges to stabilize APP on the cell surface.

APP contains the Aβ sequence extending into the transmembrane domain. APP770 and APP751 contain a Kunitz protease inhibitor (KPI) subdomain following the AcD. In addition, APP770 contain an OX2 domain following the KPI domain. At the C-terminus part, all APP isoforms and homologs possess the intracellular domain, the APP intracellular domain (AICD) or the APLP1/APLP2 intracellular domain (ALID1/ALID2) and contain a YENPTY motif controlling their transport and metabolism. These different domains are reviewed in [START_REF] Coburger | The structural biology of the amyloid precursor protein APPa complex puzzle reveals its multi-domain architecture[END_REF].

Function

APP can form homodimers or heterodimers with its homologues APLP1 and APLP2. These dimers promote cell adhesion, synaptogenesis, and synapse stabilization and can act as cell surface receptors for adaptors and signaling proteins [START_REF] Baumkotter | Amyloid precursor protein dimerization and synaptogenic function depend on copper binding to the growth factor-like domain[END_REF][START_REF] Deyts | APP Receptor? To Be or Not To Be[END_REF][START_REF] Soba | Homo-and heterodimerization of APP family members promotes intercellular adhesion[END_REF]. Due to its cysteine-rich domain (CRD) within the E1, APP was recently recognized as a Wnt receptor, and this function actually regulates the levels and trafficking of APP protein. When APP binds to Wnt3a, it promotes the recycling of APP full length (APP-FL), whereas binding to Wnt5a enhances the targeting of APP to lysosomal compartments [START_REF] Liu | The amyloid precursor protein is a conserved Wnt receptor[END_REF].

APP dimerizes primarily in the ectodomain via the E1 domain and disulfide bridges that stabilize the dimerization; the E2 domain can also dimerize but with lower affinity. The APP and its mammalian homologues APLP1 and APLP2 share on overall a similar domain architecture. The Aβ-peptide and the extension domain (ED) are only present in all isoforms of APP, the KPI-insertion only in APP751, the KPI/OX2-insertion only in APP770 and APLP2 and the E1domain is less conserved in APLP1 as indicated by its light green color. Additional functional units in APP isoforms are indicated by brackets [heparin binding domain (HBD); growth factor like domain (GFLD)]. Adapted from [START_REF] Coburger | The structural biology of the amyloid precursor protein APPa complex puzzle reveals its multi-domain architecture[END_REF].

transmembrane domain, on the other hand, contains three consecutive GXXXG motifs that would promote dimerization via helix-helix interaction.

The study of mice invalidated for the APP gene yielded important insights into the in vivo functions of APP. Single-knockout (KO) APP mice (APP-/-) exhibited subtle phenotypes such as reduced brain, body weight, and age-dependent impairments in neuronal morphology, synaptic plasticity, and behavior while double (APP-/-and APLP2-/-) or triple-(APP-/-, APLP1-/-and APLP2-/-) KO are lethal after birth. Intriguingly, the APP-/-mice remained viable, most likely due to APP/APLP redundancy. However, despite the fact that its homologs can compensate for some of its function, APP clearly appears to have a physiological function of its own. Aged APP KO mice showed synaptic defects, LTP and memory impairments, suggesting the involvement of APP in the synaptic plasticity and memory. Moreover, it participates in the development of the nervous system at the embryonic stage, particularly at the level of neuronal migration, neurogenesis and axonal guidance [START_REF] Müller | Not just amyloid: physiological functions of the amyloid precursor protein family[END_REF][START_REF] Muller | Physiological functions of APP family proteins[END_REF]. Lastly, it has been recently highlighted that Wnt pathway plays an important role in regulating neurite outgrowth through CRD of the APP (Liu et al, 2022).

Given that each proteolytic fragment of APP has one or more distinct roles of its own (see chapters Aβ peptides, AICD, C99, sAPPβ, sAPPα, C83, P3), all the precise potential functions of APP are challenging to determine and require further investigation.

B) CANONICAL APP-PROCESSING PATHWAY

For many years, cleavages of APP have been investigated to better understand Aβ generation. These cleavages can be classified into two main canonical proteolytic pathways: the amyloidogenic pathway, which produces Aβ following β-secretase and γ-secretase and the nonamyloidogenic pathway, in which APP is first cleaved by α-secretase then by γ-secretase, thus precluding Aβ production (Figure 5).

Numerous non-canonical APP cleavage pathways have been identified and were found to generate novel fragments; some of these are emerging as new players in the pathophysiology of AD.

The Amyloidogenic Pathway

In the amyloidogenic pathway where Aβ is formed, β-secretase cleaves APP at the N-terminus of Aβ sequence, releasing sAPPβ, a 100 kDa soluble amino-terminal (N-terminal) fragment, and C99, a 12 kDa carboxy-terminal (C-terminal) fragment that remains membrane bound. C99 can be further cleaved by γ-secretase, resulting in the production and secretion of Aβ, and a cytosolic Cter fragment AICD (Figure 5-B). p

Aβ peptides

The cleavage of γ-secretase results in the generation of several Aβ species ranging from 37 to 42 amino acids. The majority of the Aβ generated under physiological conditions is Aβ40, with only a small percentage attributed to Aβ42. Although Aβ40 is the most prominent form, Aβ42 is thought to be the most pathogenic due to its high tendency for aggregation.

Until 1992, it was assumed that Aβ generation was exclusively pathological. Nonetheless, despite the fact that amyloid plaques are neuropathological characteristic of AD, Aβ is produced throughout life in physiological levels under entirely normal cellular conditions. In fact, picomolar doses of Aβ, appear to physiologically promote LTP, and improve synaptic plasticity, learning, and memory [START_REF] Morley | A Physiological Role for Amyloid-β Protein: Enhancement of Learning and Memory[END_REF][START_REF] Puzzo | Endogenous amyloid-β is necessary for hippocampal synaptic plasticity and memory[END_REF][START_REF] Puzzo | Picomolar Amyloid-Positively Modulates Synaptic Plasticity and Memory in Hippocampus[END_REF].

Other physiological effects of Aβ include lowering neuronal apoptosis and oxidative damage as well as enhancing blood-brain barrier function [START_REF] Morley | What is the Physiological Function of Amyloid-Beta Protein? The journal of nutrition[END_REF]. A) represents the non-amyloidogenic pathway in which APP is first cleaved by the α-secretase producing the soluble fragment sAPPα and the membrane-embedded Cterminal fragment, C83 that is then cleaved by γ-secretase to release the cytosolic AICD fragment and p3 peptide. B) represents the amyloidogenic pathway in which APP is first cleaved by the β-secretase to liberate the soluble fragment sAPPβ and the membrane-embedded C99 fragment, respectively. Then γ-secretase cleavage releases the cytosolic AICD fragment and Aβ peptides (Checler et al., 2021).

However, in pathological conditions, Aβ peptides accumulate abnormally in AD brains due to either an increase in production or a problem with their clearance. Aβ monomers can aggregate into soluble oligomers, protofibrils, and eventually insoluble amyloid fibrils that assemble into amyloid plaques [START_REF] Serpell | Alzheimer's amyloid fibrils: structure and assembly[END_REF]. Aβ accumulation and aggregation, through an amyloid cascade, induce synaptic alterations, microglial and astrocytic activation, progressive neuronal death, and cognitive impairment [START_REF] Haass | Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide[END_REF].

Although Aβ40 and Aβ42 have gained the most attention, N-and C-terminally shortened Aβ peptides have received far less attention. These shortened Aβ molecules are produced by either direct enzymatic cleavage of APP or subsequent processing of Aβ by exopeptidases, and many of these peptides have been shown to be involved in the pathology of AD (Dunys et al., 2018).

For instance, human CSF fluids and blood plasma samples from AD patients have been found to contain the C-terminally truncated Aβ37, Aβ38, and Aβ39.

In addition to full-length Aβ1-42, research has shown the presence of N-truncated Aβ variants in amyloid plaques [START_REF] Bayer | N-Truncated Abeta Starting at Position Four-Biochemical Features, Preclinical Models, and Potential as Drug Target in Alzheimer's Disease[END_REF]; the predominant variants were Aβ4-42 and AβpE3-42, a pyroglutamate-modified Aβ where the first two residues are removed and the glutamate in position three is converted to pyroglutamate. Both Aβ1-42 and AβpE3-42 have been found to cause neurodegeneration and memory impairments. Given the toxicity of these N-truncated Aβ variants, their capacity to aggregate, and their role in AD pathogenesis, therapies targeting these variants are being studied. For example, targeting aminopeptidase A (APA) or dipeptidyl peptidase 4 (DPP4), both enzyme contributing to the synthesis of AβpE3-42, leads to reduced levels of plaque-associated AβpE3-X peptides, as well as improved memory deficits in 3xTg-AD mice (Valverde et al., 2021a;Valverde et al., 2021b).

AICD

AICD is the cytosolic counterpart of Aβ produced in the amyloidogenic pathway but also theoretically generated in the non-amyloidogenic pathways when γ-secretase cleaves C99 or C83 respectively. However, research has shown that the transcriptionnaly active form of AICD is mostly generated via the amyloidogenic pathway, implying its possible contribution to AD pathology (Flammang et al., 2012;[START_REF] Goodger | Nuclear signaling by the APP intracellular domain occurs predominantly through the amyloidogenic processing pathway[END_REF]. After being highly debated for several years, AICD's ability to act as a transcription factor has now been established (Pardossi-Piquard and Checler, 2012). In fact, (von Rotz et al., 2004) showed that AICD may interact with the APP-adaptor protein Fe65, and the AICD-Fe65 complex can translocate into the nucleus, where it assembles into the AFT trimeric complex with histone acetyl-transferase Tip60 (AICD-Fe65-Tip60). In this study, they discovered an AICD-mediated increase in APP and β-site APP cleaving enzyme (BACE) transcripts, showing that AICD might indirectly enhance C99 and Aβ production. However, another study from my laboratory found that AICD could help eliminate Aβ by promoting the transcription of the Aβ-degrading enzyme neprilysin (Pardossi-Piquard et al., 2005).

On the other hand, AICD has been found to produce pathological features of AD in an Aβindependent manner, where AICD up-regulates the glycogen synthase kinase-3β (GSK-3β), the latter being involved in neuronal apoptosis and responsible for hyperphosphorylation and agedependent aggregation of tau and neurodegeneration [START_REF] Ghosal | Alzheimer's diseaselike pathological features in transgenic mice expressing the APP intracellular domain[END_REF]Kim et al., 2003;Ryan and Pimplikar, 2005).

Additionally, it has been demonstrated that AICD physically interacts with the p53 promoter, leading to p53-dependent cell death (Alves da Costa et al., 2006). The AICD-mediated cell death has been proved to be neuron-specific [START_REF] Ohkawara | The amyloid precursor protein intracellular domain alters gene expression and induces neuron-specific apoptosis[END_REF], or both, neuronal and nonneuronal specific when promoting FoxO-induced transcription of the pro-apoptotic gene Bim (Wang et al., 2014).

Many more target genes of AICD, that are involved in DNA repair, cell cycle re-entry, cytoskeletal dynamics, and synaptic activity are reviewed and discussed in some articles [START_REF] Bukhari | Small things matter: Implications of APP intracellular domain AICD nuclear signaling in the progression and pathogenesis of Alzheimer's disease[END_REF][START_REF] Müller | The amyloid precursor protein intracellular domain (AICD) as modulator of gene expression, apoptosis, and cytoskeletal dynamics-Relevance for Alzheimer's disease[END_REF].

C99

The C99 transmembrane fragment includes both the Aβ and AICD sequences. However, C99 is not only the precursor of two fragments known to be involved in AD, but it may also have its own pathogenicity, especially since the fragment's aggregation can be found in individuals with the disease. In fact, a recent study found that the accumulation of C99, rather than Aβ, correlated with neuronal death in AD patients (Pulina et al., 2020).

Several research conducted by my team highlighted the role of C99 in the etiology of AD. First, C99 was found to accumulate in the hippocampus of 3xTgAD mice, in the early stages, even before Aβ, raising the possibility that C99 could be an early contributor to the disease (Lauritzen et al., 2012). C99 were detected in the endosomal-lysosomal network, and by pharmacologically inhibition of -secretase, in absence of Aβ formation, the accumulation of C99 was linked to lysosomal dysfunction (Lauritzen et al., 2019;Lauritzen et al., 2016). Since C99 can be degraded by the lysoso-autophagic system, its accumulation is both, the cause and the consequence of impaired lysosomal-autophagic function (Lauritzen et al., 2016).

Furthermore, C99 was discovered to promote endocytic pathway impairment in DS and AD patients (Jiang et al., 2010).

Importantly, C99 accumulation has also been associated to endolysosomal dysfunction in more physiological cellular models devoid of any overexpression such as induced pluripotent stem cells (iPSCs) harboring AD-related APP or PS1 mutations (Kwart et al., 2019) and iPSCs derived neurons obtained from AD patients harboring APP or PS1 mutations (Hung and Livesey, 2018).

In addition to endosomal and lysosomal alterations, high levels of C99 expression were related with inflammatory responses and synaptic dysfunctions in C99 expressing mice when Aβ formation was also prevented (Lauritzen et al., 2016). In addition to synaptic dysfunction, the intraneuronal accumulation of C99 was also found to contribute to spatial learning deficits and apathy-like behavior, in 3xTgAD mice, and more importantly in double transgenic AD mouse model (2xTgAD)(APPswe, TauP301L) that show the same accumulation of C99 as 3xTgAD mice without any Aβ accumulation (Bourgeois et al., 2018). Oligomers of C99 were not only detected in the endo-lysosomal network (ELN), but in exosomes as well, which indeed originate from endosomes (Lauritzen et al., 2019). Exosomes are known to transmit neurotoxic molecules from cell to cell in the brain, and since microglia were shown to take neuron-derived extracellular vesicles (EVs) (Lauritzen et al., 2019), C99 might be undergoing neuron-neuron propagation causing LTP impairment and behavioral defects, or neuron-glial cell propagation implying the neuroinflammation caused by accumulation of C99.

In addition to these findings, and with mitochondrial dysfunction being one of the key characteristics of AD, a recent study from our team envisioned that C99 accumulation causes mitophagy disruption by interfering with multiple steps in the mitophagic process (Vaillant-Beuchot et al., 2021). 1.4. sAPPβ Only a few studies were interested in this soluble fragment of APP generated by the β-secretase cleavage but it has been demonstrated that sAPPβ stimulates the differentiation of human embryonic stem cells toward a neuronal fate [START_REF] Freude | Soluble Amyloid Precursor Protein Induces Rapid Neural Differentiation of Human Embryonic Stem Cells[END_REF]. Additionally, it has been shown that sAPPβ binds to the gamma-aminobutyric acid type B receptor subunit 1a (GABABR1a), leading to a suppressed synaptic transmission and an enhanced short-term facilitation in hippocampal synapses [START_REF] Rice | Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission[END_REF].

As reviewed in the article from Chasseigneaux and colleagues [START_REF] Chasseigneaux | Functions of Aβ, sAPPα and sAPPβ : similarities and differences[END_REF], sAPPβ can perform similar roles to sAPPα, such as: promoting axon outgrowth both during development and in response to brain injury [START_REF] Chasseigneaux | Functions of Aβ, sAPPα and sAPPβ : similarities and differences[END_REF], and triggering microglial activation [START_REF] Barger | Microglial activation by Alzheimer amyloid precursor protein and modulation by apolipoprotein E[END_REF]. However, since sAPPβ lacks the last 16 residues of sAPPα, its neuroprotective properties are significantly reduced compared with sAPPα [START_REF] Copanaki | sAPPα antagonizes dendritic degeneration and neuron death triggered by proteasomal stress[END_REF][START_REF] Furukawa | Increased activity-regulating and neuroprotective efficacy of alpha-secretase-derived secreted amyloid precursor protein conferred by a C-terminal heparin-binding domain[END_REF][START_REF] Taylor | Endogenous secreted amyloid precursor protein-alpha regulates hippocampal NMDA receptor function, long-term potentiation and spatial memory[END_REF].

The Non-Amyloidogenic Pathway

The predominant and physiological cleavage mechanism of APP is the non-amyloidogenic pathway. α-secretase first cleaves APP, resulting in the soluble N-terminal fragment sAPPα and the 83 amino acid C-terminal transmembrane protein C83. The latter can be then cleaved by γsecretase, generating a 3kDa Nter peptide, p3, and AICD (Figure 5-A).

sAPPα

The sAPPα fragment released after the α-secretase cleavage of APP has been found to exhibit neurotrophic and neuroprotective characteristics, contributing to neuron physiology (Müller et al., 2017). In fact, neurons produce sAPPα as a survival and defense strategy in response to stress caused by elevated Aβ levels [START_REF] Rose | Transient increase in sAPPalpha secretion in response to Abeta1-42 oligomers: an attempt of neuronal self-defense?[END_REF]. Interestingly, sAPPα inhibits BACE1 by direct interaction, shifting and modulating APP processing, and thus decreasing Aβ production. Indeed, the overexpression of sAPPα in an AD mouse model reduced amyloid plaques, as well as Aβ oligomeric forms [START_REF] Obregon | Soluble amyloid precursor protein-alpha modulates beta-secretase activity and amyloid-beta generation[END_REF]. Another study that demonstrates how sAPPα prevents the loss of dendritic spines and protects neurons from Aβ and phosphotau-dependent toxicity in AD provides more evidence for the neuroprotective properties of sAPPα [START_REF] Tackenberg | The secreted APP ectodomain sAPPalpha, but not sAPPbeta, protects neurons against Abeta oligomer-induced dendritic spine loss and increased tau phosphorylation[END_REF]. Furthermore, sAPPα reduces phospho-tau-dependent toxicity by decreasing GSK3β activity, an enzyme that is responsible for Tau phosphorylation [START_REF] Deng | Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3beta signaling pathway[END_REF]. sAPPα appears also to be capable of binding to APP, interrupting its dimerization [START_REF] Gralle | Neuroprotective secreted amyloid precursor protein acts by disrupting amyloid precursor protein dimers[END_REF], and activating the PI3K/Akt/mTor pathway via coupling to a G-protein, a pathway mostly known for its function in cell survival by blocking apoptosis [START_REF] Milosch | Holo-APP and G-protein-mediated signaling are required for sAPPalpha-induced activation of the Akt survival pathway[END_REF]. Along with its neuroprotective qualities, sAPPα promotes axonal outgrowth via a separate mechanism that involves the recruitment of α3-NKA to the neuronal surface [START_REF] Dorard | Soluble Amyloid Precursor Protein Alpha Interacts with alpha3-Na, K-ATPAse to Induce Axonal Outgrowth but Not Neuroprotection: Evidence for Distinct Mechanisms Underlying these Properties[END_REF].

C83

C83's functions have not been fully investigated. What is well known is that C83 can be produced directly from the α-secretase cleavage of APP full length but also by cleaving C99 with α-secretase, lowering C99 levels and limiting Aβ production [START_REF] Jager | alpha-secretase mediated conversion of the amyloid precursor protein derived membrane stub C99 to C83 limits Abeta generation[END_REF]. C83 can be then cleaved by γ-secretase, resulting in p3 and AICD. However, it has been demonstrated that the latter is primarily derived from the amyloidogenic pathway (Flammang et al., 2012;[START_REF] Goodger | Nuclear signaling by the APP intracellular domain occurs predominantly through the amyloidogenic processing pathway[END_REF], and that C83's interaction with γ-secretase limits the latter's catalytic ability via feedback inhibition, thereby down-regulating Aβ production [START_REF] Tian | Dual role of alpha-secretase cleavage in the regulation of gamma-secretase activity for amyloid production[END_REF].

P3

More research is needed to better understand the P3 peptide's characteristics and effects, as studies on this peptide are often inconsistent. P3 lacks Aβ's first 16 residues but includes the 17-21 residues essential for fibril assembly and neurotoxicity [START_REF] Higgins | P3 beta-amyloid peptide has a unique and potentially pathogenic immunohistochemical profile in Alzheimer's disease brain[END_REF]. Despite the fact that it contains residues involved in the formation of β sheets, a previous study indicated that p3 is unlikely to form stable soluble oligomers [START_REF] Dulin | P3 peptide, a truncated form of A beta devoid of synaptotoxic effect, does not assemble into soluble oligomers[END_REF]. However, a more recent work published in 2020 found that p3 aggregated to form fibrils and oligomers with a comparable size and distribution as Aβ (Kuhn et al., 2020).

Furthermore, p3 has been detected in some dystrophic neurites, implying that this peptide may play a pathogenic or neurotoxic role [START_REF] Higgins | P3 beta-amyloid peptide has a unique and potentially pathogenic immunohistochemical profile in Alzheimer's disease brain[END_REF]. Nonetheless, a further investigation found that only Aβ oligomers, and not p3, are synaptotoxic in AD [START_REF] Walsh | Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo[END_REF].

All of these contradicting findings lead to reassess the p3 and α-secretase pathways' categorization as "non-amyloidogenic" (Kuhn and Raskatov, 2020).

APP Cleaving Enzymes

α-secretase

The α-secretase cleavage is carried out by three members of the ADAM (a disintegrin and metalloproteinase) family: ADAM9, ADAM10, and ADAM17, also known as TNF-α converting enzyme (TACE) [START_REF] Allinson | ADAMs family members as amyloid precursor protein alpha-secretases[END_REF]. It has been determined that ADAM10 is the essential enzyme responsible for cleaving APP at the -secretase site both physiologically and functionally in neurons [START_REF] Kuhn | ADAM10 is the physiologically relevant, constitutive α-secretase of the amyloid precursor protein in primary neurons[END_REF]. Therefore, ADAM10 functions as the constitutive neural α-secretase under non-stimulated conditions, whereas ADAM17 is known to be the "regulated α-secretase". This signifies that stimuli such as protein kinase C (PKC) activation or M1 muscarinic receptor induction is required for ADAM17 to cleave APP [START_REF] Buxbaum | Evidence That Tumor Necrosis Factor α Converting Enzyme Is Involved in Regulated α-Secretase Cleavage of the Alzheimer Amyloid Protein Precursor[END_REF][START_REF] Caccamo | M1 Receptors Play a Central Role in Modulating AD-like Pathology in Transgenic Mice[END_REF]. Considering that α-secretase is involved in the non-amyloidogenic pathway, ADAM17 may, in theory, have a preventative effect on the development of AD.

However, due to microglial activation and TNFα induced neuroinflammation, ADAM17 may have the opposite consequences, as reviewed in [START_REF] Hsia | Functions of 'A disintegrin and metalloproteases (ADAMs)' in the mammalian nervous system[END_REF]. ADAM9 is also involved in the α-secretase cleavage but indirectly by activating ADAM10 [START_REF] Cissé | The Disintegrin ADAM9 Indirectly Contributes to the Physiological Processing of Cellular Prion by Modulating ADAM10 Activity[END_REF][START_REF] Moss | ADAM9 Inhibition Increases Membrane Activity of ADAM10 and Controls α-Secretase Processing of Amyloid Precursor Protein[END_REF].

ADAMs are type I integral membrane proteins that belong to the zinc metalloproteinases. In a nutshell, they are synthesized as inactive precursors where the prodomain acts as an inbuilt inhibitor, and the enzyme becomes active only when the inhibitory prodomain is processed by a proprotein convertase in the Trans-Golgi network (TGN) [START_REF] Allinson | ADAMs family members as amyloid precursor protein alpha-secretases[END_REF][START_REF] Endres | Tumor necrosis factor-alpha converting enzyme is processed by proprotein-convertases to its mature form which is degraded upon phorbol ester stimulation[END_REF]. After being activated, ADAM10 seems to cleave APP within the TGN compartment or predominantly on the plasma membrane [START_REF] Sisodia | Beta-amyloid precursor protein cleavage by a membrane-bound protease[END_REF][START_REF] Tan | The trans-Golgi network is a major site for alpha-secretase processing of amyloid precursor protein in primary neurons[END_REF]. These ADAMs are not only linked to Alzheimer's disease; for example, ADAM10 and ADAM17 are known to cleave not only APP but over 40 distinct type I and II integral membrane proteins, contributing to neurological diseases, cancers, prion diseases, and inflammatory diseases [START_REF] Vincent | alpha-Secretase in Alzheimer's disease and beyond: mechanistic, regulation and function in the shedding of membrane proteins[END_REF].

β-secretase

The type 1 transmembrane aspartic protease BACE1 (β-site APP cleaving enzyme 1), also known as aspartyl protease 2 and membrane-associated aspartic protease 2, is considered as the main enzyme in charge of the β-secretase cleavage of APP. It cleaves APP695 at the bond between Met596 and the first Aβ residue, Asp597 (β-site) to generate C99, also known as β-CTF (C-terminal fragment). BACE1 also cleaves APP at another bond between Tyr606 and the 11th residue of Aβ, Glu607 (β'-site), generating the non-amyloidogenic C89 fragment, favored in physiological conditions, and in individuals with the protective Icelandic APP mutation [START_REF] Kimura | Alternative Selection of beta-Site APP-Cleaving Enzyme 1 (BACE1) Cleavage Sites in Amyloid beta-Protein Precursor (APP) Harboring Protective and Pathogenic Mutations within the Abeta Sequence[END_REF] (Figure 6). Additionally, it has been shown that BACE1 can cleave Aβ40 and Aβ42 to generate a C-terminally truncated Aβ34 form. Aβ34 levels are significantly increased in people with MCI and correlate with the overall Aβ clearance rates in amyloid positive individuals. In the early stages of the disease, it is believed that the increase in Aβ34 help to protect against Aβ-mediated toxicity; however, it may not be sufficient to counteract the toxicity brought on by increased levels of Aβ in the advanced stages of the pathology (Caillava et al., 2014;[START_REF] Liebsch | Abeta34 is a BACE1-derived degradation intermediate associated with amyloid clearance and Alzheimer's disease progression[END_REF].

BACE1 is produced as a pro-enzyme and is present in the ER in its immature state, already Nglycosylated. It is then delivered to the TGN to be cleaved by furins for maturation [START_REF] Capell | Maturation and pro-peptide cleavage of beta-secretase[END_REF]. Nevertheless, research has revealed that even pro-BACE1 can cleave APP and act as a β-secretase in the ER and early Golgi [START_REF] Creemers | Processing of beta-secretase by furin and other members of the proprotein convertase family[END_REF]. BACE1 can then target the plasma membrane and be internalized to the endosomal system, or it can directly target endocytic compartments from the TGN, completely bypassing the plasma membrane. The Cterminal dileucine motif of BACE is essential for its trafficking to endosomes, where its activity is highest at an acidic pH (4.5). The deletion of this motif results in a twofold increase in recycling to the cell surface and accumulation on the plasma membrane [START_REF] Huse | Maturation and endosomal targeting of beta-site amyloid precursor protein-cleaving enzyme. The Alzheimer's disease betasecretase[END_REF].

While much focus has been given to its role in the amyloidogenic process, APP is not the only substrate of BACE1; in fact, about 70 BACE1 substrates have been identified to date. Many of the BACE1 substrates are associated with neurological function. The most extensively researched substrates, for example, are Neuregulin 1 (NRG1), cell adhesion molecule L1-like protein (CHL1), Jagged 1 (Jag1), and Seizure protein 6 (SEZ6) which are required for myelination and remyelination, axonal growth and migration, astrogenesis and neurogenesis, and synaptic plasticity, respectively [START_REF] Yan | Physiological Functions of the beta-Site Amyloid Precursor Protein Cleaving Enzyme 1 and 2[END_REF].

Through a variety of its non-neuronal substrates, BACE1 has also been linked to inflammation, cardiovascular health, glucose homeostasis, and insulin signaling [START_REF] Taylor | BACE1: More than just a betasecretase[END_REF].

According to a 2018 study, BACE1 regulates the amount of active insulin receptors in the liver by cleaving their ectodomain in a glucose concentration-dependent manner; and that in diabetes patients, high glucose levels contribute to lower active insulin receptor levels and impair insulin signaling [START_REF] Meakin | The beta secretase BACE1 regulates the expression of insulin receptor in the liver[END_REF].

BACE2 was discovered to be a close homologue of BACE1, exhibiting 64% amino acid sequence identity. In contrast to BACE1, BACE2 is highly expressed in the heart, kidney, and placenta, but modestly in the brain. Furthermore, BACE 2 does not contribute as much as BACE1 to the formation of Aβ because it cuts APP essentially in the Aβ sequence, resulting in the C80 fragment. However more recently BACE2 has been described as a conditional βsecretase cleaving APP at the β-site when its juxtamembrane helix domain is mutated as it is the case for the Flemish and Arctic mutations [START_REF] Wang | BACE2, a conditional beta-secretase, contributes to Alzheimer's disease pathogenesis[END_REF].

γ-secretase

γ-Secretase belongs to a new class of proteases, intramembrane-cleaving proteases (I-CliPs) characterized by an unusual cleavage process occurring in the lipid bilayer of membranes. Like β-secretase, γ-Secretase is an aspartyl protease highly active at acidic pH but with a particularity to require the formation of a high molecular weight proteolytic complex.

After being cleaved by β-secretase, the membrane-bound C99 is cleaved by γ-secretase in a sequential manner in order to generate Aβ. Cleavage at ε-site is the first, releasing AICD49 or AICD50 and Aβ48 or Aβ49 respectively, followed by cleavage at ζ-site giving rise to Aβ45 or Aβ46, and Aβ43, then Aβ40 or Aβ42 generated at γ-site. A final cleavage can occur, producing even shorter species: Aβ37 and Aβ38, the latter being with Aβ42, the predominant forms after Aβ40 (Zhang et al., 2014a). Thus, in a more straightforward manner, C99 is cleaved at the εsite to produce AICD along with Aβ48 and Aβ49, which are then, trimmed every three amino acids along two pathways: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→Aβ38 (Figure 7).

The γ -secretase complex consists of four essential subunits: PS1 or PS2 constitute the catalytic core of γ-secretase, and three interacting proteins partner, anterior pharynx-defective 1 (APH-1), Nicastrin, and Presenilin Enhancer Protein 2 (PEN2), crucial for complex maturation and stabilization (Lu et al., 2014;Wolfe, 2020) (Figure 8-A). PS1 and PS2 are two homologues of intramembrane cleaving proteases that are produced as precursor proteins and mature through autocatalysis. In addition to their endoproteolysis, their proper assembly into the multiprotein complex is required for γ-secretase activity to occur [START_REF] De Strooper | Presenilins and gamma-secretase: structure, function, and role in Alzheimer Disease[END_REF]. Composition of γ-secretase complex is of high importance for its activity and can differ depending on its cellular localization. In fact, in addition to the two possible PS, there are two homologs of Aph-1 (Aph-1a and Aph-1b) identified in humans, as well as splice variants for . Only one of the two PS and one of the three Aph1 isoforms can coexist in a complex, resulting in at least six distinct γ-secretase complexes in humans. A recent study found that APH-1b-containing γ-secretase complexes produce longer Aβ peptides than APH-1a-containing complexes (Zhang et al., 2014a). γ-secretase is located in multiple subcellular compartments, including the plasma membrane, ER, TGN, endosomes, and lysosomes. Interestingly, it has been shown that complexes containing PS2 have a distinct distribution than those containing PS1. PS1/γ-secretase, for example, is localized to the plasma membrane and is more widely distributed in the cell, releasing extracellular Aβ, whereas PS2/γ-secretase is primarily restricted to the TGN, as well as late endosomes and lysosomes, thus creating Aβ in the endosomal pathway (Meckler and Checler, 2016;Sannerud et al., 2016).

Cryo-EM single-particle analysis revealed the three-dimensional structure of the human γsecretase complex, which includes a horseshoe-shaped transmembrane domain, which contains 20 transmembrane segments (TMs), and a large extracellular domain (ECD) from Nicastrin [START_REF] Bai | An atomic structure of human gamma-secretase[END_REF]Guner and Lichtenthaler, 2020;Lu et al., 2014) (Figure 8-B). This structure is in accordance with Nicastrin's alleged substrate recruitment function, which depends on Glu333 and Tyr337 that are hidden in a hydrophilic pocket. In the absence of a substrate, this pocket is covered by a "Lid" that is positioned directly above the concave side horseshoe, while the cleaving site is found on the convex side. The "Lid" opens upon substrate binding, reorienting the substrate for cleavage, possibly via lining up the hydrophilic pocket and the active site of PS1. The subunits are mostly buried within the membrane. An exception is Nicastrin. It has a large extracellular domain that acts as a gatekeeper which only allows proteins with short ectodomains to be transferred to the active site and provides a mechanism to anchor the substrates in the active site. While the majority of substrates undergo ectodomain shedding to fit under the Nicastrin lid-like structure, a substrate with a naturally short ectodomain may be directly cleaved by γ-secretase without prior shedding (Figure 8-B).

Indeed, γ-secretase cleavage is reliant on shedding of the substrate's ectodomain; the enzyme appears to cleave substrates within the transmembrane domain near the cytoplasmic membrane border, releasing a cytosolic fragment, the intracellular domain (ICD), capable of entering the nucleus and controlling the transcription of different genes; a process generally referred to as regulated intramembrane proteolysis (RIP).

APP is just one of more than 140 type 1 transmembrane proteins that are substrates candidates for γ-secretase (Guner and Lichtenthaler, 2020). Notch was the second, widely known substrate of γ-secretase [START_REF] Ray | Cell surface presenilin-1 participates in the gamma-secretase-like proteolysis of Notch[END_REF]. Notch is cleaved by γ-secretase, releasing the Notch IntraCellular Domain (NICD), which can bind to transcription factors and regulate genes implicated in neural differentiation, which is especially crucial during embryonic development.

Moreover, Notch is critical cell differentiation and tissue homeostasis in healthy adults and plays an important role in the regulation of inflammatory responses in many diseases.

Aside from APP and Notch, several of the γ-secretase substrates appear to produce an ICD that regulates cell fate determination, adhesion, migration, neurite outgrowth, axon guidance, or formation and maintenance of synapses, angiogenesis and tumorigenesis, immune responses, etc… (Guner and Lichtenthaler, 2020;Jurisch-Yaksi et al., 2013).

Given the diverse variety of γ-secretase substrates, some may serve as signaling molecules, while others may only be intermediates heading for degradation. It has been hypothesized that γ-secretase cleavage could possibly serve as the membrane's "proteasome", maintaining the membrane homeostasis (Kopan and Ilagan, 2004). According to this theory, ectodomain shedding prepares the γ-secretase to cleave within the membrane-tethered CTF, either producing a biologically active protein fragment or releasing peptides for further degradation by soluble proteases. This is analogous to how ubiquitin conjugation prepares target proteins for proteasomal degradation.

APP Localization

APP Trafficking and Co-Residence with Canonical Secretases

At cellular level, APP is located in the endoplasmic reticulum (ER), the TGN, the plasma membrane, endosomes and lysosomes [START_REF] Thinakaran | Amyloid Precursor Protein Trafficking, Processing, and Function[END_REF]. APP has also been detected in mitochondria, cytosol or nucleus. At the neuronal level, it is found in the soma-dendritic compartment but also at the level of the axon.

After being synthesized in the membrane-bound polysomes, the signal peptide of APP gets cleaved during its translocation in the ER. APP then transports to the Golgi apparatus, the trans-Golgi network where it undergoes a large number of post-translational modifications (N and Oglycosylations, phosphorylations and sulphations), necessary to obtain its mature form and its three-dimensional structure. Only 10% of APP approximately reaches the plasma membrane, whereas the majority is predominantly localized at steady state in the Golgi apparatus and TGN (Figure 9). APP at the plasma membrane is mainly cleaved by α-secretase (Figure 9-1). Uncleaved APP on the cell surface is internalized into the early endosomes thanks to its YENPTY motif [START_REF] Haass | Trafficking and Proteolytic Processing of APP[END_REF][START_REF] Lai | Characterization of sorting signals in the beta-amyloid precursor protein cytoplasmic domain[END_REF]

(Figure 9-2).
From endosomes, APP can be sorted into different pathways through the ELN: the majority is directed to the endosome-lysosomal pathway for degradation in lysosomes (Figure 9-7) or incorporation into exosomes (Figure 9-8), the remaining part is recycled to the membrane (Figure 9-3) or directed towards the Golgi apparatus via a so-called retrograde transport (Figure 9-5). Finally, the APP present in the TGN can also reach the endosomes directly, without passing through the plasma membrane, via so-called anterograde transport (Figure 9-

4).

While α-secretase cleavage takes place at the plasma membrane, β-cleavage mainly occurs in the endosomes and lysosomes where the acidic pH is optimal for the activity of β-secretase.

And since this acidic pH also suits γ-secretase complex, amyloidogenic cleavage of APP mainly occurs in the endosomal compartments. However, in addition to early endosomes, late endosomes, lysosomes and autophagic vesicles, the γ-secretase complex have been found also in the plasma membrane.

In order to properly understand APP trafficking and metabolism, it is crucial to understand the complex biology of the ELN.

Endo-Lysosomal Network (ELN) -Generalities

The ELN consists of complex and highly dynamic vesicles distinguished by their morphology, their pH and by the expression of specific markers, in particular the small GTPases, Rabsfor "Ras-associated binding".

Cargos are internalized and then subsequently sorted by sorting complexes to either reach the cell surface, to be degraded by lysosomes, or to be transported to the trans-Golgi for further After synthesis, APP transits along the secretory pathway through the ER to the TGN, then from the TGN to the plasma membrane where it is further metabolized by α-secretase (1). APP can be also endocytosed into early endosomes vesicles (2) or directly transported from the TGN to early endosomes (4). Early endosomes can be recycled back to the plasma membrane (3) or to the Golgi compartment (5). The maturation of early endosomes generates multivesicular bodies (MVB)/late endosomes ( 6) containing multiple vesicles. Once in the MVB/endosomes, APP can be degraded in lysosomes (7) or released through exosomes (8). Non-amyloidogenic processing mainly occurs at the cell surface where α-secretases are present (1). Amyloidogenic processing involves transit through the endocytic organelles where APP encounters βand γ-secretases (6 to 8). Figure created with BioRender.com free software.

sorting. Furthermore, the ELN engages in exocytosis, which allows cellular contents to be released into the extracellular environment. Thus, this system enables the recycling or destruction of dysfunctional, potentially harmful internal materials. This system also regulates the amount and topography of surface receptors, including those that mediate neurotransmission and synaptic plasticity, through internalization and recycling back to the cell surface. ELN plays an equally significant role in cell signaling by transmitting vast information from the surface and external environment to the nucleus.

Extracellular and plasma membrane proteins are internalized through clathrin-mediated or clathrin-independent endocytosis (Figure 9-2). The internalization, or the endocytosis starts with the collecting of the membrane-bound cargos, the formation of endocytic vesicles from the plasma membrane, and the fission of these vesicles, which merge with early endosomes.

Early endosomes have a slightly acidic pH values of ≈6.2, and they are mainly marked by Rab5 and by its effector protein early endosome antigen 1 (EEA1) which is a tethering protein required for fusion of endocytic vesicles with early endosomes.

Once cargos reach early endosomes, they can be recycled back to the plasma membrane (Figure 9-3), or routed by retrograde transport to the TGN (Figure 9-5) [START_REF] Langemeyer | Rab GTPase Function in Endosome and Lysosome Biogenesis[END_REF][START_REF] Scott | Endosome maturation, transport and functions[END_REF].

Endosome maturation is the transformation of early endosomes into late endosomes (Figure -6) by a sequence of biochemical and morphological changes particularly Rab7 takes the place of Rab5 in late endosomes. In addition, luminal invaginations of the early endosomes membrane can be formed and then detach to form cargo-containing intraluminal vesicles (ILVs). This ILV formation that starts in early endosomes is mainly dependent of the endosomal sorting complex required for transport (ESCRT) machinery. During this ILV formation, some proteins are incorporated into the invaginating membrane, while the cytosolic components are engulfed and enclosed within the ILVs. Vacuolar areas harboring these ILVs also detach and mature from early endosomes, resulting in the formation of free MVBs (multivesicular bodies) (Figure 9-6). Late endosomes/MVBs can either fuse with lysosomes for protein degradation (Figure 9-7), or with the plasma membrane, releasing ILVs into the extracellular space and resulting in the secretion of exosomes (Figure 9-8) [START_REF] Elkin | Endocytic pathways and endosomal trafficking: a primer[END_REF][START_REF] Langemeyer | Rab GTPase Function in Endosome and Lysosome Biogenesis[END_REF][START_REF] Scott | Endosome maturation, transport and functions[END_REF].

C) APP AND APP-FRAGMENTS DEGRADATION AND ELIMINATION

The APP protein and its resulting fragments have been described to be degraded by both the proteasome and the lysosomal and autophagic pathways, and many studies have also revealed the elimination of these fragments via exosomal secretion.

1. Proteasome and its dysfunction in AD 1.1. The ubiquitin-proteasome system (UPS)

In eukaryotic cells, the ubiquitin-proteasome system (UPS) is the principal system for the degradation of short-lived proteins as well as the elimination of damaged soluble proteins.

Mostly, intracellular soluble proteins are degraded by the UPS, but transmembrane proteins can occasionally be degraded by this system if released from the membrane into the cytosol [START_REF] Dederer | Transmembrane dislocases: a second chance for protein targeting[END_REF][START_REF] Nakatsukasa | Dissecting the ER-associated degradation of a misfolded polytopic membrane protein[END_REF].

This process starts with the adenosine tri-phosphate (ATP)-dependent insertion of ubiquitin chains covalently linked to lysine residues in substrate proteins. This step serves as a recognition signal and is then followed by proteolytic degradation of ubiquitinated proteins by the proteasome. The ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin ligases (E3) are three types of enzymes that contribute to the multistep process of ubiquitination. First, E1 forms a thiol-ester bond with the carboxyl-terminal glycine of ubiquitin. E1 then passes the ubiquitin to E2 that also binds to the carboxyl terminus of the ubiquitin by a transthiolation reaction. Finally, E3 catalyzes the transfer of ubiquitin from the E2 enzyme to the substrate's lysine residue. The same process attaches additional ubiquitin molecules to the initial one via internal ubiquitin lysines, resulting in the formation of a ubiquitin chain (Figure 10-A).

The proteasome, also known as the 26S proteasome, is a hollow barrel-shaped multiprotein structure consisting of two major assemblies: the core particle (CP), or 20S proteasome, which is a central cylindrical structure with peptide hydrolysis activity, and the regulatory particle (RP), also referred to as 19S proteasome, which borders the CP from one or both sides (Figure 10-B). The cylindrical structure of the CP is composed by two types of subunits, α and β, which are organized in four stacked heptameric rings around a central cavity; seven α subunits constitute the cylinder's two outer rings, while seven distinct β subunits form the two inner rings. Three of the inner ring's β-type subunits have proteolytic active sites facing the inside of the cylinder: β1 has caspase-like specificity, cleaving after acidic residues; β2 is trypsin-like, cleaving after basic residues; and β5 is chymotrypsin-like, cleaving after hydrophobic residues (Figure 10-B).

Once poly-ubiquitinated with at least four ubiquitins, the protein is detected, de-ubiquitinated, and then unfolded in an ATP-dependent process by the RP, allowing ubiquitin molecules to be recycled and the target protein to be delivered to the CP for degradation. The protein gets then processively degraded by the catalytic core to produce heterogeneous mixture of peptides. The proteasome does, however, occasionally subject regulatory proteins to partial proteolysis, activating them by removing their inhibitory domains [START_REF] Finley | Recognition and processing of ubiquitin-protein conjugates by the proteasome[END_REF].

Not only APP is degraded by the proteasome [START_REF] Hiltunen | Ubiquilin 1 modulates amyloid precursor protein trafficking and Abeta secretion[END_REF][START_REF] Jung | Acute ER stress regulates amyloid precursor protein processing through ubiquitin-dependent degradation[END_REF][START_REF] Kaneko | Loss of HRD1mediated protein degradation causes amyloid precursor protein accumulation and amyloid-beta generation[END_REF] but also the secretases related to the amyloidogenic pathway, β-, and γ-secretases, are potentially degraded by the UPS [START_REF] Flood | Proteasomemediated effects on amyloid precursor protein processing at the gamma-secretase site[END_REF][START_REF] Haapasalo | Emerging role of Alzheimer's disease-associated ubiquilin-1 in protein aggregation[END_REF][START_REF] Qing | Degradation of BACE by the ubiquitin-proteasome pathway[END_REF]. As a result, the UPS's activity contributes to the clearance of Aβ peptide. Additionally, some studies suggest that the proteasome may degrade the C99 as well [START_REF] Bustamante | Turnover of C99 is controlled by a crosstalk between ERAD and ubiquitinindependent lysosomal degradation in human neuroglioma cells[END_REF][START_REF] Evrard | Contribution of the Endosomal-Lysosomal and Proteasomal Systems in Amyloid-beta Precursor Protein Derived Fragments Processing[END_REF][START_REF] Nunan | The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase[END_REF][START_REF] Nunan | Proteasomemediated degradation of the C-terminus of the Alzheimer's disease beta-amyloid protein precursor: effect of C-terminal truncation on production of beta-amyloid protein[END_REF].

Dysfunctions of Proteasome in AD

Since aggregated Aβ and tau proteins accumulate, AD is also considered as a proteinopathy and it was speculated that impairments in the UPS play a significant role in the disease's pathogenesis through various mechanisms [START_REF] Cao | Endo-lysosomal pathway and ubiquitinproteasome system dysfunction in Alzheimer's disease pathogenesis[END_REF][START_REF] Gadhave | The ubiquitin proteasomal system: a potential target for the management of Alzheimer's disease[END_REF]. Indeed, as a consequence of processing and degrading critical proteins involved in the pathogenesis of AD, including APP, BACE1, PS1, PS2, and C99, UPS indirectly reduces the produced levels of Aβ. Moreover, Aβ has also been discovered to be a substrate of this enzymatic complex, which enables UPS to directly degrade and eliminate Aβ peptides [START_REF] Lopez Salon | Relationship between beta-amyloid degradation and the 26S proteasome in neural cells[END_REF][START_REF] Zhao | Amyloid-beta peptide is a substrate of the human 20S proteasome[END_REF]. However, as it turned out, there is a two-way link between Aβ and UPS, as Aβ is degraded by UPS while also having an impact on this system [START_REF] Hong | Relationship between amyloid-beta and the ubiquitinproteasome system in Alzheimer's disease[END_REF]. In fact, Aβ may operate as a UPS inhibitor or induce a UPS impairment in AD due to competition of natural proteasomal substrates with rising amounts of toxic oligomeric Aβ peptides. As a consequence of alterations in the proteasome in AD, abnormal accumulations of Aβ42 and tau occur, both of which are degraded by the proteasome. Proteasome dysfunction, for example, has been found to take place early in the development of pathological events in 3xTg-AD mice, right when Aβ accumulates, Aβ plaques first appear, and tau pathological alterations begin [START_REF] Tseng | Abeta inhibits the proteasome and enhances amyloid and tau accumulation[END_REF]. Along with Tau's accumulation, hyperphosphorylated tau oligomers were found accumulated in AD synapses due to the proteasome's impairment, suggesting that these 2. Lysosomal and autophagic pathway and their dysfunctions in AD

Lysosomal Degradation

As already mentioned (see chapter Endo-Lysosomal Network (ELN) -Generalities), in the ELN, cargos that are destined for degradation are delivered by late endosomes to lysosomes.

Late endosomes merge with lysosomes, exchanging membrane components and solutes to generate a transitory hybrid endo-lysosome, which is later re-converted into secondary lysosomes, where hydrolases are retained. This fusion and exchange often make it difficult to distinguish between late endosomes and lysosomes at the molecular level. The main components of their membranes are lysosomal-associated membrane proteins 1 and 2 (LAMP1 and LAMP2) which create a glycocalyx-like layer that shields the rest of the cell from the aggressively acidic environment of the lumen.

Endosome-to-lysosome maturation is the gradual decrease of internal pH to ≈ 5, which is required for several physiological processes such as ligand-receptor uncoupling, and lysosomal enzyme activity. This acidification needs the presence of a proton-pumping V-type ATPase that uses the energy released by ATP hydrolysis to pump protons into the lysosomal lumen.

Soluble hydrolases carry out the degradation process in lysosomes; in fact, the lysosomal lumen contains 60 different soluble hydrolases. These enzymes include sulphatases, glycosidases, phosphatases, lipases, nucleases and proteases; with cathepsins being the most abundant lysosomal proteases.

To sum up, hydrolases, the acidification machinery, and membrane proteins must all work together to allow lysosome-mediated cellular clearance activities [START_REF] Gruenberg | Life in the lumen: The multivesicular endosome[END_REF][START_REF] Settembre | Signals from the lysosome: a control centre for cellular clearance and energy metabolism[END_REF].

Interestingly, recent studies demonstrated that ILVs can also be found in lysosomes, where they are preserved from degradation and released as exosomes via a mechanism known as lysosomal exocytosis.

In fact, lysosomal exocytosis is a process in which lysosomes merge with the plasma membrane and secrete their stored material, and it is highly regulated by the transcription factor EB (TFEB). This mechanism is important not just for cellular clearance, but also for plasma membrane repair, as lysosomes migrate and reseal damaged membrane regions [START_REF] Settembre | Signals from the lysosome: a control centre for cellular clearance and energy metabolism[END_REF][START_REF] Tancini | Insight into the Role of Extracellular Vesicles in Lysosomal Storage Disorders[END_REF].

In addition, increases in released EVs were observed when cells were treated with bafilomycin A (BafA1) or chloroquine, two treatments that alkalinize lysosomal pH and hence impair lysosomal activity. As a result, studies have confirmed the hypothesis stating that the production of EVs is a response to lysosomal dysfunction induced by an excess of unneeded or damaged molecules [START_REF] Buratta | Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: The Autophagic-and Endo-Lysosomal Systems Go Extracellular[END_REF].

Autophagic pathway

Autophagy is considered as a "self-eating" mechanism that is completely reliant on lysosomal activity. Unlike the endo-lysosomal system, which recycles or degrades extracellular molecules or cellular proteins trafficking along the endolysosomal pathway, the lysosomal-autophagic pathway digests and recycles unneeded or harmful intracellular components and damaged cytosolic organelles (mitochondria, ribosomes, ER fragments, etc.).

The three types of autophagy that have been described are microautophagy, chaperonemediated autophagy (CMA), and macroautophagy. Microautophagy is identified by the direct invagination of the lysosomal membrane to integrate the cytosolic elements to be degraded, whereas CMA is characterized by the internalization of these materials mediated by receptors or chaperone proteins. The last type is macroautophagy, which involves encapsulating cytoplasmic particles and/or damaged portion of cytosolic organelles in autophagosomes intended to fuse with lysosomes. Since macroautophagy is the most common form, it is usually referred to as autophagy.

The autophagic process begins with cytosolic nucleation that progress to an open structure known as a phagophore. Phagophores engulf cytoplasmic proteins and organelles before sealing their membranes to create autophagosomes. Autophagosomes later fuse with lysosomes to generate autolysosomes where engulfed contents are degraded (Figure 11).

Interestingly, in addition to lysosomal exocytosis, research has demonstrated that the autophagic pathway can also be implicated in the secretory pathway termed as "autophagydependent secretory pathway," which is a process for discarding waste material and permitting the extracellular release of certain signaling molecules.

At the organelle level, autophagosomes can merge with MVBs to generate amphisomes, which represent an intersection of the exosome and autophagy pathways (Figure 11). Amphisomes are thought to fuse with lysosomes, which destroy their components; nonetheless, these components can also end up being released extracellularly.

Furthermore, both exosomes and autophagy are important in maintaining cellular homeostasis, and one route may compensate for a defect in the other. Defective MVBs and their contents, for example, may be prone to autophagic degradation, and inhibiting autophagy may save exosome release from MVBs that would otherwise be degraded. [START_REF] Gruenberg | Life in the lumen: The multivesicular endosome[END_REF][START_REF] Settembre | Signals from the lysosome: a control centre for cellular clearance and energy metabolism[END_REF][START_REF] Xu | The interplay between exosomes and autophagypartners in crime[END_REF].

Numerous studies have demonstrated that the endosomal-lysosomal-autophagic pathway is responsible for the degradation of APP, APP-CTFs, and Aβ [START_REF] Asai | An alternative metabolic pathway of amyloid precursor protein C-terminal fragments via cathepsin B in a human neuroglioma model[END_REF]Boland et al., 2010;[START_REF] Gallwitz | Cathepsin D: Analysis of its potential role as an amyloid beta degrading protease[END_REF]Tian et al., 2011;Vingtdeux et al., 2007) (Figure 11). This route has a large number of cellular and molecular actors. Therefore, an alteration in one of the actors of this quite complicated route may result in the dysfunction of this pathway and consequently lead to the accumulation of toxic players in AD. Adapted from [START_REF] Gonzalez | Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway[END_REF].

Dysfunctions of ELN and Autophagy in AD

The detection of accumulating immature autophagic vacuoles in AD brains led to the discovery of links between autophagy and AD [START_REF] Nixon | Extensive involvement of autophagy in Alzheimer disease: an immuno-electron microscopy study[END_REF]. In the early stages, neurons exhibit an increase in the expression of autophagy activating transcription factors, which leads to the hyperactivation of genes involved in autophagy induction, autophagosome formation, and autophagosome fusion with lysosomes. However, this process in these neurons appears to be gradually reduced as disease progresses, demonstrated by increased autolysosome size and number at advanced stages. These abnormalities are caused by a serious lysosomal clearance deficiency, which results in the accumulation of autophagic vacuoles [START_REF] Bordi | Autophagy flux in CA1 neurons of Alzheimer hippocampus: Increased induction overburdens failing lysosomes to propel neuritic dystrophy[END_REF]. The accumulation of amphisomes and autophagic vesicles can possibly be explained by a defect in axonal transport, which is required for autophagosome maturation and degradation [START_REF] Morel | Levels of kinesin light chain and dynein intermediate chain are reduced in the frontal cortex in Alzheimer's disease: implications for axoplasmic transport[END_REF].

Since it has been demonstrated that autophagy is also implicated in Aβ degradation (Tian et al., 2011), defects in the autophagic pathway therefore cause an accumulation of Aβ and contribute to the intracellular toxicity of the peptide. However, as another early AD phenotype, it is challenging to determine if autophagic defects lead to Aβ accumulation or the opposite.

In fact, the increased number of autophagosomes observed in AD due to the enhanced autophagy leads to increased Aβ secretion and consequently has a direct impact on Aβ plaque formation. On the other hand, increased intracellular Aβ levels result from impaired autophagosome clearance [START_REF] Nilsson | Aβ Secretion and Plaque Formation Depend on Autophagy[END_REF].

PS1 mutations and AD-risk factors contribute to ELN dysfunctions

The endosomal-lysosomal network and autophagy collaborate to play critical roles in maintaining neuronal homeostasis. Growing evidence suggests that changes to cell systems involved in protein sorting and trafficking may have an impact on neurodegeneration particularly in AD brains by limiting clearance of disease-associated pathologic proteins.

Indeed, FAD-associated presenilin mutations and many SAD-associated risk factors are associated to endosomal-lysosomal network disruption and have an effect on the cellular dysfunction seen in AD [START_REF] Szabo | The role of Alzheimer's disease risk genes in endolysosomal pathways[END_REF].

PS1 for example, was found to play crucial role in the maturation and trafficking of the v-ATPase, which is in charge of acidifying the lysosomes. Besides from leading to enhanced cleavage of APP and Aβ production causing early onset FAD, mutations of PS1 were found responsible for disrupting lysosomal and autophagic function, and PS1-FAD mediated increases in Aβ. Importantly PS1 deletion that is PS1-loss of function leads to a similar lysosomal and autophagic dysfunction [START_REF] Lee | Lysosomal Proteolysis and Autophagy Require Presenilin 1 and Are Disrupted by Alzheimer-Related PS1 Mutations[END_REF]. Additionally, a number of GWAS -confirmed risk genes for AD, including APOE4, Sorla, Bin1, and CALM (or PICALM), have been reported as being involved in this degradation pathway (Figure 12).

For instance, the endocytosis of APP and the γ-secretase complex is increased by CALM loss of function, although these observations are controverted [START_REF] Kanatsu | Decreased CALM expression reduces Abeta42 to total Abeta ratio through clathrin-mediated endocytosis of gamma-secretase[END_REF][START_REF] Xiao | Role of phosphatidylinositol clathrin assembly lymphoid-myeloid leukemia (PICALM) in intracellular amyloid precursor protein (APP) processing and amyloid plaque pathogenesis[END_REF]. BACE1 is retained in endosomes as a result of BIN-1's loss of function, which impairs membrane recycling of BACE1 and reduces their degradation through lysosomes [START_REF] Miyagawa | BIN1 regulates BACE1 intracellular trafficking and amyloid-beta production[END_REF][START_REF] Ubelmann | Bin1 and CD2AP polarise the endocytic generation of beta-amyloid[END_REF]. And the loss of Sorla function reduces retrograde transport of APP to TGN, causing it to accumulate in endosomes [START_REF] Willnow | Sorting receptor SORLA--a trafficking path to avoid Alzheimer disease[END_REF]. Overall, the loss of function of Bin1, CALM, and Sorla cause dysregulation in intracellular trafficking of APP and/or secretases, as well as an increase in their retention in sorting endosomes, which potentiate Aβ42 synthesis. Other pathways, however, including inefficient clearance of Aβ42 via the BBB due to impaired endocytosis/transcytosis via sorting endosomes and defective Aβ42 lysosomal clearance, have been identified as being affected by the loss of function of the genetic risk factors (Guimas [START_REF] Almeida | Impact of late-onset Alzheimer's genetic risk factors on beta-amyloid endocytic production[END_REF]. Regarding APOE4, it enhances both, the endocytosis of APP and the amount of APP in the secretory pathway due to increased APP transcription. It has also been linked to endosome enlargement in neurons and endosomallysosomal network disruption that is unrelated to Aβ and tau pathology [START_REF] Cataldo | Endocytic pathway abnormalities precede amyloid beta deposition in sporadic Alzheimer's disease and Down syndrome: differential effects of APOE genotype and presenilin mutations[END_REF][START_REF] Peng | Apolipoprotein E4 genotype compromises brain exosome production[END_REF].

C99 accumulation contribute to ELN and autophagic dysfunctions

As explained previously, the pH of the endosomes, as well as the presence of APP, β and γsecretases, allow for the production of C99 and Aβ in this compartment. In fact, enlarged endosomes, increased endocytic activity, and greater ability to produce Aβ under pathological circumstances were observed in sporadic AD patients [START_REF] Cataldo | Increased neuronal endocytosis and protease delivery to early endosomes in sporadic Alzheimer's disease: neuropathologic evidence for a mechanism of increased beta-amyloidogenesis[END_REF].

It was later discovered that also elevated levels of C99 in AD patients, independent of Aβ, cause a pathological overactivation of Rab5, resulting as well in accelerated endocytosis, and endosome enlargement (Kim et al., 2016). Two other studies using iPSC that express FAD mutations have confirmed the accumulation of C99 in human AD cell models without any overexpression, and the involvement of C99 in early endosomal dysfunction, and lysosomalautophagic pathology (Hung and Livesey, 2018;Kwart et al., 2019).

Similar to those seen in endosomes, dysfunctions of the lysosomal degradation pathway are also described to be one of the earliest clinical phenotypes of AD to be identified.

It is believed that there is a bidirectional link between APP, its cleavage products Aβ and C99, and lysosomal dysfunction. APP, C99 and Aβ are degraded in lysosomes under normal circumstances, but in AD, lysosomal dysfunctions enable the formation of misfolded aggregates of APP products, which in turn impair lysosomal activity [START_REF] Colacurcio | Dysfunction of autophagy and endosomal-lysosomal pathways: Roles in pathogenesis of Down syndrome and Alzheimer's Disease[END_REF]).

Aβ, for example, has been shown to permeabilize lysosomal membranes, allowing the release of lysosomal enzymes and calcium into the cytoplasm, inducing apoptosis, and explain the mislocalisation of APP and Aβ to the cytosolic compartment [START_REF] Guicciardi | Lysosomes in cell death[END_REF][START_REF] Yang | Loss of endosomal/lysosomal membrane impermeability is an early event in amyloid Abeta1-42 pathogenesis[END_REF]. In addition, our laboratory has reported that, C99 is found accumulated in the subiculum of 3xTgAD mice earlier than Aβ (Lauritzen et al., 2012) and this C99 accumulation contributes to lysosomal pathology independently of Aβ (Lauritzen et al., 2016).

3. Secretion of exosomes and their dysfunction in AD [START_REF] Mobius | Immunoelectron microscopic localization of cholesterol using biotinylated and non-cytolytic perfringolysin O[END_REF].

With EVs being a term used to point to all membrane-limited vesicles released from cells, exosomes in particular are a subset of EVs with a diameter ranging from 30 to 150 nm and distinguished by a set of specific molecular markers. Microvesicles, apoptotic bodies, and ectosomes that originate from the plasma membrane can also be classified as EVs but ranging in size from 200 to 2000 nm in diameter.

The production of ILVs is the first event in the biogenesis of exosomes, and the size, number, and cargos of exosomes are all reliant on this initial step. As previously stated, ILV synthesis is mostly dependent on the ESCRT machinery, which consists of four protein complexes (ESCRT 0, I, II, III) in addition to accessory proteins (ALIX, Tsg101, VSP4). The coordinated and sequential association of ESCRT machinery's proteins with the endosomal membrane regulates the creation of ILVs and the recruitment of exosomal cargo components through the binding to ubiquitinilated proteins.

ILVs may also be formed via ESCRT-independent pathways that involve membrane microdomains rich in tetraspanins. In fact, tetraspanin-enriched membrane microdomains can contribute to the entry of proteins and oligonucleotides into exosomes. So in addition to proteins, exosomal cargos can include oligonucleotides such as DNA, mRNAs and species of small RNAs that could also enter exosomes by interacting with RNA-binding proteins or lipid rafts in MVBs.

The molecular composition of exosomes is influenced by the tetraspanins such as, CD63 and Tspan8. Furthermore, the levels of expression of CD63, together with levels of CD9 and CD81, regulate the total amount of exosomes released [START_REF] Chairoungdua | Exosome release of betacatenin: a novel mechanism that antagonizes Wnt signaling[END_REF][START_REF] Escola | Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes[END_REF][START_REF] Hurwitz | Nanoparticle analysis sheds budding insights into genetic drivers of extracellular vesicle biogenesis[END_REF][START_REF] Kumar | Biomolecular characterization of exosomes released from cancer stem cells: Possible implications for biomarker and treatment of cancer[END_REF]. Unfortunately, CD9, CD63, and CD81 were formerly thought to be exosome-specific indicators; however, these proteins have then been suggested to be present in apoptotic bodies and microvesicles [START_REF] Crescitelli | Distinct RNA profiles in subpopulations of extracellular vesicles: apoptotic bodies, microvesicles and exosomes[END_REF].

Tetraspanins are not the only factor associated to exosome synthesis; depending on the cell type, lipids and lipid-related enzymes are also involved. For instance, ILVs that are intended to be secreted as exosomes include large amounts of ceramide. Moreover, lysobisphosphatidic acid (LBPA)-rich endosomal membrane domains may potentially regulate the biogenesis of exosomes; and ALIX, an LBPA partner, is also present in exosomes and participates in their biogenesis.

After being released into the extracellular space, exosomes can be internalized by the secreted cell or by other cells. Surface integrins are known to facilitate exosome attachment and entry into their intended recipient cells. The uptake of exosomes by the acceptor cell has been reported to occur via various mechanisms: the cargo can be released into the cytosol after the EVs either fuse to the plasma membrane, or internalized by endocytosis. Cargos may then be recycled and re-secreted or routed to the endo-lysosomal pathway for degradation [START_REF] Buratta | Lysosomal Exocytosis, Exosome Release and Secretory Autophagy: The Autophagic-and Endo-Lysosomal Systems Go Extracellular[END_REF][START_REF] Gruenberg | Life in the lumen: The multivesicular endosome[END_REF][START_REF] Tancini | Insight into the Role of Extracellular Vesicles in Lysosomal Storage Disorders[END_REF][START_REF] Xu | The interplay between exosomes and autophagypartners in crime[END_REF].

For a long time, it was assumed that exosomes' sole function was to maintain cellular homeostasis by clearing the cell of toxic waste. Nonetheless, exosomes are now considered to be a new participant in cell communication, transferring biological molecules from one cell to another. Furthermore, it is well established that exosomes play an important role in the transfer of mRNA and microRNA (miR) across cells, allowing genetic exchange (Valadi et al., 2007), and the attachment of these exosomes to a recipient cell allows for cell-specific signaling.

Exosomes can be secreted by all cell types in the CNS, and these exosomes play a role in physiological processes such synaptic activity, vascularization, and neuronal regeneration (Rajendran et al., 2014). A neuronal exosomal miR-mediated genetic regulation of astrocyte functions has also been suggested by the discovery of exosomal miR-mediated neuron to glial transmission [START_REF] Men | Exosome reporter mice reveal the involvement of exosomes in mediating neuron to astroglia communication in the CNS[END_REF].

Exosomal dysfunction in AD

A 2006 study initially identified Aβ as being present in exosomes [START_REF] Rajendran | Alzheimer's disease beta-amyloid peptides are released in association with exosomes[END_REF]. In reality, APP itself, APP-CTFs, and AICD are also present in exosomes and discharged into the extracellular space in addition to Aβ (Perez- [START_REF] Perez-Gonzalez | The Exosome Secretory Pathway Transports Amyloid Precursor Protein Carboxyl-terminal Fragments from the Cell into the Brain Extracellular Space[END_REF][START_REF] Sharples | Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes[END_REF]Vingtdeux et al., 2007;[START_REF] Yuyama | Exosomes as Carriers of Alzheimer's Amyloid-ss[END_REF]. In fact, according to Dr. Vingtdeux's research, lysosome alkalinization enhances AICD enrichment in exosomal vesicles released by APP wild-type (APPwt)-overexpressing neuroblastomas while γ-secretase inhibition promotes the secretion of of exosome-associated APP-CTFs [START_REF] Sharples | Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes[END_REF]Vingtdeux et al., 2007).The results of Dr. Laulagnier's most recent study show that exosomes release APP and APP-CTFs; the exosomes isolated from N2a cells overexpressing APPwt are concentrated in C83 and ηCTF, whereas those from N2a cells overexpressing APPswe are enriched in C83 and C99 [START_REF] Laulagnier | Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons[END_REF]. The identification of Aβ and APP-CTFs in exosomes has led to several hypotheses regarding their role in the disease. Recent findings indicate that exosomes have a dual function in AD pathogenesis, assisting in Aβ clearance while simultaneously playing potentially harmful roles that worsen and spread the pathogenesis [START_REF] Xiao | The role of exosomes in the pathogenesis of Alzheimer' disease[END_REF].

Homeostasis is maintained by a concerted effort of the ELN, autophagy, and exosomes. It was proposed for this reason that activating exosome secretion in AD patients with deficiencies in autophagy or lysosomal function would be advantageous by sending endosomal-lysosomal material into the extracellular space, where macrophages may assist in the degradation of neuronal waste [START_REF] Mathews | Exosome Production Is Key to Neuronal Endosomal Pathway Integrity in Neurodegenerative Diseases[END_REF]. As a matter of fact, the decrease in exosome secretion reported in APOE4 carriers, together with changes in the neuronal endosomallysosomal pathway, may be consistent with the concept that activating exosome secretion may reduce neuronal vulnerability and the risk of AD [START_REF] Peng | Apolipoprotein E4 genotype compromises brain exosome production[END_REF]. Very interestingly, a study recently found a strong correlation between endosomal dysfunction and the release of exosomes enriched in APP-CTFs (Miranda and Di Paolo, 2018). In fact, it has been established that AD brains lack PI3P, a master regulator of endolysosomal and autophagic functions (Morel et al., 2013a). Indeed, the research team led by Dr. Di Paolo has demonstrated that endolysosomal-autophagic dysfunction induced by genetic invalidation or pharmacological inhibition of PI3 kinase VSP34 (the enzyme that synthesizes phosphatydilinositol-3-phosphate (PIP3)) results in the secretion of exosomes abnormally enriched in APP-CTFs, as well as in autophagic substrates, such as polyubiquitinylated proteins and substrates that were ineffectively degraded. Furthermore, exosomes released following Vsp34 inhibition have a distinct lipid composition; they are rich in ceramides and Bis-Monoacylglycero-Phosphate (BMP) phospholipids, which are normally lacking in exosomes but present in late endosome membranes (Miranda et al., 2018).

Greater levels of phosphorylated Tau, toxic oligomeric Aβ, and Aβ1-42 have been observed in the exosomes of AD patients even up to 10 years before the onset of clinical symptoms [START_REF] Fiandaca | Identification of preclinical Alzheimer's disease by a profile of pathogenic proteins in neurally derived blood exosomes: A case-control study[END_REF]. Exosomes also contained APP, its metabolites (C99, C83, and AICD), and members of the secretase family of proteases (BACE, PS1, PS2, and ADAM10) (Perez- [START_REF] Perez-Gonzalez | The Exosome Secretory Pathway Transports Amyloid Precursor Protein Carboxyl-terminal Fragments from the Cell into the Brain Extracellular Space[END_REF][START_REF] Sharples | Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes[END_REF]Vingtdeux et al., 2007). For example, in Dr. Di Paolo's study, which established that cells tend to discharge undesired and undigested endolysosomal cargoes in exosomes in an attempt to eliminate them, C99 was found to be abundant in these exosomes, along with other toxic proteins or lipids (Miranda and Di Paolo, 2018). Intriguingly, our laboratory discovered that C99 monomers are mostly found at the trans-Golgi level, whereas oligomeric forms are more abundant in exosomes and exceed monomers in this compartment. Additionally, γ-secretase inhibition (D6 treatment or presenilin invalidation) promotes the formation of not only dimers and oligomers of C99, but also heterodimers with C83, and their presence in exosomes (Lauritzen et al., 2019).

As a result, when exosome secretion attempts to compensate for the dysfunction of other degradation pathways in AD, it ironically promotes the spread and progression of the pathology when nearby neurons collect these exosomes [START_REF] Xu | The interplay between exosomes and autophagypartners in crime[END_REF].

D) THERAPEUTIC APPROACHES AND CLINICAL TRIALS FOR AD 1. Targeting Aβ

The amyloid cascade theory has been at the center of AD research for many years since it is supported by genetic, biochemical, and anatomical evidence (see chapter The Amyloid Cascade Hypothesis). With this theory proposing that the aggregation of Aβ in oligomers, fibrils and deposition into amyloid plaques are the cause of the pathological phenotypes of AD, significant effort is placed into finding therapeutic strategies to reduce Aβ levels, either by preventing its production or by neutralizing it once it has been formed.

Neutralizing the formed Aβ

Immunotherapy has been the most extensively studied Aβ-targeted therapy for removing preexisting amyloid plaque loads. Active immunotherapy involves injecting a vaccine containing the Aβ-antigen into the patient in order to trigger an immune response against the peptide, whereas passive immunotherapy consists of directly administering monoclonal Aβ-specific antibodies to stimulate Aβ clearance. Prevention of Aβ aggregation: The most neurotoxic type of Aβ is soluble oligomers, and these are more closely associated with the severity of AD than are amyloid plaques. Anti-aggregation drugs are thus a reasonable approach in disease-modifying therapy. They bind to soluble Aβ peptides, preventing oligomerization and consequent neurotoxicity. While some of the studies on these molecules are still in progress, the majority have been discontinued due to their ineffectiveness or toxicity, as in the cases of scyllo-inositol and tramiprosate (Panza et al., 2019;[START_REF] Vaz | Alzheimer's disease: Recent treatment strategies[END_REF].

1.2. Preventing Aβ production -targeting β and γ-secretases

Inhibiting β and γ-secretases to reduce Aβ production is another extremely well researched approach.

BACE1 inhibitors

Although the inhibition of BACE1 is thought to have therapeutic potential for AD, the effectiveness of its inhibitors is greatly limited. In addition to the fact that BACE1's physiological functions could be disrupted by its inhibition, BACE1's amino acid sequence shares 64% similarity with that of BACE2, which has distinct biological roles that could be impacted if BACE1's inhibitors are not highly selective. Furthermore, designing a small molecule that can occupy and inhibit the relatively large active site of BACE1 while be able to cross the BBB is a further challenge in the development of BACE1 inhibitors. Over the past few years, several BACE1 inhibitors have been developed, but only five reached phase III of clinical trials. All five, however, were discontinued due to liver damage in the case of atabecestat, ineffectiveness in the case of verubecestat and lanabecestat, an unfavorable riskbenefit ratio in the case of elenbecestat, or even more cognitive decline following umibecestat treatment [START_REF] Vaz | Alzheimer's disease: Recent treatment strategies[END_REF].

γ-secretase inhibitors/modulators

Similar to the β-secretase, reducing the activity of γ-secretase also offers a potential therapeutic treatment. The diversity of γ-secretase substrates, including Notch, has made therapeutically effective inhibitor development difficult. All clinical trials based on these inhibitors, such as on semagacestat and avagacestat, were suspended due to adverse effects such as an increased risk of skin cancer and infection, gastrointestinal bleeding, and even worsening of cognitive function. To avoid such side effects, therapeutic development changed to γ-secretase modulators, which were expected to be safer because they do not block the enzyme's activity, but instead shift the cleavage of C99 toward shorter Aβ species such as Aβ37 and Aβ38, thus selectively inhibiting the aggregation-prone Aβ42 production while sparing Notch processing and avoiding the accumulation of APP-CTFs. Despite the fact that clinical trials of these modulators, particularly Tarenflurbil, the most well-known, indicated a favorable safety profile, they did not slow cognitive or functional decline due to the drugs' inability to cross the BBB [START_REF] Vaz | Alzheimer's disease: Recent treatment strategies[END_REF].

Since no cognitive improvements were observed in any of these trials, even when Aβ levels were greatly reduced, it appears evident that decreasing total Aβ alone was insufficient to restore cognitive function. More intriguingly, findings from preclinical studies revealed a link between the accumulation of APP-CTFs brought on by γ-secretase inhibition and the lack of improvement or even worsening of cognitive function in these trials.

In light of the toxic role C99 may play in AD (see chapter C99), clinical studies for γ-secretase inhibitors may have failed in part because γ-secretase can be regarded of as a beneficial C99inactivating enzyme that prevents the accumulation of C99. Inhibiting γ-secretase may therefore, independently of Aβ, contribute to the pathology of AD carried on by the accumulation of C99. We discussed this important point of view in a review in which I participated and published in the Journal of Biological Chemistry. This review will be presented in Annex.

"Is γ-secretase a beneficial inactivating enzyme of the toxic APP C-terminal fragment C99?"

Frédéric Checler, Elissa Afram, Raphaëlle Pardossi-Piquard, and Inger Lauritzen.

Modulation of the ELN

Every treatment strategy for AD has as its major goal reducing toxic accumulating proteins.

Besides the most advanced strategy targeting Aβ, one of the potential therapeutic approaches to treat AD would be to restore the function of the endosomal-lysosomal-autophagic degradation pathway as a part of the accumulation is brought on by a dysfunction of this system (see chapters Dysfunctions of ELN and Autophagy in AD and Exosomal dysfunction in AD).

As noted previously, lysosomal activity proteolysis is impaired in AD due to a lack of acidity of the lysosomes, which prevents hydrolases activity. This highlights the importance of finding possible therapies that enhance the activity of these hydrolases or can maintain lysosomes' pH.

Activating hydrolases

The first strategies were to either genetically or pharmacologically activate cathepsins. Both of these techniques have been found to be effective in numerous AD models. In the first study, lentivirus-mediated expression of cathepsin B in aged mice with neuronal expression of FAD mutant human APP, reduced amyloid plaques in the brain, demonstrating the potential of this technique in clearing pre-existing amyloid plaques (Mueller- [START_REF] Mueller-Steiner | Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease[END_REF]. These findings were confirmed by injecting an adeno-associated virus expressing cathepsin B into the hippocampus of another AD model, which revealed not only a reduction in intracellular and extracellular levels of Aβ, but also an improvement in AD-related memory deficits [START_REF] Embury | Cathepsin B Improves ss-Amyloidosis and Learning and Memory in Models of Alzheimer's Disease[END_REF]. Other research took a different strategy, consisting of invalidating endogenous inhibitors of cysteine protease inhibitor, such as cathepsin B; these inhibitors are known as cystatin B or C. Likewise, this indirect cathepsin B activation decreased Aβ levels, and improved synaptic, and cognitive functions in various FAD models [START_REF] Sun | Cystatin C-cathepsin B axis regulates amyloid beta levels and associated neuronal deficits in an animal model of Alzheimer's disease[END_REF][START_REF] Yang | Reversal of autophagy dysfunction in the TgCRND8 mouse model of Alzheimer's disease ameliorates amyloid pathologies and memory deficits[END_REF][START_REF] Yang | Defective macroautophagic turnover of brain lipids in the TgCRND8 Alzheimer mouse model: prevention by correcting lysosomal proteolytic deficits[END_REF]. Interestingly, this approach leads to the same effects on Aβ in mice overexpressing the human APPwt [START_REF] Wang | Cathepsin B degrades amyloid-beta in mice expressing wild-type human amyloid precursor protein[END_REF]. Unfortunately, these genetic techniques are difficult to envision in humans, and given the non-permeability of the BBB, a pharmacological approach based on enzyme supplementation does not appear suitable either.

However, pharmacological methods based on lysosomal modulators have been investigated

since the BBB of AD patients would be altered as the disease advances. A systemic injection of the modulator Z-Phe-Ala-diazomethylketone (PADK) in AD mice models, for example, drastically elevated cathepsin B activity in the brain, resulting in clearance of intracellular Aβ peptide, a reduction in extracellular deposits, and an improvement in cognitive abilities [START_REF] Bahr | Positive lysosomal modulation as a unique strategy to treat age-related protein accumulation diseases[END_REF][START_REF] Butler | Protective effects of positive lysosomal modulation in Alzheimer's disease transgenic mouse models[END_REF]. It also seems more strategic to target cathepsin B, rather than cathepsin D for example because its action pH is higher than that of cat D and therefore it would be more easily activated in neurons where there is a V-ATPase dysfunction.

Normalization and restoration of lysosomal activity

Because lysosomal enzymes require an acidic pH, an increase in enzyme levels in the absence of pH correction seems unnecessary. One method for directly restoring lysosomal pH has been studied mostly in animal models of Parkinson's disease and has exhibited promising results. In this approach, Poly(lactic-co-glycolic acid) (PLGA) acidic nanoparticles (aNP) are internalized into cells and trafficked to lysosomes, where they re-acidify and rescue defective lysosomes (Bourdenx et al., 2016). Since researchers are seeking to find non-invasive therapies procedures where treatments can cross the BBB, PLGA nanoparticles (PLGA NPs) are being intensively studied in the interest of treating neurodegenerative illnesses. PLGA NPs provided numerous advantages, including improved drug solubility, resistance to enzymatic digestion, higher targeting efficiency, and improved cellular internalization [START_REF] Cunha | PLGA-Based Nanoparticles for Neuroprotective Drug Delivery in Neurodegenerative Diseases[END_REF]. This approach of using acidic nanoparticles was also performed in PS1 KO cells (Lee et al., 2015) and in cell models of DS patients (Jiang et al., 2019), improving lysosomal deficiencies and restoring normal lysosomal proteolysis.

Another method for restoring the acidic pH of lysosomes is to increase cyclic-AMP (cAMP) levels by either blocking phosphodiesterases (PDEs; the enzymes that degrade cAMP) or activating adenylyl cyclases (the enzymes that produce cAMP). As cAMP levels rise, protein kinase A (PKA) is activated, increasing the amount of free Zn2+ in lysosomes. Lysosomes will become acidified once again due to the increased levels of free Zn2+. Hence, another potential therapeutic strategy to re-acidify lysosomes is increasing the levels of free Zn2+ intracellularly or in the lysosomes [START_REF] Koh | Lysosomal dysfunction in proteinopathic neurodegenerative disorders: possible therapeutic roles of cAMP and zinc[END_REF][START_REF] Lai | Endosomal-lysosomal dysfunctions in Alzheimer's disease: Pathogenesis and therapeutic interventions[END_REF].

The "molecular tweezer" CLR01, is an inhibitor of abnormal protein self-assembly that selectively binds lysine residues, disrupting molecular interactions that are necessary for the toxic aggregation of multiple amyloidogenic proteins. These molecules are being researched as potential therapeutics for amyloidosis and many other proteinopathie [START_REF] Attar | Safety and pharmacological characterization of the molecular tweezer CLR01 -a broad-spectrum inhibitor of amyloid proteins' toxicity[END_REF][START_REF] Shahpasand-Kroner | Molecular tweezers -supramolecular hosts with broadspectrum biological applications[END_REF]. In AD in particular, CLR01 has been demonstrated to inhibit Aβ and tau aggregation, in vitro and in in vivo models, and to improve behavioral deficits [START_REF] Attar | Protection of primary neurons and mouse brain from Alzheimer's pathology by molecular tweezers[END_REF][START_REF] Di | The molecular tweezer CLR01 improves behavioral deficits and reduces tau pathology in P301Stau transgenic mice[END_REF][START_REF] Zheng | Amyloid beta-protein assembly: The effect of molecular tweezers CLR01 and CLR03[END_REF]. More impressively, the CLR01 compounds were shown to be mostly localized in lysosomes after being internalized in cells, where they unfold aggregated proteins, reducing lysosomal enlargement and reactivating the autophagylysosomal pathway. This, in turn, promotes the enzymatic degradation of misfolded proteins by lysosomal cathepsins [START_REF] Li | Lysine-selective molecular tweezers are cell penetrant and concentrate in lysosomes[END_REF][START_REF] Monaco | The Amyloid Inhibitor CLR01 Relieves Autophagy and Ameliorates Neuropathology in a Severe Lysosomal Storage Disease[END_REF].

Autophagy activation

Autophagy is one of the primary methods through which the cell eliminates aberrant proteins and protein aggregates, both of which have been linked to AD (see chapter Autophagic pathway). Thus, inducing autophagy could be used to eliminate such protein aggregates.

Several autophagy-stimulating treatments have already shown significant therapeutic potential for AD in clinical trials. The majority of the identified drugs promote autophagy by blocking the mammalian target of rapamycin (mTOR) and/or activating AMP-activating protein kinase (AMPK).

Rapamycin, a selective inhibitor of target-of-rapamycin complex 1 (TORC1) and hence regulator of mTOR pathway activity, is the most well-known mTOR inhibitor. When rapamycin is administered to AD mouse models, neurofibrillary tangles and Aβ protein aggregation are significantly reduced, and cognitive abilities including learning and memory are enhanced [START_REF] Ozcelik | Rapamycin attenuates the progression of tau pathology in P301S tau transgenic mice[END_REF]Spilman et al., 2010). Beneficial effects of mTOR pathway inhibition have also been demonstrated in studies with temsirolimus, a rapamycin prodrug. In a mouse model of AD, temsirolimus was demonstrated to enhance autophagy-dependent Aβ clearance and to improve learning and memory [START_REF] Jiang | Temsirolimus promotes autophagic clearance of amyloid-beta and provides protective effects in cellular and animal models of Alzheimer's disease[END_REF]. Additionally, SMER28, a small molecule rapamycin enhancer, reduces levels of Aβ peptide and APP-CTFs by activating autophagy through an Atg5-dependent route (Tian et al., 2011).

The anti-psychiatric medicine lithium has been shown to promote autophagy in an AMPKdependent manner [START_REF] Bao | Lithium targeting of AMPK protects against cisplatin-induced acute kidney injury by enhancing autophagy in renal proximal tubular epithelial cells[END_REF], enhancing cognition in AD patients and those with MCI [START_REF] Matsunaga | Lithium as a Treatment for Alzheimer's Disease: A Systematic Review and Meta-Analysis[END_REF]. Also via AMPK activation, resveratrol, a deacetylase activator and anti-aging drug from the sirtuin family SIRT1, has been shown to activate autophagy, decreasing Aβ accumulation [START_REF] Vingtdeux | Novel synthetic small-molecule activators of AMPK as enhancers of autophagy and amyloid-beta peptide degradation[END_REF].

Metformin, which is extensively used to treat type 2 diabetes, stimulates autophagy by activating AMPK and/or inhibiting mTORC1, lowers amyloid plaques and tauopathies, and has shown promising outcomes in clinical trials in enhancing cognitive abilities [START_REF] Koenig | Effects of the Insulin Sensitizer Metformin in Alzheimer Disease: Pilot Data From a Randomized Placebo-controlled Crossover Study[END_REF]. A multicenter, Phase 2/3 prevention trial is slated to begin in August 2020 on early or late MCI overweight or obese without diabetes and will run through 2024 (Metformin in Alzheimer's Dementia Prevention (MAP) study, Alzforum).

Memantine, an FDA-approved AD therapy medicine, and carbamazepine, an FDA-approved antiepileptic drug, have both been demonstrated to promote autophagy. Carbamazepine has intriguing therapeutic potential for AD by activating the autophagy, along with a number of other drugs, such as trehalose, which activates autophagy via the transcription factor TFEB;

however, these treatments are not yet in human clinical trial phases. All of these medications are discussed in detail in the review [START_REF] Zhang | New therapeutics beyond amyloid-beta and tau for the treatment of Alzheimer's disease[END_REF].

Exosomes as drug carriers and biomarkers

Exosomes as drug carriers

Despite their multiples roles in inter-cellular communications, clearing the cell of toxic waste or spreading toxic molecules responsible for the propagation of disease (see chapter Secretion of exosomes), exosomes can serve as effective carriers for therapeutic drugs. As was highlighted for a range variety of treatments, crossing the BBB is a significant obstacle for drug development. Exosomes, which are biocompatible, self-derived, and less prone to elicit immunological responses, can cross the BBB, making them an attractive choice for drug delivery into the CNS. In addition, because of their RNA transport potential, studies are trying to employ exosomes as carriers to transfer nucleic acid fragments such as miRNA and siRNA for the treatment of AD. For example, miR-124a delivered via exosomes is internalized by astrocytes, regulating synaptic activity, increasing glutamate uptake, as well as decreasing neuronal apoptosis in AD through increased expression of excitatory amino acid transporter 2 (GLT1) [START_REF] Morel | Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1[END_REF]. In AD mice, exosomes containing siRNA were tested as a potential therapy, and they significantly reduced BACE1 levels and Aβ deposition [START_REF] Alvarez-Erviti | Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes[END_REF]. This supported the use of exosomes as gene therapy carriers for diffuse distribution to the brain in the treatment of neurodegenerative diseases. Furthermore, a study from Chen and colleagues demonstrated the therapeutic performance of exosomes derived from mesenchymal stem cells (MSC-exosomes) in the treatment of AD, proving that these exosomes encapsulating a variety of MSC factors reduced Aβ expression, cleared brain glucose metabolism, and improved neuronal plasticity, memory, and cognitive function (Chen et al., 2021). Conforming to this study, neprilysin was discovered to be highly concentrated in exosomes isolated from adipose tissue-derived mesenchymal stem cells (ADSCs), and internalization of these exosomes resulted in a decrease in extracellular and intracellular

Aβ levels [START_REF] Katsuda | Human adipose tissue-derived mesenchymal stem cells secrete functional neprilysin-bound exosomes[END_REF]. Exosomes enriched in a neprilysin variant with improved specificity and efficacy in degrading Aβ, reduced Aβ40 levels in vitro with greater potency than exosomes derived from ADSCs, and were found to selectively target the hippocampus in vivo, lowering proinflammatory gene expression [START_REF] Yu | Genetically engineered exosomes display RVG peptide and selectively enrich a neprilysin variant: a potential formulation for the treatment of Alzheimer's disease[END_REF].

Exosomes as biomarkers

Another major challenge in AD today is the discovery of an early biomarker that would allow patients to be treated as early as possible, ideally preventing or significantly slowing the disease's progression. As already mentioned, the analysis of t-Tau, p-Tau and Aβ1-42 in the CSF has widely been studied as diagnostic biomarkers (see chapter Biomarkers). However, other AD biomarker candidates are being investigated due to the invasiveness of the CSF sampling method. Blood-based biomarkers could be a promising alternative to CSF testing in the diagnosis of AD because they are far less invasive. Exosomes are being evaluated for use as a biomarker to track the pathological development of AD since it has been established that they are produced, released from the brain, and found in peripheral blood [START_REF] Zhuang | Treatment of brain inflammatory diseases by delivering exosome encapsulated antiinflammatory drugs from the nasal region to the brain[END_REF].

Interestingly, a recent study revealed that exosomal Aβ1-42, T-Tau, and p-Tau levels were higher in AD patients and correlated with those in CSF, suggesting that these exosomal proteins have the same diagnostic utility as those in CSF [START_REF] Jia | Concordance between the assessment of Abeta42, T-tau, and P-T181-tau in peripheral blood neuronalderived exosomes and cerebrospinal fluid[END_REF]. What's more intriguing is that a different study recently used the amplified plasma exosomes (APEX) technology to measure different populations of blood-circulating Aβ. This study revealed that the exosomebound Aβ population can strongly reflect brain plaque deposition and correlate with PET imaging more effectively [START_REF] Lim | Subtyping of circulating exosome-bound amyloid beta reflects brain plaque deposition[END_REF]. Neuroinflammation-related proteins in astrocytederived blood exosome extracts have been found to be substantially greater in AD patients [START_REF] Winston | Complement protein levels in plasma astrocyte-derived exosomes are abnormal in conversion from mild cognitive impairment to Alzheimer's disease dementia[END_REF], and a large number of small miRNAs found in exosomes are being investigated as a potential diagnostic method in AD [START_REF] Manna | Exosomal miRNAs as Potential Diagnostic Biomarkers in Alzheimer's Disease[END_REF]. Furthermore, several additional proteins known to be affected in AD, such as lysosomal, autolysosomal, presynaptic, and postsynaptic proteins, are detectable in neuron-derived exosomes. All these mentioned studies provide evidence for the use of exosomes as AD biomarkers.

E) NON-CANONICAL APP-PROCESSING PATHWAYS

Although cleavages of APP by α, β and γ-secretases are complicated enough, additional APP cleavages have been described in recent years, making the comprehension of APP processing even further complex. As an alternative to the classical amyloidogenic enzymes, new proteases have been identified as APP-processing enzymes. Among them are δ-secretase, meprin-β, cathepsins, caspases, and η-secretase.

δ-secretase

This APP cleavage by δ-secretase was initially described when a cleavage activity 12 residues N-terminal to the β-site was discovered (Simons et al., 1996). The cleavage's responsible enzyme was later revealed to be asparagine endopeptidase (AEP or legumain), a lysosomal cysteine protease cleaving APP695 at N585 (δ-site) [START_REF] Zhang | Delta-secretase cleaves amyloid precursor protein and regulates the pathogenesis in Alzheimer's disease[END_REF]. The same study also discovered a second cleavage site for APP695 at N373, mediated by AEP. These cleavages produce two C-terminal fragments (CTF374-695 and CTF586-695) that can both be processed by α, β, and γ-secretases. However, CTF374-695 was observed to increase Aβ levels by acting as a substrate for β-secretase more effectively than APP-FL, indicating that δ-secretase may be involved in AD pathogenesis. Additionally, the toxicity and neurodegeneration brought on by the N-terminal ectodomain sAPPδ18-373, as well as the accumulation of AEP and its enhanced cleavage of APP with aging, were both found to support the pro-amyloidogenic feature of AEP.

AEP may serve as a possible target for therapeutic intervention given that the KO of AEP in the mouse AD model repaired synaptic loss, Aβ buildup, and behavioral impairments [START_REF] Zhang | Delta-secretase cleaves amyloid precursor protein and regulates the pathogenesis in Alzheimer's disease[END_REF][START_REF] Zhang | Asparagine endopeptidase is an innovative therapeutic target for neurodegenerative diseases[END_REF]. Recent research supports this notion by demonstrating that CTF586-695 interacts with CCAAT/enhancer binding protein beta CEBPB, an inflammatory transcription factor, and increases its transcriptional activity on APP, MAPT, δ-secretase, and inflammatory cytokines, inducing AD pathology and cognitive deficits that were reversible after elimination of APP C586-695 [START_REF] Yao | A deltasecretase-truncated APP fragment activates CEBPB, mediating Alzheimer's disease pathologies[END_REF]. In addition to APP, AEP has also been described to cleave tau protein inducing microtubule destabilization and neurofibrillary tangles formation (Zhang et al., 2014b).

Meprin-β

Meprin-β is a type I transmembrane zinc metalloprotease that cleaves APP at several sites in both N-terminal ectodomain and around β-site. First, the Meprin-β cleavages described on APP695 N-terminal ectodomain (Ala124/Asp125, Glu380/Thr381, and Gly383/Asp384) generate soluble non-toxic fragments with a molecular mass of 11 kDa to 20kDa that are detected in both healthy and AD brains [START_REF] Jefferson | Metalloprotease Meprin β Generates Nontoxic N-terminal Amyloid Precursor Protein Fragments in Vivo[END_REF]. Although the enzyme is primarily membrane-bound, these APP N-terminal cleavages do occur when meprin-β is in its soluble form. In contrast, membrane-anchored meprin-β cleaves APP more close to its transmembrane domain, at the Met596-Asp597 site (β-site), as well as the proximal Asp597-Ala598 and Ala598-Glu599 sites (APP695) [START_REF] Bien | The Metalloprotease Meprin β Generates Amino Terminal-truncated Amyloid β Peptide Species[END_REF]. Unlike BACE1, Meprin-β cleaves APP at plasma membrane before endocytosis and these cleavages are reliant on subsequent γ-secretase cleavage producing Aβ peptides with different size. When compared to non-truncated Aβ species, Aβ2-X and Aβ3-X originating from meprin-β and γ-secretase activity are more prone to aggregate and are commonly found in AD brains [START_REF] Schonherr | Generation of aggregation prone N-terminally truncated amyloid beta peptides by meprin beta depends on the sequence specificity at the cleavage site[END_REF][START_REF] Wiltfang | Elevation of β-Amyloid Peptide 2-42 in Sporadic and Familial Alzheimer's Disease and Its Generation in PS1 Knockout Cells[END_REF]. Of particular interest, the glutamate residue at position 3 of Aβ3-X generated by meprin-β can be converted into pyroglutamate, producing the neurotoxic pGlu-3Aβ [START_REF] Bayer | N-Truncated Abeta Starting at Position Four-Biochemical Features, Preclinical Models, and Potential as Drug Target in Alzheimer's Disease[END_REF].

Furthermore, the direct interaction with ADAM10 promotes the shedding of meprin-β, which in its soluble form loses its β-secretase function [START_REF] Marengo | Meprin β knockout reduces brain Aβ levels and rescues learning and memory impairments in the APP/lon mouse model for Alzheimer's disease[END_REF], and it was recently discovered that meprin-β deletion decreased brain Aβ levels and enhanced learning and memory in an AD mice model [START_REF] Scharfenberg | Regulation of the alternative β-secretase meprin β by ADAM-mediated shedding[END_REF].

Caspases

Caspases are cysteine aspartyl-specific proteases that are essential for the initiation and execution of apoptosis. Caspases are classified into three functional groups: initiator caspases (caspases 2, 8, 9, and 10) that initiate the apoptosis signal, executioner caspases (caspases 3, 6, and 7) that carry out the mass proteolysis that leads to apoptosis, and inflammatory caspases (caspases 1, 4, 5, 11, and 12) that are involved in inflammatory cytokine signaling but do not function in apoptosis [START_REF] Chang | Proteases for cell suicide: functions and regulation of caspases[END_REF].

Caspases (predominantly caspase-3) have been shown to cleave mature APP695 at Asp664, behind the VEVD sequence, similar to the consensus caspase-3 ''DEVD'' motif. This cleavage occurs in the AICD domain, resulting in the intracellular peptide C31. This cleavage is considered to occur in apoptotic neuronal cells [START_REF] Gervais | Involvement of caspases in proteolytic cleavage of Alzheimer's amyloid-beta precursor protein and amyloidogenic A beta peptide formation[END_REF][START_REF] Weidemann | Proteolytic processing of the Alzheimer's disease amyloid precursor protein within its cytoplasmic domain by caspase-like proteases[END_REF].

Moreover, C31 has been demonstrated to exhibit cytotoxicity, to be linked to Aβ-induced synaptic damage, and to form APP/31 complexes that activate cellular toxicity signals [START_REF] Galvan | Reversal of Alzheimer's-like pathology and behavior in human APP transgenic mice by mutation of Asp664[END_REF][START_REF] Lu | Caspase cleavage of the amyloid precursor protein modulates amyloid beta-protein toxicity[END_REF][START_REF] Park | Caspase Activation and Caspase-Mediated Cleavage of APP Is Associated with Amyloid beta-Protein-Induced Synapse Loss in Alzheimer's Disease[END_REF][START_REF] Park | Mechanism of cytotoxicity mediated by the C31 fragment of the amyloid precursor protein[END_REF]. JCasp fragment which is produced following -secretase and caspase cleavages, has also been proved to inhibit APP, thus blocking the release of excitatory synaptic vesicles [START_REF] Fanutza | APP and APLP2 interact with the synaptic release machinery and facilitate transmitter release at hippocampal synapses[END_REF].

Cathepsins

Cathepsins are the most abundant hydrolases in lysosomes, and their proteolytic activity is responsible for the degradation of proteins in these compartments. As described before, lysosomes are involved in APP degradation (see chapter Lysosomal and autophagic pathway and their dysfunctions in AD), and it has also been shown that cathepsin D is primarily engaged in Aβ clearance [START_REF] Hamazaki | Cathepsin D is involved in the clearance of Alzheimer's beta-amyloid protein[END_REF][START_REF] Mcdermott | Degradation of Alzheimer's beta-amyloid protein by human cathepsin D[END_REF]Nixon, 2017).

Cysteine (Cathepsin B) and aspartyl (Cathepsin D) cathepsins are also thought to have the ability to cleave APP at β-site, in addition to their critical role in the pathogenesis of AD and in the degradation of APP, APP-CTF and Aβ [START_REF] Haque | New insights into the roles of endolysosomal cathepsins in the pathogenesis of Alzheimer's disease: cathepsin inhibitors as potential therapeutics[END_REF]Hook et al., 2008a).

Cathepsin D has been discovered to be approximately 280-fold more expressed in human brains than BACE 1, and exhibiting BACE 1-like β-secretase specificity, but with better activity towards the β-secretase site on APP with the Swedish mutation than APPwt [START_REF] Schechter | Kinetic properties of cathepsin D and BACE 1 indicate the need to search for additional beta-secretase candidate(s)[END_REF]. Cathepsin B, on the other hand, prefers to cleave the wild-type β-secretase site over the Swedish mutant β-secretase site (Hook et al., 2008b).

Furthermore, in AD brains, cathepsin B and cathepsin D were detected in high concentrations in early endosomes, where APP is primarily trafficked to be cleaved by the amyloidogenic pathway, in contrast to normal brains, where both cathepsins were present in only a minimal proportion of endosomes [START_REF] Cataldo | Increased neuronal endocytosis and protease delivery to early endosomes in sporadic Alzheimer's disease: neuropathologic evidence for a mechanism of increased beta-amyloidogenesis[END_REF].

Cathepsin B and D present a dual role in AD pathology: cleaving APP at β-site, they contribute to Aβ production but on the contrary, they could have a neuroprotective function through Aβ and C99 degradation.

η-secretase

η-secretase cleaves APP between residues Asn504 and Met505 (APP695 numbering),to generate a soluble N-terminal fragment, sAPPη, and ηCTF, a fragment of approximately 30 kDa (Willem et al., 2015). ηCTF undergoes subsequent cleavages by α, and β-secretases,

producing Aηα and Aηβ fragments, respectively (Figure 14) The activity of this η-secretase is ensured by the matrix metalloproteinases (MMPs): MT1-MMP (MMP14), MT3-MMP (MMP16) and MT5-MMP (MMP24) have been described to be able to cleave APP at this ηsite (detailed below) [START_REF] Ahmad | Cleavage of Amyloid-β Precursor Protein (APP) by Membrane-Type Matrix Metalloproteinases[END_REF]Baranger et al., 2016;[START_REF] Higashi | Novel processing of beta-amyloid precursor protein catalyzed by membrane type 1 matrix metalloproteinase releases a fragment lacking the inhibitor domain against gelatinase A[END_REF]Willem et al., 2015).

F) BIOLOGY OF MATRIX METALLOPROTEINASES A) MATRIX METALLOPROTEINASES (MMPS) OVERVIEW

The metalloproteinases are the largest of the five primary families into which all proteases are commonly classified. Metzincins, in particular, are a metalloproteinase superfamily with a conserved methionine and 3 histidines residues in their catalytic site and whose activity is reliant on a zinc ion. Rivera's team [START_REF] Rivera | Metalloproteinases and their tissue inhibitors in Alzheimer's disease and other neurodegenerative disorders[END_REF][START_REF] Rivera | Metzincin Proteases and Their Inhibitors: Foes or Friends in Nervous System Physiology[END_REF]. Due to their ability to degrade Aβ, some MMPs, particularly MMP-2,7,9 and MT1-MMP, have been linked to AD. MMP-9 can cleave Aβ40, whereas MMP-2 was found to be more effective at cleaving Aβ42; MMP-7 and MT1-MMP were also reported to be able to cleave both, preventing the formation of neurotoxic β-sheet and thus amyloid plaques [START_REF] Backstrom | Matrix metalloproteinase-9 (MMP-9) is synthesized in neurons of the human hippocampus and is capable of degrading the amyloid-beta peptide (1-40)[END_REF][START_REF] Hernandez-Guillamon | Sequential Amyloid-beta Degradation by the Matrix Metalloproteases MMP-2 and MMP-9[END_REF][START_REF] Liao | Degradation of soluble and fibrillar amyloid beta-protein by matrix metalloproteinase (MT1-MMP) in vitro[END_REF][START_REF] Taniguchi | MMP-7 cleaves amyloid beta fragment peptides and copper ion inhibits the degradation[END_REF].

In addition to being a substrate for MMPs, Aβ also modulates their expression, perhaps indicating the presence of regulatory feedback mechanisms. Aβ was reported to enhance the expression of MMP-2, -3, and -9 in neuronal and astrocyte cultures, and to increase the levels of MMP-1, -3, -9, -10, -12, and -19 in human postmortem brain microglia [START_REF] Deb | Increased production of matrix metalloproteinases in enriched astrocyte and mixed hippocampal cultures treated with beta-amyloid peptides[END_REF][START_REF] Walker | Gene expression changes by amyloid beta peptide-stimulated human postmortem brain microglia identify activation of multiple inflammatory processes[END_REF]. Similar to these in vitro findings, MMP-2, -9, and MT1-MMP expression were elevated in mouse AD models, particularly in Aβ-enriched areas [START_REF] Py | Differential spatio-temporal regulation of MMPs in the 5xFAD mouse model of Alzheimer's disease: evidence for a pro-amyloidogenic role of MT1-MMP[END_REF][START_REF] Yin | Matrix metalloproteinases expressed by astrocytes mediate extracellular amyloid-beta peptide catabolism[END_REF]. Human studies have mostly observed an increase in MMP levels, such as MT1-MMP and MMP-1, -3, and 14, in AD patients as when compared to healthy controls [START_REF] Langenfurth | Membrane-type 1 metalloproteinase is upregulated in microglia/brain macrophages in neurodegenerative and neuroinflammatory diseases[END_REF][START_REF] Leake | Brain matrix metalloproteinase 1 levels are elevated in Alzheimer's disease[END_REF][START_REF] Mroczko | Concentrations of matrix metalloproteinases and their tissue inhibitors in the cerebrospinal fluid of patients with Alzheimer's disease[END_REF]. MMP-9 levels, on the other hand, have been found to be reduced in the CSF and plasma of AD patients in certain studies [START_REF] Horstmann | Matrix metalloproteinases in peripheral blood and cerebrospinal fluid in patients with Alzheimer's disease[END_REF][START_REF] Mroczko | Concentrations of matrix metalloproteinases and their tissue inhibitors in the cerebrospinal fluid of patients with Alzheimer's disease[END_REF] and increased in others [START_REF] Lorenzl | Increased plasma levels of matrix metalloproteinase-9 in patients with Alzheimer's disease[END_REF][START_REF] Lorenzl | Profiles of matrix metalloproteinases and their inhibitors in plasma of patients with dementia[END_REF][START_REF] Stomrud | Alterations of matrix metalloproteinases in the healthy elderly with increased risk of prodromal Alzheimer's disease[END_REF].

While an increase in MMPs may be considered to be beneficial due to their Aβ-degrading activity, MMPs are also dependent on certain substrates that may be involved in pathogenic processes in AD. For example, MMP-2 and MMP-9 have been related to the disrupted bloodbrain barrier (BBB) found in AD patients and mice, which contributes to inflammatory and neurodegenerative processes [START_REF] Hernandez-Guillamon | Matrix metalloproteinase 2 (MMP-2) degrades soluble vasculotropic amyloid-beta E22Q and L34V mutants, delaying their toxicity for human brain microvascular endothelial cells[END_REF][START_REF] Spampinato | Astrocytes contribute to Abeta-induced blood-brain barrier damage through activation of endothelial MMP9[END_REF]. MMP-9 has been linked to neuroinflammatory processes and the death of hippocampal neurons [START_REF] Jourquin | Neuronal activity-dependent increase of net matrix metalloproteinase activity is associated with MMP-9 neurotoxicity after kainate[END_REF], and MMP-3 has been linked to the disruption of the blood-CSF barrier (BCSFB), which limits the restriction of the passage of unwanted molecules and pathogens into the brain [START_REF] Brkic | Amyloid beta Oligomers Disrupt Blood-CSF Barrier Integrity by Activating Matrix Metalloproteinases[END_REF]. Aside from its link with Aβ, MMP-9 can cleave Tau, resulting in fragments that are susceptible to the formation of toxic tau oligomers [START_REF] Nubling | Limited cleavage of tau with matrix-metalloproteinase MMP-9, but not MMP-3, enhances tau oligomer formation[END_REF].

B) MT-MMP AND APP CLEAVAGE

MT-MMPs in particular, have also been demonstrated to have Aβ-degrading and APP cleaving activity, implying that they play key roles in the regulation of APP functions in tissues such as the CNS [START_REF] García-González | Emerging Alternative Proteinases in APP Metabolism and Alzheimer's Disease Pathogenesis: A Focus on MT1-MMP and MT5-MMP[END_REF].

MT1-MMP, like MMP-2, -3, -7 and -9, was identified as an Aβ-degrading enzyme. First, Higashi and colleagues described the MT1-MMP cleavage site on APP770 (Asn579-Met580) in a fibrosarcoma cell line corresponding to the latter called η-site [START_REF] Higashi | Novel processing of beta-amyloid precursor protein catalyzed by membrane type 1 matrix metalloproteinase releases a fragment lacking the inhibitor domain against gelatinase A[END_REF]. Further research revealed that this cleaving site, along with the cleaving sites Ala463-Met464, Asn579-Met580, His622-Ser623, and His685-Gln686, are a target for MT3-MMP also. The His685-Gln686 cleavage occurs within the Aβ sequence, but unlike MT1-MMP, MT3-MMP does not change Aβ levels in HEK293, implying that MT3-MMP processes APP in cell compartments that do not affect amyloidogenesis [START_REF] Ahmad | Cleavage of Amyloid-β Precursor Protein (APP) by Membrane-Type Matrix Metalloproteinases[END_REF]. The same study on HEK293 cells revealed that MT5-MMP may cleave APP at the same sites as MT1-MMP and MT3-MMP.

Indeed, it has been demonstrated that MT5-MMP can cleave the shorter neuronal isoform of APP695 at the N504-M505 (η-site), resulting in the production of a soluble APP fragment of sAPP95 in HEKswe with the release of a complementary transmembrane fragment of ∼30 kDa (CTF-30) (Baranger et al., 2016).

Interestingly, in both Rivera's and Willem's study, MT5-MMP, was identified as η-secretase in vivo, cleaving at this N504-M505/η-site of APP695 (Baranger et al., 2016;Willem et al., 2015).

In addition, among all MMP, MT5-MMP is the most abundant MMP expressed in the brain with important function in brain development and synaptic plasticity.

C) MT5-MMP

Overview

The MMP24 gene, which is located on chromosome 20q11.22, encodes MT5-MMP (also known as MMP-24) (https://www.proteinatlas.org/ENSG00000125966-MMP24#gene_information).

MT5-MMP can be present in the kidneys, and pancreas, although the neurons of the PNS and CNS have the highest expression levels [START_REF] Komori | Absence of mechanical allodynia and Aβ-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase[END_REF]. During embryonic and early postnatal development, MT5-MMP is highly expressed in the nervous system [START_REF] Komori | Absence of mechanical allodynia and Aβ-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase[END_REF]. Adults maintain this expression primarily in the cerebellum, with a reduced levels of expression in the hippocampus, dentate gyrus, and olfactory bulb [START_REF] Jaworski | Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system[END_REF].

Structure

MT5-MMP's domain structure is similar to that of other MT-MMPs, consisting of a signal peptide, prodomain, RXKR activation motif, catalytic domain, hinge region hemopexin-like domain. MT5-MMP is referred to as a membrane-type MMP with a transmembrane domain and a short cytosolic tail (Pei, 1999) (Figure 16).

The signal peptide at the N-terminus directs the MT-MMP to the secretory pathway and is followed by a cysteine-containing prodomain. This cysteine residue of the prodomain, binds to the Zn2+ of the catalytic domain, causing the prodomain to fold to the catalytic site, inhibiting the proteolytic activity of the MT-MMPs and leaving them in an inactive form known as "zymogens." Therefore, until activated by enzymes that cleave the prodomain or release the cysteine bond, these metzincin proteases remain in a latent condition. MT5-MMP belonging to Furin-activable MMPs is activated in the trans-Golgi network where furin, the proprotein convertase, cleaves its RXKR motif [START_REF] Rivera | Metzincin Proteases and Their Inhibitors: Foes or Friends in Nervous System Physiology[END_REF]. The ability of furin to activate and shed MT5-MMP provides cells with a mechanism to balance the ratio of activated/membranebound and activated/shed MT5-MMP by modulating the concentration of the convertases in the TGN [START_REF] Wang | Shedding of Membrane Type Matrix Metalloproteinase 5 by a Furin-type Convertase[END_REF].

The catalytic domain is highly conserved and binds and provides stability to Zn2+ via a consensus motif with three histidine residues (HExxHxxGxxH). The catalytic domain of all MT-MMP contains also the MT-loop, an 8 amino acid loop structure that allows the enzyme to be localized to focal adhesion locations and effectively degrading the underlying matrices [START_REF] Itoh | Membrane-type matrix metalloproteinases: Their functions and regulations[END_REF].

A small hinge region downstream of the catalytic site marks a shift to the hemopexin domain, the latter being important for their specificity for substrates and inhibitors [START_REF] Rivera | Metzincin Proteases and Their Inhibitors: Foes or Friends in Nervous System Physiology[END_REF].

MT5-MMP has a type I transmembrane domain that anchors it to the plasma membrane and hence controls, together with an intracytoplasmic domain, its trafficking and proteolytic activity [START_REF] Wang | Shedding of Membrane Type Matrix Metalloproteinase 5 by a Furin-type Convertase[END_REF]

Physiological Role

The fact that MT5-MMP's expression is highest during embryonic development and is mostly restricted to the cerebellum in adults, with a lower level in the hippocampus, dentate gyrus, and olfactory bulb, which are known to be neuronal plasticity regions, supports MT5-MMP's involvement in brain development and synaptic plasticity [START_REF] Jaworski | Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system[END_REF].

Proteoglycans, including chondroitin sulfate proteoglycans, are primary ECM components in the brain that have been shown to inhibit neurite growth and axonal regeneration (Jin et al, 2017). These proteoglycans were also discovered to be the preferred substrates of MT5-MMP [START_REF] Jaworski | Developmental regulation of membrane type-5 matrix metalloproteinase (MT5-MMP) expression in the rat nervous system[END_REF], implying that this metalloproteinase enhances neurite outgrowth.

Another study found that MT5-MMP, along with ADAM10, has a function in post-lesion synaptic structure modification following traumatic brain injury [START_REF] Warren | MT5-MMP, ADAM-10, and N-Cadherin Act in Concert To Facilitate Synapse Reorganization after Traumatic Brain Injury[END_REF].

N-cadherin is a non-ECM substrate of MT5-MMP that promotes neural stem cells (NSCs)

quiescence and adherence to their niches. MT5-MMP mediates the release and activation of NSCs in tissue renewal and repair by shedding N-cadherin [START_REF] Porlan | MT5-MMP regulates adult neural stem cell functional quiescence through the cleavage of N-cadherin[END_REF].

N-cadherin is also a synaptic protein that is involved in synapse remodeling and maintenance.

It was discovered that MT5-MMP and γ-secretase work together to provide synaptic autoregulation by cleaving N-cadherin on both sides of the synapse [START_REF] Restituito | Synaptic Autoregulation by Metalloproteases and -Secretase[END_REF].

Despite MT5-MMP's multiple roles and predicted developmental importance, MT5-MMP deficient mice are viable. However, when these mice were subjected to sciatic nerve injury, no sprouting nor invasion of Aβ-fibers were observed, along with the absence of mechanical allodynia caused by such lesions. This favors the fact that of MT5-MMP plays critical role in neuronal plasticity and remodeling [START_REF] Komori | Absence of mechanical allodynia and Aβ-fiber sprouting after sciatic nerve injury in mice lacking membrane-type 5 matrix metalloproteinase[END_REF]. genes with MT5-MMP-/-mice. In the cortical and hippocampus regions, 5xFAD/MT5-/-mice showed a significant decrease in soluble and oligomeric Aβ40 and Aβ42, as well as C99. This intriguing reduced amyloidosis is accompanied by a decline in pro-inflammatory cytokines, interleukine-1β (IL-1β) and tumour necrosis factor-α (TNF-α), as well as a decrease in glial reactivity, all while preserving LTP, spatial learning, and memory (Baranger et al., 2016).

MT5-MMP in AD

The same team then discovered a second mechanism through which MT5-MMP exerts proamlyoidogenic action. They demonstrated that MT5-MMP can be internalized in endosomes while interacting with APP, increasing APP sorting in endosomes, a major site of Aβ production [START_REF] Baranger | MT5-MMP Promotes Alzheimer's Pathogenesis in the Frontal Cortex of 5xFAD Mice and APP Trafficking in vitro[END_REF]Vassar et al., 1999).

MT5-MMP as η-secretase

Independent studies from two teams have described the processing of APP by MT5-MMP through its η-secretase activity

In 2015, Willem and colleagues identified the cleavage site N504-M505 of APP695 as η-site, and MT5-MMP, responsible for this cleavage in vivo, as η-secretase (Willem et al., 2015).

They characterized the fragments produced by this processing as "η-CTF," a new CTF with an estimated molecular weight of 30 kDa, and "sAPPη, a soluble N-terminal APP fragment of 80 kDa. These two fragments match with the descriptions of sAPP95 and CTF-30 reported in the same time by Baranger and collaborators (Baranger et al., 2017;Baranger et al., 2016).

The implication of sAPPη in AD is unknown; however, a recent study found that sAPP, including sAPP, sAPP and sAPPη, can bind to GABABR1a, leading to decreased presynaptic release and therefore decreasing hippocampal synaptic plasticity and neurotransmission in vivo This could represents a negative-feedback mechanism to maintain proper homeostatic control of neural activity and prevent any hyperexcitability [START_REF] Rice | Secreted amyloid-β precursor protein functions as a GABABR1a ligand to modulate synaptic transmission[END_REF].

ηCTF was discovered to be accumulated in dystrophic neurites of APP-PS1mice brains and human AD brains (Willem et al., 2015). Another study found increased levels of ηCTF in the CSF of patients with PSEN1 familial mutation, sporadic AD, and Down Syndrome compared to age-matched controls, implying that this fragment could be a possible diagnostic biomarker of AD [START_REF] Garcia-Ayllon | C-terminal fragments of the amyloid precursor protein in cerebrospinal fluid as potential biomarkers for Alzheimer disease[END_REF]. Additionally, a CTF of 26 kDa was found in exosomes from rat cortical neurons and was characterized in this work to most likely correspond to ηCTF, implying that this fragment may contribute to the disease after being endocytosed by neurons [START_REF] Laulagnier | Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons[END_REF]. They also observed that exosomes released by N2a cells expressing APPwt are enriched in both -CTF (C83) and ηCTF while those purified from N2a cells expressing APPswe are rather enriched in both -CTF (C83) and -CTF (C99); however, inhibition of BACE1 in the latter model results in increased exosomal levels of ηCTF [START_REF] Laulagnier | Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons[END_REF]. This could be explained by the Swedish mutation, which enhances APP cleavage by -secretase in amyloidogenic processing, which may compete with ηsecretase cleavage,or possibly explained by extensive cleavage of ηCTF at C99 when this mutated form of APP is expressed. Willem and colleagues further stated that the transmembrane η-CTF undergoes subsequent cleavages by α and β-secretases generating Aηα and Aηβ respectively and also investigated the impact of these new soluble fragments in AD. In their first study, they exposed hippocampal slices to 100 nM of synthetic Aηα and Aηβ and discovered that only Aηα, not Aηβ, displays neurotoxic properties and impairs LTP at the CA3-CA1 synapse (Willem et al., 2015). However, the same team published contradicting results years later, reporting that both Aηα and Aηβ, even at low nanomolar concentrations of 5 nM, impair LTP ex vivo [START_REF] Mensch | Aη-α and Aη-β peptides impair LTP ex vivo within the low nanomolar range and impact neuronal activity in vivo[END_REF]. Further studies are needed to elucidate synaptic function and pathological contribution of each η-secretase-derived fragment in AD pathology.

AD is by far the most common cause of dementia and a major global health concern. In the absence of effective treatments, AD has significant financial, physical, and mental health consequences on individuals and society. This explains why searching for therapeutic strategies is therefore one of the greatest challenges that scientists have been facing for decades. The Aβ peptide, according to the amyloid cascade hypothesis, is the origin of AD. However, the failure of Aβ-targeted therapies highlights the multifactorial nature of the disease. Indeed, several studies, including research from my team, have shown for example that independently of Aβ, the C99 fragment accumulates very early in AD models causing several impairments associated with AD. Due to the fact that AD is a complex disease, my team is interested in investigating APP-derived fragments other than Aβ that may be implicated in the pathology and may contribute in the discovery of new therapeutic targets in the future.

With all the focus of the research in the field of AD aiming to better understand the APP processing pathways; the recent discovery of η-secretase, as new APP cleaving enzyme has revealed the existence of novel fragments that may have toxic potential, complicating understanding of the AD pathophysiology.

With just approximately ten papers describing this cleavage and some of the resulting fragments, more eyes must be opened and greater focus should be placed on this pathway. As a consequence, the objectives of my PhD research were to principally concentrate on the ηCTF fragment: the η-secretase-derived APP membrane embedded fragment; especially given how little is known about this fragment. My first priority was to characterize it, and define its role in the pathophysiology associated with AD, which will serve to better understand the complexity of the APP metabolism.

Importantly, my laboratory had developed all the tools, plasmids, and methodological approaches to express these fragments not only in cellular models but also ex vivo and in vivo using Adeno-Associated Virus10 (AAV10). The purpose of my study consisted in characterizing the newly discovered ηCTF fragment using biochemical, pharmacological, and immunohistochemical approaches, and then thoroughly evaluating its functional influence on AD by electrophysiological and behavioral experiments. For this goal, I used various study models with their strengths and weaknesses described below.

 In vitro:

First, in order to study the expression, fate, processing and cellular localization of ηCTF, I start to express it in the SH-SY5Y neuroblastoma cells; the advantage of these cells is that they are immortalized human neuronal-like cell line, but not differentiated into mature neuronal cells.

Human neuroblastoma SH-SY5Y stably overexpressing human wild-type APP (SH-SY5Y

APPwt) were also used to attempt the detection of the APPwt-derived ηCTF, as endogenous ηCTF is difficult to detect. In addition, mouse embryonic fibroblasts naturally devoid of APLP1 and knocked out for APP and APLP2 (MEF APP KO) served also as important cell line in my study, although they are not a human nor neuronal-like cell line, they were a crucial model for identifying fragments that are genuinely derived from ηCTF transiently transfected in these cells, eliminating the possibility of them being mistaken for endogenous APP-derived fragments. MEF expressing endogenous wild-type APP (MEF-APPwt) served as controls.

Finally, due to the small size of SH-5YSY cells and in order to better characterizing the specific intracellular localization of ηCTF, we decided to use the human cervical cancer cells (HeLa), for immunocytochemistry, despite the fact that they are non-neuronal cells.

 Ex-vivo:

My team recently developed a study model that consists of the culture of wild-type mouse (C57BL/6) organotypic hippocampal slices which have advantages over the in vitro models, because it conserves the composition, structure, and synaptic organization of the hippocampus while avoiding cell isolation and lack of contact (Valverde et al., 2021b). This model enabled us to infect the slices with virus expressing ηCTF (AAV-CTF), which allows expressing and analyzing the localization of this fragment of interest in a more integrated model.

 In vivo:

-Mouse models:

To investigate the functional impact of ηCTF expression in vivo models, we developed the "AAV-CTF" mouse model that was obtained by intraventricular injection of 1-day-old WT mice (C57BL/6) with AAV-CTF virus, which allows the expression in mouse brain of the human ηCTF fragment under the control of the neuronal promoter synapsin-1.

In order to determine if ηCTF is present in AD models, we chose the 3xTgAD triple transgenic mouse model (APPswe, TauP301L and PS1M146V). These mice overexpress the human APP with the Swedish familial mutation (APPswe) and Tau protein with the P301L mutation. These mice also express an endogenous level of PS1 with the M146V familial mutation (knock-in). It is a widely used mouse model in the field of AD since these mice replicate the majority of the pathological phenotypes, including late senile plaque formation and neurofibrillary degeneration, as well as cognitive and synaptic impairments. Interestingly, 3xTgAD mice accumulate C99 earlier than Aβ, which correlates with endolysosomal dysfunction (Lauritzen et al., 2016), mitochondrial alterations (Vaillant-Beuchot et al., 2021), apathetic behavior (Pardossi-Piquard et al., 2016) and contributes to AD-like cognitive dysfunction (Bourgeois et al., 2018).

-Human models:

To examine whether ηCTF is present in AD patients, human brain samples were also tested.

Only information on the patients' age, sex, and neuropathology were provided, while samples were anonymized. Brain samples from AD patients were isolated from post-mortem patients who had been diagnosed with the disease, and they were categorized according to their Braak and Braaks's NFT stage (I to VI), while samples from controls were taken from individuals who had a negative diagnosis for several AD-unrelated neuropathologies (Braak and Braak's NFT stage -).

Thus my results are divided into two parts:

 First, study of expression, processing, fate, and subcellular localization of ηCTF in different in vitro, ex vivo, and in vivo models.

 Second, preliminary investigations on the detection of ηCTF in human AD patients, and functional implication of ηCTF and related fragments (Aηα and Aηβ) on synaptic activity.

1) The study of expression, processing, fate, and subcellular localization of ηCTF in different in vitro, ex vivo, and in vivo models. Since the discovery of the η-secretase cleavage and the resulting fragments, dates only a few years, very little is known about ηCTF and, decades of research are certainly required to properly define its contribution to the pathology associated with AD. For this reason, the of my first-author article was to start by characterizing the ηCTF fragment by studying its expression, processing, fate, and subcellular localization in different in vitro, ex vivo, and in vivo models, aiming that these results will serve as a starting point of the research towards the understanding of the toxic potential of this fragment. It's interesting to note that we developed a new antibody named ηCTF-NTer that exclusively recognizes ηCTF, Aηα, and Aηβ. This antibody proved useful for in situ and biochemical analyses of ηCTF in both mice and human models. We confirmed that ηCTF is processed by α, β, and γ-secretases using a pharmacological approach, demonstrating for the first time that ηCTF is a genuine precursor of Aβ. Furthermore, we showed that ηCTF is degraded by both proteasomal and autophagic pathways. We then performed immunostaining experiments in different study models to identify the localization of ηCTF in the trans-golgi compartment and endosomes. In an interesting matter, and in agreement with this endosomal localization, we detected ηCTF in exosomes. Lastly, ηCTF fragments were found surrounding amyloid plaques in brain slices of 3xTgAD mice.

Overall, these finding suggest that ηCTF could be a potential AD player, and further investigation will be required to evaluate whether it can serve as a possible therapeutic target.

Introduction

Among all hypotheses proposed to explain the etiology of Alzheimer's disease (AD), the amyloid hypothesis represents the most widely accepted one since it is supported by a large number of biochemical and histopathological evidences. This amyloid cascade hypothesis postulates that accumulation and aggregation of hydrophobic amyloid  peptides (A) trigger synaptic dysfunction and neurodegeneration in Alzheimer's disease 1,2 . Genetic evidences also provided strong support to this amyloid cascade. Thus, most of APP mutations responsible for aggressive autosomal dominant familial forms of AD (FAD) lead to exacerbated production of A or yield aggregation-prone A species 3 . Conversely the "Icelandic" APP mutation (APP Ala673Thr) decreases Aβ levels by about 40% and protects against the risk of developing AD 4 .

How APP is proteolytically processed has been the matter of numerous studies. APP undergoes a complex set of proteolytic events by enzymatic activities called secretases. Two main proteolytic pathways referred to as amyloidogenic and non-amyloidogenic pathways have been extensively examined. In the amyloidogenic pathway, APP is first cleaved by the -secretase to release a C-terminal fragment, C99, which then undergoes subsequent hydrolysis by the γ-secretase to yield not only A but also the cytosolic APP Intracellular Domain (AICD) that controls the transcription of several genes 5 . However, this amyloidogenic pathway responsible for A production does not represent the major part of APP metabolism. The non-amyloidogenic pathway that precludes A formation is the major APP processing route. It involves -secretase, yielding a C83 fragment that is further cleaved by -secretase, thereby producing AICD and a small N-terminally truncated fragment named p3.

In addition to these two canonical and well described pathways, a newly η-secretase pathway has emerged. First Wang and collaborators reported in HEK293 cells an accumulation of two novel APP-CTF fragments at 15 and 25kDa unraveled upon cathepsins inhibition 6 . They were the first to name the 25kDa APP-CTF fragment as the new APP-CTFη or ηCTF fragment 6 . Using various antibodies and mass spectroscopy analyses, Willem and colleagues subsequently demonstrated that this ηCTF fragment can be cleaved by αand secretases to generate Aηα and Aη peptides respectively 7 . They proposed the membrane-type matrix metalloprotease MT5-MMP as a new η-secretase since they observed a reduction of Aηα production in brains from MT5-MMP-knockout mice 7 . By distinct approaches, concomitant studies also demonstrated the cleavage of APP by MT5-MMP and showed its contribution to AD pathology. Thus, MT5-MMP deficiency reduced amyloid pathology, neuroinflammation and cognitive decline in 5xFAD mice 8,9 . However, few data were available concerning the fate, biology and cellular localization of this fragment.

In this current study, we observed that overexpressed ηCTF undergoes proteasomal and autophagic degradations in human neuroblastoma cells (SH-SY5Y) and for the first time, we show that ηCTF behaves as a genuine precursor of A. Since, the immunological toolbox available to specifically detect ηCTF, particularly in in situ approach fell short, we designed a novel immunological probe referred to as ηCTF-NTer antibody that interacts selectively with ηCTF N-terminus thus targeting ηCTF, Aη, Aη but importantly, not C83, C99 or AICD. By using this antibody in Hela transfected cells, we were able to establish a subcellular localization of ηCTF mainly in trans-Golgi network but also, to a smaller extent, in endosomes. This endosomal localization was also observed in organotypic hippocampal slices infected with an adeno-associated-virus (AAV) expressing ηCTF.

In agreement, we detected the presence of ηCTF in small extracellular vesicles (sEVs) prepared from culture media of SH-SY5Y expressing APP or from brain tissues of AAV-ηCTF-injected mice. Of interest, microscopic analysis of ηCTF-NTer-like immunoreactivity revealed an ηCTF staining surrounding amyloid plaques associated to an age-dependent accumulation of ηCTF in cortex and hippocampus of triple transgenic mice (3xTgAD).

Materials and Methods

Design of plasmid constructs and viruses:

pcDNA3 plasmid expressing Aηα or Aηβ: Aηα and Aηβ constructs were obtained by PCR amplification using APP695 cDNA as template and the forward primer 5'-GATAAGCTTGCCACCATGATTAGTGAACCAAGGATCAGTTAC-3' for both Aηα and Aηβ. This primer contains a Hind III restriction site and a Kozak sequence. We used the reverse primer 5'-GATCTCGAGCTATTTTTGATGATGAACTTCATATCCTGAGTC-3' for Aηα and the reverse primer 5'-GATCTCGAGCTACATCTTCACTTCAGAGATCTCCTCC-3' for Aηβ. Both reverse primers contain the XhoI enzyme restriction site. The amplicon was then digested by Hind III and XhoI enzymes and subcloned in the pcDNA3 vector. pcDNA3 plasmid expressing ηCTF: the ηCTF plasmid construction contains the APP signal sequence and two additional residues (Leu-Glu) from APP695 cDNA in frame with the 5'end of the ηCTF sequence. As previously described for SPA4CT 10 , SP-C99 and SP-C83 constructs 11 , the APP signal sequence is required for a correct membrane insertion of the ηCTF fragment. In a first PCR reaction, the APP signal sequence was amplified using the primers 5'-GATAAGCTTATGCTGCCCGGTTTGGCACT-3' that contains an HindIII restriction site and 5'-TTCACTAATCATCTCCAGCGCCCGAGCC-3' containing the first nucleotides of the 5' end of ηCTF sequence. In an additional PCR, ηCTF sequence was amplified using the primer 5'-GCGCTGGAGATGATTAGTGAACCAAGGATCAGTTA-3' that contains the last nucleotides of the 3' end of the signal peptide sequence, and the primer 5'-GATCTCGAGCTAGTTCTGCATCTGCTCAAAGAA-3'

containing XhoI restriction site. Finally, the amplicons resulting from the first two PCR were incubated together in a third PCR reaction to obtain a final DNA fragment containing the APP signal peptide in frame with ηCTF.

This amplicon digested with HindIII and XhoI enzyme was then subcloned in pcDNA3 vector.

AAV-10 plasmid expressing ηCTF under the control of synapsin-1 promoter: For viral plasmid construction, the pcDNA3 plasmid expressing ηCTF described above was used as a template with the primers 5'-GATGCTAGCCCACCATGCTGCCCGGTTTGGCACTGCTCCT-3' and 5'-GATGCTAGCCTAGTTCTGCATCTGCTCAAAGAACTTGTAGGTT-3' that both contain a NheI restriction enzyme site. The PCR product was then digested with NheI and sub-cloned into AAV10 plasmid.

pGEX plasmid for expression of recombinant ηCTF: the DNA sequence encoding the ηCTF was amplified by PCR, using the forward primer 5'-GATGGATCCATGATTAGTGAACCAAGGATCAGTTA-3', which has a BamH1 restriction site and the reverse primer 5'-GATCTCGAGCTAGTTCTGCATCTGCTCAAAGAA-3', containing a XhoI restriction site. The PCR product was then sub-cloned between BamH1 and XhoI restriction enzymes sites of pGEX-4T-1 vector.

All above constructs were verified by full sequencing.

Recombinant ηCTF production:

Recombinant ηCTF fragment was obtained as previously described for the production of recombinant PrP c fragment N1 12 , with some minor changes. Briefly, pGEX-4T-1 vector containing ηCTF sequence was transformed into BL21 gold strain of Escherichia coli. After induction with Isopropyl 1-thio-D-galactopyranoside (200 μM), the medium was centrifuged. Cells pellets were resuspended with PBS supplemented with a protease inhibitor mixture, DTT (10mM) and lysozyme (0.2mg/ml), then proteins were solubilized by the addition of Triton X-100 (10%), MgCl2 (1M), and DNase (1u/μl). After centrifugation, glutathione-Sepharose beads (GE Healthcare) were added to the supernatant, pelleted, and resuspended in PBS (1 ml). Peptides were cleaved with thrombin (5 units/ml; GE Healthcare), and thrombin was removed using Sepharose benzamidine beads (GE Healthcare).

Production of the ηCTF-Nter antibody

The new ηCTF-Nter antibody is a rabbit polyclonal antibody made following the Covalab's immunization protocol. As immunogen epitope, we designed peptide corresponding to the first 16 N-terminal residues of ηCTF fragment, the C-terminal end of which was blocked by a cystein residue in order to preserve a free N-terminal part of the peptide and to obtain antibodies directed towards the N-terminal moiety (sequence: MISEPRISYGNDALM-C). Rabbit's immunoreactivity and titer were controlled by ELISA then the ηCTF-Nter antibody was purified by antigen-specific affinity with the same peptide used for immunization.

Viral production and mice ICV injection:

Virus production was performed following a protocol previously described 13 . Briefly, HEK293 cells were transfected with the adenovirus helper plasmid (pXX6), the AAV packaging plasmid (rAAV2-rh10), and the AAV10 plasmid empty vector or encoding human ηCTF under control of the synapsin-1 promoter (AAV-empty, and AAV-ηCTF). Viruses were produced, purified and vector titers were determined by real-time PCR and expressed as viral genomes per ml (vg/ml). Four µl of AAV virus (5.5x10 12 vg/ml) were administrated in 1-dayold C57Bl6JRj mice (Janvier Labs, France) through intra-cerebroventricular (ICV) injection as described previously 13 then mice brains were analyzed at 3-months of age by western-blot and immunochemistry.

Animals:

Pregnant C57Bl6JRj females (Janvier Labs, France) were ordered for new born mice viral injection (see above).

In addition wild-type and 3xTg (APPswe; TauP301L; PS1M146V) mice were maintained from breeding pairs provided by Dr F. LaFerla

Cell Culture and treatments

Human neuroblastoma (SH-SY5Y, ATTC), human epitheloid cervix carcinoma (HeLa, ATCC) and mouse embryonic fibroblasts either wildtype (MEF-APPwt), or naturally devoid of APLP1 and knocked out for APP and APLP2 15 (referred to as MEF APP KO) were cultured in Dulbecco's modified Eagle's medium supplemented with fetal calf serum (10%), penicillin (100 U/ml) and streptomycin (50 μg/ml), and incubated at 37°C in a 5%

CO2 atmosphere. Human SH-SY5Y cells stably expressing wild type full-length APP (SH-SY5Y-APPWT) were generated as already described 16 , and maintained in the presence of G-418 (400 µg/ml). Transient transfections of cells were carried out using Lipofectamine 2000 (Life Technologies) for SH-SY5Y and MEFs and JetPrime (Polyplus transfection) for HeLa cells, according to the manufacturer's instructions. Twenty-four hours posttransfection, cells were treated with pharmacological agents: Lactacystin (5 µM; Sigma-Aldrich), Epoxomicin (1 µM; Enzo), MG132 (5 µM), Bafilomycin A1 (100 nM), Smer28 (50 µM), or with secretases inhibitors: -secretase inhibitor, Bi (30 µM, Elan Pharmaceuticals), α-secretase inhibitor, Gi (GI254023X; 10 µM; Sigma-Aldrich) or γsecretase inhibitor D6 (1 µM; Elan Pharm/Imago). Cells were analyzed 48 h post-transfection.

Western-blot analysis

Cells were lysed in RIPA buffer (Tris (50 mM); pH 7,4; NaCl (150 mM); EDTA (1mM); Triton X100 (1%); deoxycholate (0,5%); SDS (0,1%)) supplemented with a cocktail of protease inhibitors (Roche diagnostics). Cell homogenate proteins (50 µg) were separated on 16% tris-tricine gels, and sEVs proteins were separated on 16% tris-tricine gels or Bio-Rad 12% stain-free TM TGX FastCastTM acrylamide gels. It should be noted that in few cases, when protein of interests and loading control proteins have similar molecular weights, in order to achieve accurate normalizations, gels were run separately in strictly identical conditions. All full gels are provided as indicated in the legends of figures showing gels. Tris-tricine gels were directly transferred to nitrocellulose membranes using a conventional transfer system and boiled in PBS before saturation. Bio-Rad gels were photoactivated for the visualization of proteins before being electrophoretically transferred to nitrocellulose membranes using the Bio-Rad Trans-Blot® TurboTM Transfer System. Membranes were saturated with skimmed milk, and incubated overnight with the following primary antibodies: APPCter rabbit polyclonal (1:5000, gift from Paul Fraser); WO2 mouse monoclonal (1:5000, Sigma Aldrich); ηCTF-Nter rabbit polyconal (1:1000, homemade with Covalab, see above); α-HSC70 mouse monoclonal (1:1000, Santa Cruz) or α-GAPDH mouse monoclonal

(1:5000, EMD Millipore). After probing with primary antibodies, immunological complexes were revealed with anti-mouse or anti-rabbit HRP-conjugated antibodies (1:5000, Jackson ImmunoResearch) followed by electrochemiluminescence (Westernbright™ Sirius™ and Quantum™ chemiluminescent HRP substrate, Advansta, France). Peak height of signal intensities from protein bands were quantified with MultiGauge software.

For western-blot analysis of mice brains, following intracardiac perfusion with PBS, hemibrains were extracted then enzymatically and mechanically lysed using "the adult brain dissociation kit" and a GentleMACS Dissociator (Miltenyi Biotec). PBS was added to the cell suspension, which was then filtered through a 70 μm Smartstrainer and centrifuged at 300 x g for 10 min. The cell pellets were lysed in RIPA buffer supplemented with protease inhibitors and then loaded on Bio-Rad 12% stain-freeTM TGX FastCastTM acrylamide gels.

Sandwich ELISA analysis

MEF APP KO cells were transiently transfected with pcDNA.3 or ηCTF constructs using Lipofectamine 2000 and treated or not with secretases inhibitors Bi, Gi and D6. The concentrations of human secreted Aβ40 were measured in the culture medium by using the ELISA kit (Invitrogen) following the manufacturer's instructions.

Aηα Immunoprecipitation

MEF APP KO cells were plated on 6 wells plates, transiently transfected with pcDNA.3 or ηCTF constructs using Lipofectamine 2000 and treated or not with secretases inhibitors Bi, Gi and D6, as described above. Culture media were collected, centrifuged at 14000 x g for 5 min to remove cell debris then supernatants were supplemented with RIPA buffer 5X and precleared with 10 μl of Protein A Sepharose (GE Healthcare) for 1h at 4 °C. Resulting supernatants were incubated for 1 h with WO2 mouse monoclonal antibody (1 μl) then 20 μl of Protein A Sepharose beads were added and left for overnight incubation. After three washes with RIPA buffer (and 1 time in PBS), the beads were resuspended with 20 μl of loading buffer, loaded on a tris-tricine gel (16%) and subjected to western blot analysis as described above.

Cells immunostaining:

Hela cells grown on coverslips were fixed in paraformaldehyde 4% solution for 10 min, permeabilized with Triton-X 100 (0.1%) for 10 min, saturated in BSA (5%)/Tween20 (0.1%), and probed for 1h with appropriate primary antibodies: APPCter rabbit polyclonal (1:5000), APPCter, mouse monoclonal (1:5000, Biolegend), WO2 mouse monoclonal (1:5000), or ηCTF-Nter rabbit polyconal (1:1000) for ηCTF detection, α-Lamp2 mouse monoclonal

(1:1000, Santa Cruz), α-EEA1 rabbit monoclonal (1:200, Cell Signaling Technology), α-CD63 mouse monoclonal

(1:1000, Santa Cruz) and α-TGN46 (Serotec, AHP500G, 1:1000) for lysosomes, early and late endosomes and golgi specific markers, respectively. After washes, coverslips were incubated for 1 h with Alexa Fluor-488 and Alexa Fluor-590 conjugated antibodies (Molecular Probes, 1:1000) and DAPI (1:20,000, Roche) staining. Finally, the sections were washed with PBS, then mounted onto glass slides and cover-slipped. The stained slices were kept at 4•C before analysis with confocal microscopy (Zeiss LSM 780 with 63X Objective).

DAB and Immunofluorescence staining of tissue

For immunohistochemistry, mice were anesthetized by an intraperitoneal injection of a combination of ketamine (150 mg/kg), xylazine (10 mg/kg) and acepromezine (10 mg/kg) then perfused intra-cardiacally with PBS followed by paraformaldehyde (4%) before collecting the brains. Paraformaldehyde-fixed brains were embedded in paraffin and cut on a microtome in 8 μm thick sections (Thermoscientific, France). Brain sections were deparaffined in xylen bath and rehydrated by successive 5 min baths of EtOH (100%, twice), 90%, and then 70%. Antigens were unmasked in a 90% formic acid bath for 5 min and then in citric acid solution at 90-100°c for 20 min (Pressure cooker, Vector Laboratories). For DAB staining, sections were treated with H2O2 3% for 15 mins. Non-specific binding sites were blocked for 1h in BSA (5%)/Tween20 (0.1%) and brain sections were then incubated at 4 °C overnight with primary antibodies ηCTF-Nter rabbit polyclonal (1: 800) or WO2 mouse monoclonal (1:1000).

Sections were incubated for 1h with Alexa Fluor-488 and Alexa Fluor-594 conjugated antibodies (1:1000) and DAPI (1:20,000) for immunofluorescence, or with horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch, 1:1000) then revealed with the DAB-ImmPACT system (Vector Laboratories) for DABstaining. Images were taken with a confocal Leica TCS SP5 microscope (Immunofluorescence) or with DMD108

Leica microsystem (DAB) and processed using ImageJ software. For ηCTF-Nter staining, signal was amplified with Avidin Biotin Complex (ABC) method (VECTASTAIN Original ABC Kit, Vector Laboratories).

Purification of exosomes (sEVs)

SH-APPWT cells were cultivated in DMEM for 24h, rinsed twice with PBS, and treated or not with the -secretase inhibitor Bi or Bafilomycin A1 as described above, for another 24 h in OptiMEM to allow vesicle secretion. When overexpressing ηCTF, cells were transiently transfected 1 day before secretion in OptiMEM. sEVs were purified by ultracentrifugation as previously described 17 . Briefly, media were first harvested and centrifuged at 2000 x g for 20 min, then filtered through a 0.22 μm filter. The supernatant was then sequentially centrifuged at 10000 x g for 30 min and 100,000 x g for 125 min. The obtained pellet containing sEVs and contaminating proteins was washed in ice-cold PBS and centrifuged for another 120 min. The final sEVs pellet was resuspended in RIPA buffer and sonicated in an ultrasonic bath for 15 min before western-blot analysis.

sEVs isolated from the extracellular space of AAV-CTF mice brain were purified according to the protocol previously described 17 . Briefly, intracardiac perfusion with PBS was performed then hemibrains were extracted and enzymatically and mechanically lysed using "the adult brain dissociation kit" and a GentleMACS Dissociator (Miltenyi Biotec). PBS was added to the homogenate suspension, which was then filtered through a 70 μm

Smartstrainer and centrifuged at 300 x g for 10 min. The supernatant was used for sEVs purification and the cell pellet was kept for further analysis by western blot as described above. For sEVs purification, the supernatant was sequentially centrifuged several times as previously described 16 , and the last pellet was loaded on a sucrose gradient and centrifuged at 200000 x g for 16h. The fractions containing the sEVs were collected, diluted with PBS and centrifuged at 100000 x g for 90 min. The final sEVs pellet was resuspended in PBS, lysed in RIPA buffer complemented with protease inhibitors and ultrasonicated before western-blot analysis. Before loading, nanoparticle analysis was performed on each fraction using the ZetaView instrument (Particle-Metrix) in order to determine particle size distribution and concentration.

Membrane Fraction Preparation

Dissected hippocampi of 3xTgAD (AD) and wild-type (WT) mice were homogenized in hypotonic buffer (HEPES (5mM), pH 7.4, EDTA (1 mM), sucrose (0.25 M) containing a proteases inhibitor cocktail). The homogenate was centrifuged at 1000 x g for 5 min at 4°C and the supernatant was centrifuged at 100000 x g for 1h. Membrane pellets were then solubilized in RIPA buffer complemented with protease inhibitor, and centrifuged at 100000 x g for 20 min. Supernatants were recovered as the soluble membrane fractions and loaded on 16% tris-tricine gels.

Statistical Analyses

Statistical analyses were performed using Prism software (GraphPad Prism 7)

Results

Proteasomal and autophagic degradation of ηCTF in human SH-SY5Y neuroblastoma cells.

A newly described η-secretase cleavage occurs on APP695 between residues 504 and 505, yielding a 95kDa soluble fragment (sAPPη or sAPP95) and a 25-30kDa transmembrane fragment called ηCTF, which is subsequently cleaved by αor -secretases to generate Aηα and Aη peptides, respectively (Fig. 1a). In order to understand the biology and fate of the ηCTF fragment, we expressed it in human neuroblastoma cells (SH-SY5Y) and analyzed its expression by western-blot using APP-Cter and the WO2 antibodies targeting, the carboxy terminal end of APP and the amino-terminal part of A peptides, respectively (Fig. 1a). In SH-SY5Y cells transiently transfected with the ηCTF-pcDNA coding vector, a specific band around 30kDa is detected by both APP-Cter and WO2 that displays a similar migrating pathway than recombinant ηCTF (Fig. 1b,c), thus firmly supporting the identity of ηCTF. As expected, in C99-transfected SH-SY5Y cells, C99 is also recognized by these two antibodies (Fig. 1b,c) while APP-Cter antibody, but not WO2, detects endogenous C83 (Fig. 1b). In both C99 and ηCTF overexpressing cells, the C83 levels are increased as compared to control cells, indicating that both of these fragments undergo an α-secretase-mediated cleavage (Fig. 1b). Similarly, in ηCTF overexpressing cells, the C99 levels are increased as compared to control cells indicating that ηCTF also behaved as a -secretase substrate (Fig. 1b). Of note, as expected, WO2 but not APP-Cter recognizes the Aηα peptide (Fig. 1c). This set of experiments that aimed at characterizing ηCTF expression in SH-SY5Y cells indicates that ηCTF mostly undergoes and secretase cleavages giving rise to C99 and C83 as previously described 7 while the ηCTF-derived production of Aηα remains poorly detectable in cell homogenates.

To uncover the cellular pathways by which ηCTF is degraded, we used a pharmacological approach and treated ηCTF expressing SH-SY5Y cells with several well characterized inhibitors targeting either proteasomal or autophagic degradative processes. All proteasome inhibitors namely, lactacystin, epoxomicin or MG132 lead to significant increases of ηCTF expression (Fig. 2a,b), thus demonstrating the involvement of the proteasomal machinery in ηCTF degradation in SH-SY5Y cells. In addition, two lines of evidences indicated significant additional contribution of the autophagic pathway. First, ηCTF immunoreactivity is increased by the potent and selective autophagy blocker bafilomycin A1 (Fig. 2c,d). Second, ηCTF expression is decreased by the activator of autophagy SMER28 (Fig. 2c,d). Inhibition of both proteasome and autophagy also leads to an accumulation of endogenous and ηCTF-derived C99 and C83 fragments (Fig. 2a,c), which have already been described to be degraded by these two protein degradation systems 13,18-21 . Overall, both proteasomal and autophagic pathways contribute to ηCTF degradation overexpressed in human neuronal cells. However, since the increase in endogenous C83 expression is more importantly obtained by bafilomycin A1 treatment (Fig. 2c), this suggests that the autophagic process likely remains the main degradative process of endogenous APP-CTF. Accordingly, C83, C99 and a band corresponding to ηCTF are also increased following bafilomycin A1 treatment of SH-SY5Y cells stably expressing wild-type APP protein (SH-APPWT, Sup Fig. 1a).

We aimed at characterizing the influence of secretases inhibitors on CTF processing but since in native SH-SY5Y cells, it is difficult to detect endogenous ηCTF immunoreactivity even after blockade of the proteasomal and autophagic pathways (Fig. 2a,c), we used SH-APPWT cells and treated them with α-, or -secretase inhibitors. We analyzed all APP-Cter fragments produced using APP-Cter, WO2 and 82E1 antibodies, the latter targeting specifically the free N-terminal of C99, and thus, is unable to detect ηCTF. As expected, the levels of C83 and C99 are reduced upon αand -secretase inhibitions by Gi and Bi, respectively, while both C83 and C99

immunoreactivities were increased when -secretase is blocked by its selective and potent inhibitor D6 (Sup Fig. 1b). Interestingly, a band migrating around 30kDa detected by WO2 but not 82E1 antibody is noticeably increased following -secretase inhibition in pcDNA3-transfected cells (Sup Fig. 1b, pcDNA3 lane Bi, see long exposures) while, as expected, -secretase inhibition fully abolishes APPWT-derived C99 formation detected by both antibodies. This 30kDa band increased by Bi and bafilomycin A1 treatments in pcDNA3 mock-transfected cells appears with a similar migrating pathway than specific ηCTF-like immunoreactivity obtained in ηCTF-expressing SH-APPWT cells and thus might correspond to the ηCTF fragment derived from the stably expressed wild-type APP protein (Sup Fig. 1a,b). Of note, none of the secretase inhibitors significantly affected the level of ηCTF in CTF-transfected SH-APPWT-cells (Sup Fig. 1b,c).

Expression of ηCTF in fibroblasts devoid of APP leads to Aηα and A productions.

APP-CTF fragments (C99 and C83) may theoretically derive from both APP-full length or ηCTF in SH-SY5Y cells (see above). In order to delineate those genuinely derived from ηCTF, we expressed this fragment in mouse embryonic fibroblasts devoid of endogenous APP and its family members APLP1 and APLP2 (MEF APP KO, Fig. 3a). As expected, ηCTF is readily expressed in fibroblasts and detected by both the APP-Cter and the WO2 antibodies (Fig. 3a). We treated the ηCTF expressing APP KO fibroblasts with secretase inhibitors and examined the Aηα and A productions. Although α-, or -secretase inhibitions do not significantly modulate ηCTF expression in MEF APP KO (Fig. 3b), we provide evidence that ηCTF is indeed processed by α-and -secretase activities. First, ηCTF expression in MEF APP KO leads to the production of an APP-CTerbut not WO2immunoreactive fragment corresponding to C83 (compare lower panels in Fig. 3a). Second, we specifically immunoprecipitate a secreted fragment labeled by WO2 which is fully sensitive to the -secretase inhibitor Gi, thus corresponding to Aηα (Fig. 3c). Third, for the first time, we were able to measure by ELISA an increased level of A40 in ηCTF expressing APP KO fibroblasts compared to pcDNA3 control fibroblasts, and as expected,

we observed a drastic decrease of A40 upon cells treatment with either or -secretase inhibitiors (Fig. 3d).

Although A production indicates that ηCTF is processed by -secretase, in our conditions, we were unable to detect C99, suggesting that this fragment is either poorly generated or, alternatively, rapidly degraded or further processed into C83 as was demonstrated in other cell models 11,17 . Taken together, our results demonstrate that ηCTF expressed in APP KO fibroblasts is indeed cleaved by α-and -secretases to yield C83, Aηα and A peptides as major detectable catabolites.

Subcellular localization of ηCTF in trans-Golgi network and in endosomes.

Although the immunological tools available, combined to migration pattern and molecular weight analysis, allow us to firmly identify ηCTF in vitro by western blot, these antibodies do not allow a definitive identification of the fragment in situ and thus, have strong limitations for immunohistochemical analysis. In this context, we aimed at designing a new antibody referred to as ηCTF-Nter that would specifically label the N-terminus of ηCTF (Fig 4 a). By western blot analysis, we show that this new antibody recognizes not only ηCTF, but also, as expected, the ηCTF N-terminal sequences corresponding to Aηα and Aη peptides (Fig. 4b). In addition, similar increase in ηCTF-Nter-like immunoreactivity was obtained in SH-SY5Y treated with proteasomal/autophagic inhibitor (Sup The ηCTF-Nter-like immunoreactivity obtained using the new ηCTF-Nter antibody is found mainly intracellular (Fig. 4c). and as expected, this specific ηCTF staining co-localizes with a part of the APP-Cter-like immunoreactivity that likely corresponds to η-CTF, C83 and C99 (Fig. 4d). Similarly, the ηCTF-Nter labeling partly overlaps with WO2-like immunoreactivity (Sup Fig. 3).

To further identify the intracellular compartments in which ηCTF-Nter-like immunoreactivity is detectable, we performed co-immunostaining using antibodies labeling different organelles. We found that ηCTF co-localizes with TGN-46, which is a marker of the Golgi apparatus and trans-Golgi network in HeLa cells (Fig. 5a). In addition, a small but significant amount of ηCTF-immunoreactivity is observed in EEA1-and CD63-positive structures corresponding to early and late endosomal compartments, respectively (Fig. 5b) while little if any costaining was observed with the lysosomal marker Lamp2 (Fig. 5c,d).

A similar localization of ηCTF was observed in wild-type mouse organotypic hippocampal slices infected with an adeno-associated-virus (AAV) expressing ηCTF (Sup Fig. 4). We confirmed the presence of ηCTF-Nter-like immunoreactivity in EEA1-positive endosomal compartments and observed a characteristic perinuclear trans-Golgi staining and a strong overlap with APP-Cter labeling (Sup. Fig. 4). It is of note that the antibody does not raise significant background even in situ. Overall, the above set of data demonstrates the usefulness of our novel immunological probe to follow ηCTF expression in various models expressing ηCTF fragment in both in vitro (HeLa cells) and ex vivo (organotypic mouse hippocampal slices) and leads to the consistent conclusion of a main Golgi and endosomal localizations of the ηCTF fragment.

The specific ηCTF-staining was also examined in vivo, in brain slices of mice expressing ηCTF through a previously described AAV-viral strategy 13 . Western-blot analysis of ηCTF expression (Fig. 6a) and immunohistochemical assessment with η-CTF-Nter (Fig. 6b c) revealed that ηCTF is highly expressed after viral infection in brain homogenate (Fig. 6a) and is specifically detected in situ in cortex and hippocampus of AAV-ηCTF mice (Fig. 6b c).

Detection of ηCTF fragment in small extracellular vesicles (sEVs).

The presence of the ηCTF fragment in endosomes led us to investigate whether ηCTF fragment could be found in exosomes which originate from endosomes. These small extracellular vesicles (sEVs) are currently envisioned as organelles mediating toxic spreading in various neurodegenerative diseases 22-24 . Our data show that ηCTF fragment is detectable in sEVs isolated from η-CTF-expressing cells and mice. Indeed, we detected ηCTF in sEVs purified from both media of η-CTF-expressing SH-APPWT and of -secretase inhibitor-treated pcDNA3 SH-APPWT cells (Fig. 7a). As expected, we also observed high recoveries of ηCTF in sEVs following bafilomycin A1 treatment which is known to increase exosomal secretion (BafA1 Fig. 7b). Since exosomes contain high levels of -secretases (ADAM10 and ADAM17) 25,26 and since ηCTF was found to undergo -secretase cleavage, sEVs were purified from mouse brains in the presence (or not) of the α-secretase inhibitor (Gi) in order to enhance CTF recovery. Our data show that ηCTF can be detected in sEVs (with a medium size around 140 nm) purified from mouse brains overexpressing ηCTF (Fig. 7c,d). This agrees well with Laulagnier and collaborators who reported the presence of ηCTF in exosomes purified from primary cortical neurons or neuroblastoma N2a cells overexpressing APP wild-type 27 . ηCTF surrounds amyloid plaques and accumulates in an age-dependent manner in 3xTgAD mouse brains.

Using the ηCTF-NTer antibody, we observed ηCTF-like immunoreactivity in the hippocampus of 3xTgAD mice, a widely used AD-mice model 14 . The labeling increased progressively from 3 to 20 months of age (Fig. 8a, lower panels) while only a very faint labeling was observed in the cortex of 20-month-old wild-type mice (Fig. 8a, upper panels). This age-related accumulation of ηCTF fragment was confirmed by western-blot using the APP-Cter antibody (Fig. 8b). Interestingly, in the subiculum of 20-month-old 3xTgAD mice, the η-CTF-Nter staining obtained by peroxydase/DAB development appears more punctiform suggesting an aggregated state and is mainly surrounding plaques (Fig. 8a, lower panel). These findings were confirmed by immunofluorescence in double immunostaining experiments with ηCTF-Nter and WO2 antibodies. WO2 targeting the N-terminal part of A labeled the core of the extracellular plaque while ηCTF-Nter staining surrounded WO2-associated labeling (Fig. 8c). This set of experiments confirms the usefulness of the ηCTF-Nter antibody to detect ηCTF in vivo and indicates that ηCTF accumulates at late stage of the pathology around plaques in 3xTg-AD mice.

Discussion

The amyloid hypothesis is strongly supported by a bulk of genetic, anatomical and biochemical evidences but an increasing number of studies suggest that Aβ could likely not account for all cellular and behavioral dysfunctions taking place in AD 28 . The limited progress of A-directed clinical trials suggested that other triggers could well be involved in AD etiology. In this context, in order to reconcile genetic grounds and clinical observations, one could envision additional APP-related fragments, distinct from canonical A, as putative contributors to disease onset and/or progression. Along this line of reasoning, several tracks have been followed including numerous truncated forms of Aβ peptides 29-31 as well as the AICD transcription factor 5 . Further, our laboratory demonstrated that the -secretase-derived APP fragment C99 could account for early dysfunctions observed in AD pathology 32,33 . More recently, the disintegrin MT5-MMP was identified as a novel secretase named -secretase 7,8 . This metalloprotease triggers a cleavage of APP upstream to the one due to -secretase, thereby yielding a fragment referred to as CTF, the biology of which has been poorly examined yet. In this study, we show data on the processing, fate, subcellular localization and exosomal secretion of CTF.

In neuronal cells, as is the case for APP, CTF undergoes same and secretases cleavages yielding the Cterminal fragments C83 and C99 and their N-terminal counterparts A and A, respectively. Of interest, we report for the first time that CTF also undergoes -secretase-mediated cleavage giving rise to A peptides. This was definitively demonstrated by our data obtained in APP KO fibroblasts where the expression of ηCTF indeed leads to A40 production. In the latter cellular model, ηCTF undergoes proteolysis by -secretase as illustrated by C83 accumulation and Aη secretion. This -secretase can occur at the cellular plasma membrane as previously extensively described 34 but also inside exosomes where the sheddases ADAM10 and ADAM17 (TACE) are enriched and active 25,26 . Indeed, exosomes have been proposed to serve as a platform for ectodomain shedding of a variety of transmembrane proteins such as TNFR1, CD46, CD44 and the L1 adhesion molecule 35 . It should be noted that although -secretase cleavage takes place on CTF (since A is produced), C99 remained poorly detectable. This could be explained by different ways that would not be necessarily exclusive: 1) -secretase activity is much higher than -secretase in fibroblasts and thus, C83 is preferentially produced; 2) -secretase displays a much higher affinity for CTF than -secretase; 3) a small proportion of whole CTF meets active secretase in endosomes, 4) C99 is more rapidly degraded than C83 in fibroblasts; 5) C99 is produced but itself rapidly converted into C83 as we previously showed 11,17 . Overall, the above set of data indicates that ηCTF behaves as a direct precursor of both C99 and A peptide and thus, displays a theoretical toxic potential. This is in agreement with Baranger and colleague's studies that proposed the η-secretase as a new pro-amyloidogenic proteinase 8,9 .

Very little is known about the fate of CTF and the mechanisms by which this fragment is cleared off the cells.

By means of a pharmacological approach, we established that CTF degradation involves both proteasomal and autophagic pathways. However, the increase of endogenous C83 expression was more importantly triggered by bafilomycin A1 (Figure 2 C) than by proteasome blockers, suggesting that the autophagic process likely remains the main degradative process of endogenous APP-CTF as previously reported 13,19-21,36 . The degradation of CTF in the autophagic pathway is in agreement with previous report by Wang and collaborators who described ηCTF fragment as a new APP-Cter fragment detected in naïves HEK293 cells following cathepsins inhibition 6 . In addition to autophagy, our result demonstrated also a significant degradation of CTF by the proteasome.

However, it should be noted that transient or constitutive ectopic protein expression may lead to mis-localizations and/or artifactual protein aggregation. Thus, when misfolded proteins accumulate in the ER, the unfolded protein response (UPR) and ER-associated degradation (ERAD) mechanism are induced to avoid cellular damages. In line with this warning, Evrard and collaborators have shown that endogenous APP-CTFs are mainly processed by the endosomal/lysosomal pathway while overexpressed C99 was mainly degraded by the proteasome 37 . It could be the reason why in our experimental conditions, overexpressed ηCTF is readily degraded by the proteasome. In addition, our results indicate that the ηCTF overexpressed either in MEF APP KO fibroblasts or SH-APPWT neuronal cells, is not protected by the inhibition of BACE-1 activity unlike was previously described for the endogenous ηCTF 7 . This discrepancy could be also explained by the source of the ηCTF fragment (overexpressed vs endogenous)

Although molecular weight estimation and immunological characterization allows clear biochemical identification of CTF by western-blot, this toolbox falls short when one aims at studying CTF in situ. In this context, we envisioned the design of a novel immunological probe that would discriminate between the plethora of APP-CTFs.

Thus, we designed an antibody referred to as CTF-NTer that labels CTF N-terminus moiety and thus, would not interact with C83 and C99 or A. This was successfully achieved as illustrated in Figure 4. Thanks to this new ηCTF-Nter antibody, immunocytochemical analysis indicated that ηCTF is mostly localized in Golgi apparatus and trans-Golgi network in HeLa cells. However, a lower amount of ηCTF immunoreactivity is also observed in endosomes in both CTF transfected HeLa cells and hippocampal organotypic slices prepared from newborn mice expressing CTF.

We took advantage of our know-how in the preparation of fully characterized exosomes 17 to examine whether CTF could be detected in these small extracellular vesicles (sEVs). Indeed, we show the presence of ηCTF in sEVs purified from secretates of human neuroblastoma cells overexpressing ηCTF. This agrees well with the study by Laulagnier and collaborators who detected ηCTF in exosomes secreted by rat cortical neurons expressing endogenous APP protein as well as by mouse N2a cell line overexpressing wild-type APP protein 27 . Of note, we were also able to detect the ηCTF fragment derived from wild-type APP protein in sEVs purified from medium of SH-APPWT cells treated with -secretase inhibitor. Thus, our immunohistochemical analysis of CTF localization and biochemical analysis of exosomal content in cells all suggest that at least a part of CTF is transported through endosomes and accumulate in exosomes. Exosomes can be secreted by all cell types such as neurons, oligodendrocytes, astrocytes or microglial cells and have been described to play important physiological and pathological roles in cellular communication as well as in protein aggregates spreading 38 . In AD pathogenesis, Tau protein 39 , Aβ peptides 40,41 and C99 fragment 36,42,43 have been found in multivesicular bodies and exosomes.

Our laboratory previously established the presence of C99/C83 oligomers in exosomes and their accumulation is enhanced upon -secretase inhibition 17 . Thus, both C99 and its precursor CTF accumulate similarly in exosomes.

Finally, we also documented the usefulness of our novel immunological probe for in vivo approaches and demonstrated the presence of CTF in exosomes purified from brains of mice expressing ηCTF. Moreover, we

were able to show a high CTF expression surrounding the core of abundant senile plaques in triple transgenic mice as was also shown in APP/PS1 transgenic mice and in AD brain 7 .

A question arises as to whether CTF could be considered as a fully toxic trigger in AD or could govern more balanced physiopathological functions. In this regard, similarly to A peptides, C99 or phosphorylated Tau proteins, the presence of ηCTF fragments in exosomes could have both beneficial or deleterious consequences.

Exosomes carrying ηCTF could contribute 1) to eliminate excess of ηCTF from the cells; 2) to transport physiological intercellular signals or 3) to be responsible for neuropathological spreading 38 . Nonetheless, the presence of ηCTF in exosomes as well as its accumulation and detection around amyloid plaques may support its potential contribution to AD pathology. This postulate is strengthened by the fact that CTF acts as a direct precursor of C99 that is considered as an early trigger of AD pathology 44 . Therefore CTF could be a source of intracellular C99/A production but also an indirect way to promote the exosomal C99/A spreading resulting in an extracellular A deposits. In addition, exosomes released from neurons have been described to regulate synaptic activity 45 . Further, CTF yields A and A, the influence of which on hippocampal long-term potentiation has led to discrepant conclusions 7,46 . Apparently, Aη and Aη peptides could impair synaptic activity 46 . This adds support to the conclusion that CTF directly per se, or indirectly, as a precursor, could well contribute to AD pathology. Thus preventing CTF production could be of therapeutic relevance to act at very early stages of AD.

This newcomer on disease stage could reconcile, at least to some extent, the numerous failures of clinical trials targeting A with the well supported genetic hypothesis centered on APP.

Declarations

Ethics approval:

Legends to Figures b. SH-SY5Y cells were transiently transfected with C99, ηCTF, Aη, Aη or empty pcDNA3 vectors and analyzed by western-blot using ηCTF-Nter and APP-Cter antibodies. GAPDH is used as loading control. Note that as expected, ηCTF-Nter antibody recognizes ηCTF, Aη and Aη but neither C99 nor C83 while APP-Cter antibody recognizes ηCTF, C99 and C83 but neither Aη nor Aη.

c. Hela cells were transiently transfected with ηCTF or empty pcDNA3 vector and analyzed by immunofluorescence using ηCTF-Nter or APP-Cter antibodies as described in Methods. Note that both staining are mostly perinuclear with punctuate intracellular staining. Brains were dissected and homogenized for membrane protein purification then analyzed in western-blot using APP-Cter antibody. A specific band corresponding to ηCTF is detected around 30kDa. GAPDH is used as loading control (a).

Brain sections were immunostained with ηCTF-Nter antibody and revealed by horseradish peroxidase DAB (b) or by immunofluorescence (c). Brain regions are depicted as cortex (cxt), corpus callosum (CC), subiculum (sub), hippocampal CA1 region (CA1) and dentate gyrus (DG). Specific ηCTF-Nter immunostaining occurs in cortex, subiculum and hippocampus. Confocal images obtained with ηCTF-Nter antibody showed a perinuclear with punctuate intracellular staining (c). Nuclei were stained with DAPI. a-b. SH-APPWT cells were transiently transfected with ηCTF or empty pcDNA3 vector and treated for 24h with -secretase inhibitor (a, Bi, 30µM), or bafilomycin A1 (b, BafA1, 100nM). Cell lysates and sEVs were purified from culture media as described in methods and analyzed by western-blot using APP-Cter antibody.

c. sEVs were purified from brain homogenates of 3-month-old AAV-free and AAV-ηCTF mice in the presence or not of the -secretase inhibitor (Gi :10µM) and analyzed by western-blot using APP-Cter antibody. HSC70 is used as an exosomal marker. Whole loaded proteins were stained by photoactivation using Bio-Rad prestain method (Protein Stain) as loading control.

d. Concentration and particles size of each brain mouse exosomal purified samples were analyzed in ZetaView instrument (Particle-Metrix) before loading on gels. a-c. Brains of wild-type (WT) and triple transgenic (3xTg) females were analyzed at 3-, 6, 13-and 20-month-old by immunohistology (a), western-blot (b) or immunofluorescence (c). DAB-immunohistochemical staining is obtained using ηCTF-Nter antibody as described in the Methods. Higher magnification reveals an intracellular labeling clearly observed in cortex, subiculum and hippocampus of 3xTg mice while a weak staining was detected only in the cortex of wild-type mice. In subiculum of 20-month-old 3xTg mice, an extracellular staining is observed around amyloid plaques (a). Brains of wild-type (WT), 3xTg, AAV-free and AAV-ηCTF were homogenized for membrane protein preparation then analyzed in western-blot using -APP-Cter antibody. A specific band corresponding to ηCTF is detected around 30kDa in wild-type (WT) mice and accumulates in 3xTg mice. GAPDH is used as loading control (b). Confocal images obtained with WO2 (green) and η-CTF-Nter (red) antibodies and merged images from 20-month-old 3xTg hippocampus revealed WO2-positive core plaques surrounded by an ηCTF-Nter-like immunoreactivity (c).

2) Additional investigations on the detection of ηCTF in human AD patients and functional implication of Aηα and Aηβ on synaptic activity

Investigations on the detection of ηCTF in human AD patients

After developing and characterizing the ηCTF-NTer antibody and providing convincing findings in AD mouse models, it was time to try to detect ηCTF in AD patients.

Therefore, in the aim of detecting ηCTF in humans, Western-blot analyses from patient samples and an immunostaining experiment on paraffin-embedded brain slices of AD patients were performed. Although the results from the human samples could have been included in the paper, we preferred to test more samples first, in order to reduce variability and ensure the reliability of the findings.

Using the ηCTF-NTer antibody for peroxydase/DAB immunostaining, we observe an intracellular ηCTF-like immunoreactivity in the hippocampus of AD patients, whereas no labeling is observed in the control patient (Figure 17). I examined three controls and six AD patients for this experiment, and only the most representative images are presented in the figure. ηCTF fragment doesn't seem to accumulate neither in a stage-dependent nor in a plaquedependent manner (Figure 17, second and third lane of panels). Interestingly, in one patient (CHE in TABLE 2) with an undetermined stage of AD, ηCTF-NTer antibody appears to label cells that are surrounding the plaques, but not the core of the plaques (Figure 17, lower panels).

This patient appears to be at an advanced stage of AD; however, the ηCTF-NTer staining does not appear to be aggregated around plaques as seen in the AD mouse model (Figure 8 of the article), and the labeling looks more likely to be similar to an APP-full length (APP-FL) staining. 2) were analyzed by DAB-immunohistochemical staining and obtained using ηCTF-Nter antibody to detect ηCTF, or WO2 for the detection of amyloid plaques.

In order to determine whether the ηCTF-Nter antibody could recognize APP-FL, we compared the detection of APP-FL in different models by western-blot using APP-Cter and the ηCTF-Nter antibodies. As expected, APP-Cter antibody recognizes the endogenous APP-FL in SH-SY5Y and MEF-APPwt cells and in the wild-type mouse model, as well as overexpressed human APP-FL in SH-APPwt, and in 3xTgAD mouse models; while no signal is observed in MEF APP KO since APP is knocked out (Figure 18). Strikingly, the ηCTF-Nter antibody does also recognize the human APP, but only when its overexpressed, as in the SH-APPwt human cell model and the 3xTgAD mouse model (Figure 18). This antibody, on the other hand, does not detect endogenous APP due to its lower sensitivity compared to APP-Cter, nor does it detect murine APP.

By western-blot, we aimed at detecting the ηCTF fragment in brains of AD patients at various Braak NFT stages, compared to control patients, in two separate experiments with samples from different patients. Using the APP-Cter antibody, a band at approximately 30kDa is identified, but with a slightly different migratory pathway than the ηCTFrec (Figure 19-a, b). While this band does not appear to accumulate in a stage-related manner (Figure 19-a, b), it also does not correspond to the ηCTF fragment, as it is not detected by WO2 (Figure 19-c, d) or the ηCTF-Nter antibody (Figure 19-e, f). Moreover, the fragment migrating below 19kDa corresponds to C83 since it is recognized by APP-Cter (Figure 19-a, b), but not by WO2 (Figure 19-c, d) nor by ηCTF-Nter (Figure 19-e, f); and as expected, its expression appears to be more important in AD patients in comparison with the controls (Figure 19-a, b). Therefore, in contrast to data from Willem and colleagues, in our preliminary experiments, ηCTF seems to remain undetectable in human brains, and this could be explained by different ways: 1) ηCTF is not abundantly produced; 2) ηCTF is directly degraded by the proteasomal and autophagic pathways; or 3) ηCTF is rapidly cleaved by the secretases giving rise to its derived fragments, which is in accordance with the presence and the accumulation of C83 in AD brains (Figure 19-a, b). Brains of wild-type (WT), and 3xTg mice were homogenized for membrane protein preparation then analyzed in western-blot along with SH-SY5Y, SH APPwt, MEF APPwt and MEF APP KO cells for the detection of APP full length (APP-FL), using APP-Cter and ηCTF-Nter antibodies.

: ΗCTF IS NOT DETEC FL in different cell and mouse models, using ηCTF

Cter and ηCTF

The functional implication of Aηα and Aηβ on synaptic activity

After determining the fate, biology, and localization of ηCTF, it was time to move on to examining the role of the fragments released from the cleavage of APP by η-secretase as a first step toward the understanding of the implications of this pathway in AD. Therefore, we were interested in performing preliminary LTP experiments on wild-type mice hippocampal slices incubated with Aηα and Aηβ.

Aηα, but not Aηβ, was the first to be proven to suppress LTP after acute exposure of hippocampal slices to 100 nM this peptide (Willem et al., 2015). However, a more recent study conducted by the same team reported that both synthetic Aηα and Aηβ impair LTP ex vivo, even at low nanomolar concentrations of 5 nM [START_REF] Mensch | Aη-α and Aη-β peptides impair LTP ex vivo within the low nanomolar range and impact neuronal activity in vivo[END_REF]. While these findings are contradictory, we demonstrate in our preliminary tests that the recombinant Aηα significantly reduce LTP when incubated to hippocampal slices of C57Bl/6 mice at 100nM (Figure 20-a, c).

Materials and Methods

Ex vivo electrophysiology:

These experiments were conducted by Dr. Julie Dunys.

Three to five weeks old wild-type mice were anesthetized with isoflurane and then decapitated.

Human brain samples:

All procedures performed in studies involving human brains were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical studies. Informed consent for tissue donation for research is obtained by the Brain Bank NeuroCEB and has been declared at the Ministry of Higher Education and Research (agreement AC-2013-1887) under their approval procedures. We studied control and SAD post-mortem human brains. Cases were anonymized, but information was provided regarding sex, age at death, and neuropathology.

Western-blot analysis:

Human brain samples were homogenized in RIPA buffer (Tris 50 mM; pH 7.4 containing NaCl (150 mM), EDTA (1 mM), Triton X100 (1%), deoxycholate (0.5%), sodium dodecyl sulfate (SDS, 0,1%) and complete protease inhibitor mixture (Sigma). After homogenization with a teflon glass potter, homogenates were centrifuged (100,000 g, 1 h, 4 °C), and supernatants were kept as soluble fractions that were used for western-blot analysis. 80 µg of brain samples proteins were separated on 16% tris-tricine gels.

Cells were lysed in RIPA buffer and supplemented with a cocktail of protease inhibitors (Roche diagnostics). For western-blot analysis of mice brains, following intracardiac perfusion with PBS, hemibrains were extracted then enzymatically and mechanically lysed using "the adult brain dissociation kit" and a GentleMACS Dissociator (Miltenyi Biotec). PBS was added to the cell suspension, which was then filtered through a 70 μm Smartstrainer and centrifuged at 300

x g for 10 mins. The cell pellets were lysed in RIPA buffer supplemented with protease inhibitors. 50 µg of proteins are then loaded on Bio-Rad 7.5% tris-tricine gels.

Tris-tricine gels were directly transferred to nitrocellulose membranes using a conventional transfer system and boiled in PBS before saturation. Membranes were saturated with skimmed milk, and incubated overnight with the following primary antibodies: APPCter rabbit polyclonal (1:5000, gift from Paul Fraser); WO-2 mouse monoclonal (1:5000, Sigma Aldrich); ηCTF-Nter (1:1000, homemade with Covalab). After probing with primary antibodies, immunological complexes were revealed with anti-mouse or anti-rabbit HRP-conjugated antibodies (Jackson ImmunoResearch, 1:5000) followed by electrochemiluminescence (Westernbright™ Sirius™ and Quantum™ chemiluminescent HRP substrate, Advansta, France).

DAB and Immunofluorescence staining of tissue:

Human brain sections were deparaffined in xylen bath and rehydrated by successive 5 min baths of EtOH (100%, twice), 90%, and then 70%. Antigens were unmasked in a 90% formic acid bath for 5 min and then in citric acid solution at 90-100°c for 20 min (Pressure cooker, Vector Laboratories). Brain sections were treated with H2O2 3% for 15 mins. Non-specific binding sites were blocked for 1h in BSA (5%)/Tween20 (0.1%) and brain sections were then incubated at 4 °C overnight with primary antibodies ηCTF-Nter (1: 800) or WO2 (1:1000). Sections were incubated for 1h with horseradish peroxidase (HRP)-conjugated antibodies (Jackson ImmunoResearch, 1:1000) then revealed with the DAB-ImmPACT system (Vector Laboratories). Images were taken with DMD108 Leica microsystem (DAB) and processed using ImageJ software. For ηCTF-Nter staining, signal was amplified with Avidin Biotin Complex (ABC) method (VECTASTAIN Original ABC Kit, Vector Laboratories). In our study models, we mainly used overexpression approaches in order to study the localization of ηCTF, its processing and its degradation. In the models we tested, ηCTF was mostly localized in the trans-Golgi network, and to a lesser extent, in endosomes. When it came to ηCTF's degradation, the proteasomal and autophagic pathways were both implicated. But what exactly occurs under pathophysiological circumstances? Since MT5-MMP is actually located on the plasma membrane (Llano et al, 1999[START_REF] Klunk | Imaging brain amyloid in Alzheimer's disease with Pittsburgh Compound-B[END_REF]) η-secretase can potentially cleave APP at this level, producing endogenous ηCTF that may be subsequently either cleaved by α-secretase at the plasma membrane or internalized into early endosomes. As is generally known, the location of a fragment has a significant impact on its fate. Endosomes, for example, are a favored subcellular site of the pro-amyloidogenic pathway, where ηCTF can be selectively cleaved by β-secretease, producing C99, and γ-secreatse generating Aβ, as we highlighted, revealing a second pathway that gives rise to C99 and Aβ. Therefore, ηCTF is produced in physiological and pathological settings and is indeed partially internalized into the endo-lysosomal network, where it definitely plays a role as a precursor of fragments that are key actors in the pathology of AD, and potentially as an AD contributor itself. ηCTF is eventually either degraded by the autophagic pathway or eliminated via exosomes.

Age

2) Can ηCTF be compared to C99?

As researchers started to give their attention to APP-derived fragments other than Aβ, C99 was one of the important fragments that were focused on since they are the direct precursors of Aβ peptides. Our findings on the recently discovered ηCTF revealed some quite remarkable similarities between it and the better-defined C99. For instance, some studies have suggested that C99 can be degraded by the UPS [START_REF] Nunan | The C-terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome-dependent mechanism distinct from gamma-secretase[END_REF][START_REF] Nunan | Proteasomemediated degradation of the C-terminus of the Alzheimer's disease beta-amyloid protein precursor: effect of C-terminal truncation on production of beta-amyloid protein[END_REF]; however, these in vitro studies are mostly based on the overexpression of C99, which will be localized primarily in the ER and the Golgi apparatus, whilst amyloidogenic production occurs endogenously in the endosomal compartments. Accordingly, it has been observed that C99 produced by endogenous or overexpressed APP is primarily processed via the ELN, whereas overexpressed C99 is degraded by the proteasome [START_REF] Evrard | Contribution of the Endosomal-Lysosomal and Proteasomal Systems in Amyloid-beta Precursor Protein Derived Fragments Processing[END_REF]. Concerning ηCTF, we determined that both proteasomal and autophagic pathways contribute to the degradation of the overexpressed ηCTF in vitro, and more importantly though, that the autophagic pathway is most likely still the primary mechanism by which the ηCTF produced by overexpressed wild-type APP is degraded.

We also detected ηCTF in endosomes, the pro-amyloidogenic compartments where C99 and Aβ are generated, which is consistent with our other findings, which provided assurance that ηCTF is truly their precursor. Furthermore, and in keeping with its presence in endosomes, we located the ηCTF fragment in exosomes, as previously observed for C99 (Miranda et al., 2018;[START_REF] Sharples | Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes[END_REF]Vingtdeux et al., 2007).

Overall, ηCTF, aside from being its direct precursor, exhibits many similarities with C99 in terms of localisation, degradation, and trafficking.

3) ηCTF: a new AD player?

The first indication of η-secretase's involvement in the disease was the presence of η-secretasederived fragment in the amyloid plaques of AD patients reported by Willem and colleagues (Willem et al., 2015). In contrast, in my experiment using the new characterized antibody targeting ηCTF-Nter, I couldn't detect any ηCTF-Nter-like immunoreactivity in brain samples of AD patients assessed by both western-blot analysis and immunohistochemistry while ηsecretase-derived fragments were easily detected around plaques in old 3xTgAD mice. That being said, it appears that ηCTF is present at reduced levels. This can be attributed to a variety of factors, including limited production, direct cleavage by secretases that results in APP-CTFs, or direct processing by distinct degradation pathways.

When Dr. Rivera's team discovered that 5xFAD mice with MT5-MMP deficiency displayed a decrease in C99 as well as a decrease in Aβ, along with the number and size of amyloid plaques in cortical and hippocampal regions, they established the enzyme's pro-amyloidogenic role.

Additionally, the MT5-MMP deficiency in these AD mouse models reduced neuroinflammation while maintaining LTP, spatial learning, and memory [START_REF] Baranger | MT5-MMP Promotes Alzheimer's Pathogenesis in the Frontal Cortex of 5xFAD Mice and APP Trafficking in vitro[END_REF]. They then revealed that the pro-amyloidogenic properties of MT5-MMP are based, at least in part, on the enzyme's ability to bind with APP and C99 and enhance their trafficking into endosomes, which are pro-amyloidogenic cellular compartments [START_REF] Baranger | MT5-MMP Promotes Alzheimer's Pathogenesis in the Frontal Cortex of 5xFAD Mice and APP Trafficking in vitro[END_REF].

More interestingly, they found that the fate of C99 can also be controlled by the non-catalytic C-terminal domains of the enzyme, and that even inactive MT5-MMP promotes Aβ accumulation (Garcia- [START_REF] Garcia-Gonzalez | MT5-MMP controls APP and beta-CTF/C99 metabolism through proteolytic-dependent and -independent mechanisms relevant for Alzheimer's disease[END_REF]. Given that MT5-MMP may turn into a new therapeutic target for AD. However, inhibiting an enzyme with a wide range of physiological functions could have serious consequences, therefore acting on inhibiting its generated fragments may be a preferable strategy.

Besides the pro-amyloidogenic role of η-secretase, does ηCTF itself as a fragment, contribute to AD?

In addition to promoting the amyloidogenic pathway by enhancing the trafficking of APP into endosomes, as reported by Dr. Rivera, MT5-MMP also generates ηCTF, and throughout the course of my thesis, I gathered a weight of evidence pointing to its toxic potential. In fact, I highlighted that ηCTF produced at the plasma membrane, can be internalized into the endolysosomal pathway where it can play a role as a genuine direct precursor of the neurotoxic C99 and Aβ, hence shedding the light to a new pathway responsible for their production. Therefore, I identified a new route that generates major AD players that may represent a new, promising therapeutic target and an exciting venue for future research.

ηCTF is not only the precursor of the very well-known C99 and Aβ, but also of Aηα and Aηβ that have recently been described as being able to affect synaptic activity [START_REF] Mensch | Aη-α and Aη-β peptides impair LTP ex vivo within the low nanomolar range and impact neuronal activity in vivo[END_REF]Willem et al., 2015). Since these results were highly controverted by the same team, in our first experiments on synaptic activity, we validated the reduction of LTP and the impairment of the synaptic activity in the presence of recombinant Aηα and Aηβ peptides.

4)

What does the presence of ηCTF in exosomes lead us to believe?

Exosomes are believed to be involved in the clearance of amyloidogenic and toxic material, acting as a neuroprotective by attempting to eliminate neurotoxic molecules. However, recent evidence has gradually changed this perception since exosomes are now thought to also transmit these molecules between cells, potentially contributing to neurodegenerative diseases. Dr.

Rajendran's team conducted the first study revealing the existence of Aβ in exosomes in 2006.

Indeed, this study, which initially intended to describe the intracellular β-cleavage of APP in early endosomes, discovered the presence of Aβ in exosomes and detected that exosomal proteins accumulate in amyloid plaques in the brains of AD patients; thereby suggesting for the first time that exosomes are implicated in the propagation of amyloid pathology in AD [START_REF] Rajendran | Alzheimer's disease beta-amyloid peptides are released in association with exosomes[END_REF]. Similarly, years after the discovery of Aβ in exosomes, Dr. Saman's 2012 study confirmed the existence of phosphorylated Tau in exosomes [START_REF] Saman | Exosome-associated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid in early Alzheimer disease[END_REF].

The exosomal Tau protein is phosphorylated on various epitopes, most notably on threonine 181 in patients' CSF, which is a well-established biomarker. A subsequent study confirmed it by demonstrating that exosomal secretory pathway inhibition reduces the spread of Tau pathology both in vitro and in vivo [START_REF] Asai | Depletion of microglia and inhibition of exosome synthesis halt tau propagation[END_REF]. Along with APP, exosomes contain its metabolites also, such as C99, C83 and AICD (Miranda et al., 2018;[START_REF] Sharples | Inhibition of γ-secretase causes increased secretion of amyloid precursor protein C-terminal fragments in association with exosomes[END_REF]Vingtdeux et al., 2007). More intriguingly, oligomers of C99 as well as heterodimers with of C99 and C83 were found in exosomes after γ-secretase inhibition (Lauritzen et al., 2019).

Therefore, it appears that cells have a propensity to release undesired and undigested endolysosomal cargoes in exosomes as a result of the ELN dysfunction that develops in AD.

When adjacent neurons collect these exosomes, cells ironically promote the progression and spread of the pathology in an attempt to eliminate harmful molecules and compensate for the dysfunction of the ELN.

During my thesis, I demonstrated that ηCTF is present in exosomes recovered from SH-APPWT cells and animal brains overexpressing ηCTF in vitro and in vivo. This is consistent with the findings of Dr. Sadoul's team, who identified ηCTF in exosomes isolated from primary cortical neurons or neuroblastoma N2a cells overexpressing APP wild-type [START_REF] Laulagnier | Amyloid precursor protein products concentrate in a subset of exosomes specifically endocytosed by neurons[END_REF]. Beyond the intriguing discovery of ηCTF in exosomes, a key question is whether these ηCTF-enriched exosomes are beneficial or harmful in AD. On one hand, these exosomes could provide an alternative pathway for overcoming ELN dysfunction. Indeed, these vesicles may be a means of eliminating ηCTF and allowing its clearance, particularly by microglia. However, the capture of exosomes by microglia could have multiple conflicting consequences: 1. promote the clearance of accumulated ηCTF in neurons, and/or 2. induce neuroinflammation, as observed for C99 in AAV-C99 mice (Lauritzen et al., 2016), and/or 3. contribute to the spread of pathology. On the other hand, propagation might also occur from neurons to neurons, perhaps triggering alterations in LTP, memory and behavioral defects.

In conclusion, this opens the door to a large number of questions and perspectives. The objective of future work will therefore be to study the toxicity of these exosomes enriched in ηCTF and to determine their role in the spread of AD. During my thesis, I focused on the ηCTF fragment and analyzed if it could be considered as a new trigger involved in AD. First, we have demonstrated that ηCTF undergoes same α, β, and γ-secretases cleavages, hence ηCTF behaves as a direct precursor of C83, but more interestingly of C99 and A peptide. We also established that ηCTF degradation involves both proteasomal and autophagic pathways, with the autophagic process likely remains the main degradative process of endogenous APP-CTF including ηCTF. Furthermore, we revealed ηCTF localisation in endosomes using a new ηCTF-Nter antibody that we produced and found beneficial for both biochemical and in situ analyses of ηCTF, in mice models. Accordingly, we discovered the presence of ηCTF in sEVs in vitro as well as in-vivo. Additionally, we were able to demonstrate that 3xTg mice have significant ηCTF expression surrounding several senile plaques. Our preliminary findings proved what had been controverted, which is that Aηα and Aηβ both induce alterations in LTP. Finally, we fail to detected ηCTF in the brains of AD patients;

however, more research is required to determine the specificity of the staining and whether ηCTF accumulates in a stage-dependent manner. As a matter of fact, ηCTF appears to be present in relatively low levels, whether this is due to its direct cleavage by secretases, or to its instability and direct processing by various degradation pathways. In addition, the ηCTF-Nter antibody doesn't seem to be very sensitive for western-blots, as much as it is for immunostaining, which highlights the need to develop alternative, more sensitive ηCTF detection methods, such as the ELISA test.

Is ηCTF a new factor that can contribute to AD and another target that should be considered by researchers for AD treatments in the future?

Although our results clearly demonstrate the toxic potential of ηCTF, they are probably a characterization and the beginning of decades of research in order to entirely clarify its impact on AD.

First, it would be intriguing to determine whether the presence of ηCTF in endosomes can impair the ELN, as was shown for Aβ and C99, since the dysfunction of the ELN may also contribute to the exosomal elimination of ηCTF. Furthermore, because exosomes can mediate cell-to-cell propagation of toxic fragments in AD, the presence of ηCTF in exosomes may be associated with pathology propagation; thus, another interesting thing to consider is to culture neurons and astrocytes/microglia in a microfluidic device, where neurons and astrocytes communicate through grooves and their interaction can be analyzed. This will allow us to examine if ηCTF spreads between neurons or between neurons and astrocytes/microglia, which could trigger alterations in LTP, microglia-associated neuroinflammation, memory and behavioral defects. As a result, electrophysiological experiments comparing LTP in AAV-free and ηCTF-overexpressing mice are important, as are behavioral studies in which mice will should be examined for locomotor activity, spatial learning, and long-term memory in the Morris water maze.

V I I . A N N E X Genetic, biochemical, and anatomical grounds led to the proposal of the amyloid cascade hypothesis centered on the accumulation of amyloid beta peptides (Aβ) to explain Alzheimer's disease (AD) etiology. In this context, a bulk of efforts have aimed at developing therapeutic strategies seeking to reduce Aβ levels, either by blocking its production (γ-and βsecretase inhibitors) or by neutralizing it once formed (Aβdirected immunotherapies). However, so far the vast majority of, if not all, clinical trials based on these strategies have failed, since they have not been able to restore cognitive function in AD patients, and even in many cases, they have worsened the clinical picture. We here propose that AD could be more complex than a simple Aβ-linked pathology and discuss the possibility that a way to reconcile undoubted genetic evidences linking processing of APP to AD and a consistent failure of Aβbased clinical trials could be to envision the pathological contribution of the direct precursor of Aβ, the β-secretasederived C-terminal fragment of APP, βCTF, also referred to as C99. In this review, we summarize scientific evidences pointing to C99 as an early contributor to AD and postulate that γsecretase should be considered as not only an Aβ-generating protease, but also a beneficial C99-inactivating enzyme. In that sense, we discuss the limitations of molecules targeting γ-secretase and propose alternative strategies seeking to reduce C99 levels by other means and notably by enhancing its lysosomal degradation.

Alzheimer's disease (AD) is the most frequent age-related neurodegenerative disease. After initial clinical characterization, histopathological analysis revealed the presence of two major anatomical lesions signing this pathology: senile plaques that are extracellular protein aggregates and neurofibrillary tangles that are intracellular neuronal lesions (1)(2)(3). Senile plaques are mainly composed of the small 4 kDa amyloid beta peptide (Aβ)( 4) and neurofibrillary tangles of hyperphosphorylated and cleaved forms of the microtubuleassociated protein tau (5,6). The cloning of the Aβ precursor, the β-amyloid precursor protein (APP), in the late 80s was a key step in the understanding of the pathology. APP was found to be localized on chromosome 21 (7), thus explaining the development of early-onset dementia and the presence of senile plaques in Down syndrome patients carrying an extra copy of APP due to a duplication of this chromosome (8). Furthermore, the identification of APP mutations responsible for autosomal dominant form of AD (FAD), including the "Dutch" (9) "London" (10)a n d"Swedish" mutations (11), showed that the functional consequences of these mutations were to augment the load of Aβ and/or to shift Aβ production to more aggregating Aβ peptides (12,13). The role of Aβ in AD etiology was further confirmed by the discovery few years later of the first mutations in presenilins that were found to be involved in Aβ production and, similarly to APP mutations, seemed to exacerbate Aβ accumulation in cells, animal models as well as in AD-affected human brains (14)(15)(16). A last, but very important genetic evidence of a key role of Aβ in AD etiology was the recent findings of the Icelandic APP mutation that was shown to be protective by reducing cognitive decline and Aβ load by about 40% (17).

Thus, this set of histopathological, genetic, and biochemical data concurred to support the view that Aβ accumulation could be the etiological cause of the pathology, as stated in the amyloid cascade hypothesis that was proposed in 1992 by Hardy and Higgings (18). Indeed, mutations in three distinct proteins, namely APP, PS1, and PS2, are all responsible for FAD and have in common to modulate both APP processing and Aβ production. In this context, one can understand the huge efforts aimed at determining the mechanisms and enzymes involved in Aβ production and designing potent, specific, and bioavailable inhibitors of these enzymes (19,20)o r Aβ neutralizing antibodies. However, until so far, the outcomes of these Aβ-centered strategies have been extremely disappointing in our quest for meaningful treatments (21,22) (Table 1). This has led to question the validity of the amyloid cascade hypothesis (23) or to discuss in a more cautious and balanced manner the ins, outs, and limitations of the procedures of clinical trials (24). It remains that before "throwing out the baby and the bath water," one should try to reconcile undoubted genetic evidences linking APP processing to AD and failures of Aβ-based clinical trials. In this context, a way to reconcile these observations could be to envisage the possible contribution of other APP-derived fragments distinct from Aβ itself to AD pathology. Indeed, growing evidence proposes that the direct precursor of Aβ (see below), the β-secretase-derived fragment, C99, could be an early and main contributor to AD. Thus, in this review, we address the possibility that the failure of Aβ-centric clinical trials could be explained, at least partly, by their lack of effect on C99. To go further, we describe clues and evidences suggesting that γ-secretase should be considered as a beneficial C99-inactivating enzyme and argument against therapeutic strategies targeting this enzyme. We instead propose alternative strategies seeking to circumvent C99 accumulation, which would then have the advantage to reduce both C99 and Aβ levels.

APP processing and APP mutations

Aβ is derived from APP that undergoes sequential limited proteolysis catalyzed by proteases called "secretases" (Fig. 1A) (25). In the nonamyloidogenic pathway, cleavage by α-secretase and γ-secretase ends up with the production of three fragments: a large secreted N-terminal fragment (sAPPα), a small soluble peptide p3, and the APP intracellular domain (AICD) (Fig. 1A). In the amyloidogenic pathway, a first cleavage of APP by the β-secretase is followed by γ-secretase cleavage. Again, the cleavage by β-secretase generates a large soluble extracellular secreted domain (sAPPβ) and the remaining membrane stub, the β-secretase-derived fragment, C99, undergoes γ-secretase cleavage, thus liberating Aβ and its C-terminal counterpart AICD (Fig. 1B). Other noncanonical cleavages on APP have also been more recently described (26). Among them, the ƞ-secretase activity, carried by the matrix metalloproteinases (MT1-MMP and MT5-MMP), cleaves APP in its extracellular domain, thus producing a soluble fragment (sAPPη) and a membrane-bound C-terminal fragment, ηCTF. This latter fragment can subsequently be processed by α-orβsecretase, which will generate Anα and C83 and Anβ and C99, respectively (Fig. 1C) ( 27,28).

Until so far, 28 pathogenic APP mutations have been identified with all, except the Swedish mutation, lying within the sequence of C99 (www.alzforum.org/mutations). The Swedish variant (APPswe, KM670/671NL), although not located within C99 but two residues upstream, strongly increases the levels of this fragment, as well as those of Aβ peptides, by boosting β-secretase cleavage (12,13,29). All pathogenic APP mutations lie either close to the α-secretase cleavage site (the middle part of the Aβ domain of C99), within the γ-secretase cleavage sites, or near the β-secretase cleavage site (N-terminal part), thus modifying the cleavages of APP by either of these secretases. While the knowledge about the exact effects of the pathogenic APP mutations is still limited, it seems that mutations close to the β-secretase (such as the Swedish and Leuven (E682K) mutations) favor C99 production, those close to the α-secretase site (such as the Flemish [E693Q] or Arctic [E693G] mutations) lead to the generation of particular aggregation-prone Aβ species, while mutations lying within the γ-secretase cleavage site elevate the Aβ42/ Aβ40 ratio. In all cases, the development of AD pathology linked to these mutations is believed to be caused by an excessive Aβ deposition (30). Nevertheless, the recent study by Xu et al. (31) showed that most (20 out of 28) APP mutations not only alter Aβ production, but are also less efficiently processed by γ-secretase and thereby also enhance the levels of C99.

The β-secretase

The nature of β-secretase was described in 1999 in four independent works and was referred to as BACE1 (β-site APP cleaving enzyme), memapsin 2 or ASP2 (32)(33)(34)(35). BACE1 is an aspartic protease cleaving mainly within the Golgi and endosomes due to its optimal enzymatic activity at acidic pH (36). It cleaves APP at two distinct sites called the β and β 0 site, respectively. While only the cleavage on the β-site (Asp1) leads to Aβ production (Fig. 1B), BACE1 is considered to cut APP primarily at the β 0 -site (Glu11) (Fig. 1B), thus generating the C89 fragment and a truncated nonpathological Aβ peptide (Aβ 11-x) (36). Until so far, no mutations have been found in βsecretase, but the Swedish and the Leuven APP mutations are known to shift the cleavage from the β 0 -site to the β-site, thus leading to a much higher C99 and Aβ production (12,13,37,38). Inversely, the Icelandic mutation (A673T) seems to be protective against AD by decreasing β-secretase cleavage and thus reducing by 40% the amyloidogenic fragments sAPPβ and Aβ (17,39).

The γ-secretase and role of PS mutations

The first hint of the identity of the protease yielding the Cterminal end of Aβ, the γ-secretase, was the discovery in 1995 of presenilin 1 (PS1) (40)(41)(42) and its family member presenilin 2 (PS2) (43,44) proposed to constitute this enzyme (Fig. 2A). The definitive demonstration that presenilins could be involved in γ-secretase activity came from subsequent functional characterization showing that their depletion fully prevents Aβ production (45,46). However, later studies revealed that γ-secretase is a multiproteic complex built of not only PS1 or PS2 but also of nicastrin, the anterior pharynx defective 1 (Aph1), and presenilin enhancer 2 (PEN2) [START_REF] Edbauer | Reconstitution of g-secretase activity[END_REF][START_REF] Kimberly | g-secretase is a membrane protein complex comprised of presenilin, nicastrin, aph-1 and pen-2[END_REF][START_REF] Takasugi | The role of presenilin cofactors in the g-secretase complex[END_REF](Fig. 2B), but the exact molecular structure of this complex was revealed by crystallography only in 2014 [START_REF] Lu | Three-dimensional structure of human gamma-secretase[END_REF]. Further studies also revealed a very complex cleavage of APP-CTFs by γ-secretase, since it takes place within the membrane and successively at several sites ( 3, ζ-, γ-and γ 0 ). The first endopeptidase cleavage ( 3-cleavage) can occur at two distinct sites leading to the generation of Aβ48 and Aβ49, which is then shortened by sequential carboxypeptidase cleavages (trimming) at ζ, γ-and γ 0 -sites, occurring at each three or four residues and via two distinct product lines (Aβ49 > Aβ46 > Aβ43 > Aβ40 > Aβ37) or (Aβ48 > Aβ45 > Aβ42 > Aβ38) and the final occurrence of peptides of varying lengths but two major species of 40 or 42 amino acid residues, respectively [START_REF] Wolfe | JBC REVIEWS: γ-Secretase: a beneficial C99-inactivating enzyme 14[END_REF]. Whereas Aβ40 is the most abundant peptide, Aβ42 is considered as the most pathogenic due to its high aggregation propensity.

So far, more than 300 mutations in presenilins are proposed to be responsible for early onset and familial forms of AD (FAD) and are now believed to comprise at least 70% of all FAD cases with the vast majority on PS1 (www.alzforum. org/mutations). Although a huge effort has been made to try JBC REVIEWS: γ-Secretase: a beneficial C99-inactivating enzyme to understand the pathogenic mechanisms linked to these mutations, their exact functional consequences remain enigmatic and are probably not explained by one common mechanism. Two main, but not exclusive, hypotheses have been proposed and are still hotly debated. First, Hardy and Selkoe proposed, accordingly to the amyloid cascade hypothesis, that the effect of PS mutations should be a gain of function of γ-secretase [START_REF] Hardy | The amyloid hypothesis of Alzheimer's disease: Progress and problems on the road to therapeutics[END_REF]. However, later studies revealed that most PS mutations are actually loss-of-function mutants, and a revised version of this hypothesis became focused on the relative levels of Aβ42 to Aβ40 (Aβ42/Aβ40) rather than absolute increases in Aβ42 [START_REF] Selkoe | The amyloid hypothesis of Alzheimer's disease at 25 years[END_REF]. It was suggested that partial loss of PS mutations shifts the cleavage specificity of the mutant enzyme to favor Aβ42 production and that even a minor increase in Aβ42 levels could be sufficient for seed formation [START_REF] Selkoe | Presenilin: Running with scissors in the membrane[END_REF][START_REF] Steiner | Intramembrane proteolysis by gamma-secretase[END_REF]. In that way, loss of function should be considered as gain of "toxic function" because of a relative increase in more aggregation-prone Aβ [START_REF] Wolfe | When loss is gain: Reduced presenilin proteolytic function leads to increased Abeta42/Abeta40. Talking point on the role of presenilin mutations in Alzheimer disease[END_REF]. On the other hand, Shen and Kelleher proposed an alternative hypothesis "the presenilin hypothesis," stating that loss of general PS function should be the trigger of neurodegeneration and defects in cognitive function [START_REF] Shen | The presenilin hypothesis of Alzheimer's disease: Evidence for a loss-of-function pathogenic mechanism[END_REF]. They proposed that pathogenic PS mutations behave as dominant-negative mutation due to a mechanism in which mutant PS interferes with the activity of wild-type PS, thereby reducing their physiological functions [START_REF] Heilig | Trans-dominant negative effects of pathogenic PSEN1 mutations on gamma-secretase activity and Abeta production[END_REF][START_REF] Xia | Presenilin-1 knockin mice reveal loss-of-function mechanism for familial Alzheimer's disease[END_REF]. In agreement with this hypothesis, the extensive work of Sun and colleagues [START_REF] Sun | Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Abeta42 and Abeta40 peptides by gamma-secretase[END_REF], in which 138 pathogenic PS mutations were analyzed, revealed that more than 90% of them led to reduced production of both Aβ40 and Aβ42, and this is in a dominant negative manner [START_REF] Sun | Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Abeta42 and Abeta40 peptides by gamma-secretase[END_REF]. This conclusion of Sun and colleagues was based on purified γ-secretases harboring PS mutations and assay of C99 cleavage in vitro or in a cell-based assay. In contrast, Szaruga and colleagues ( 61)m e a s u r e dγ-secretase activity in brain extracts from patients carrying PS1 mutations (one healthy and one disease allele) and found a variable effect on endopeptidase cleavage but a consistent reduction in carboxypeptidase activity, seen as Aβ38/Aβ42. These data therefore seemed to fit with the postulate that a qualitative shift in Aβ profiles toward the generation of longer aggregation-prone peptides (>Aβ42) should be the common denominator of AD ( 61) and were in agreement with the earlier work from Saito and Saido, demonstrating a particular high toxicity of Aβ43 ( 62) and the initial statements of a role of aggregating-prone Aβ in seed formation [START_REF] Borchelt | Familial Alzheimer's disease-linked presenilin 1 variants elevate Ab1-42/1-40 in vitro and in vivo[END_REF][START_REF] Duff | Increased amyloidb42(43) in brains expressing mutant presenilin 1[END_REF][START_REF] Scheuner | Secreted amyloid bprotein similar to that in the senile plaques of Alzheimer's disease is increased in vivo by the presenilin 1 and 2 and APP mutations linked to familial Alzheimer's disease[END_REF]. However, arguing against a direct and unique role of the Aβ42/Aβ40 ratio was the findings in the work of Sun and colleagues that observed that the age of onset of FAD did not seem to correlate well with the change in this ratio [START_REF] Sun | Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Abeta42 and Abeta40 peptides by gamma-secretase[END_REF]i n contradiction to what was previous proposed [START_REF] Kumar-Singh | Mean age of onset of familial Alzheimer disease caused by presenilin mutations correlates with both increased Ab42 and decreased Ab40[END_REF]. A recent study indicated that the pathogenicity of PS1 mutations could be related to an intracellular mislocalization of the γsecretase complex composed of these mutants [START_REF] Sannerud | Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool[END_REF][START_REF] Meckler | Presenilin 1 and presenilin 2 target gamma-secretase complexes to distinct cellular compartments[END_REF]. Indeed, in contrary to wild-type PS1 having a broad cellular distribution, mutated PS1 was found to be restricted to endosomal/lysosomal compartments [START_REF] Sannerud | Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool[END_REF][START_REF] Meckler | Presenilin 1 and presenilin 2 target gamma-secretase complexes to distinct cellular compartments[END_REF]. Hence, it was postulated that mutated PS would favor intraneuronal Aβ JBC REVIEWS: γ-Secretase: a beneficial C99-inactivating enzyme production, which could be particularly toxic [START_REF] Gouras | Intraneuronal Ab42 accumulmation in human brain[END_REF][START_REF] Chui | Apoptotic neurons in Alzheimer's disease frequently show intracellular Abeta42 labeling[END_REF][START_REF] Chui | Transgenic mice with Alzheimer presenilin 1 mutations show accelerated neurodegeneration without amyloid plaque formation[END_REF]. Moreover, the acidity of endosomal/lysosomal compartments could be less propitious for carboxypeptidase activity, thus leading to the generation of longer and more aggregating Aβ peptides [START_REF] Sannerud | Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool[END_REF] and the acidic pH and high concentrations of Aβ in these compartments could be particularly propitious for Aβ aggregation and thus toxicity.

A more recent hypothesis suggests that the pathogenicity of PS mutations could also be linked, or at least partly, to their effect on C99 accumulation. Indeed, although few studies have investigated the effects of FAD mutations on C99 levels, recent studies seem to reveal a consistent increase in C99 in the presence of most pathogenic PS mutants [START_REF] Li | Effect of presenilin mutations on APP cleavage; insights into the pathogenesis of FAD[END_REF][START_REF] Kwart | A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta[END_REF][START_REF] Cacquevel | Alzheimer's disease-linked mutations in presenilin-1 result in a drastic loss of activity in purified gamma-secretase complexes[END_REF]. This was even the case in neurons derived from pluripotent stem cells (iPSCs) harboring a wide panel of pathogenic PS mutations in the absence of overexpression [START_REF] Kwart | A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta[END_REF]. Whether this effect on C99 is explained by a loss of function (reduced γ-secretase activity) and/or other PS-associated side effects remains unclear. Indeed, accumulating evidence indicates that both PS1 and PS2 are involved in essential cellular processes, independently of their role in γ-secretase, and proposed to be due notably to their function as chaperons and/or control of calcium release from the endoplasmic reticulum (for reviews see [START_REF] Duggan | Beyond gamma-secretase activity: The multifunctional nature of presenilins in cell signalling pathways[END_REF]76)). Some of these functions have been suggested to be modified by FAD mutations, although again, many contrasting findings have been reported. Indeed, most studies on noncatalytic functions of PS have been carried out on knockout models, thus in the absence of PS, whereas fewer works have investigated the impact of FAD mutations on these functions. One of the most consistently described noncatalytic functions of PS is their involvement in the regulation of both autophagy and lysosomal function [START_REF] Duggan | Beyond gamma-secretase activity: The multifunctional nature of presenilins in cell signalling pathways[END_REF]76) known also to be involved in C99 degradation (see below). Whether these γ-secretase-independent functions contribute to, or are responsible for, C99 accumulation, in cells expressing PS mutations, remains to be established.

Physiological function of γ-secretase

FAD cases correspond to only a very small subset of patients (about 1%) [START_REF] Cacace | Molecular genetics of early-onset Alzheimer's disease revisited[END_REF]. Thus, both Aβ and C99 accumulation in FAD could reflect an exacerbated process or a dysfunction occurring only in few cases. The situation seems to be different in sporadic AD (SAD) that corresponds to most cases. In SAD, Aβ accumulation is mostly believed to be linked to age-related impairment in Aβ clearance, rather than to an increased Aβ biogenesis. Thus, Aβ should be considered firstly as a physiological product derived from normal processing of APP (78,[START_REF] Haass | Normal cellular processing of the b-amyloid precursor protein results in the secretion of the amyloid b peptide and related molecules[END_REF]. Although little is still known about the exact physiological role of Aβ, the peptide is known to regulate synaptic activity, to have antimicrobial and tumor suppressing properties (for review see ( 80)), and to be neuroprotective in some pathological conditions [START_REF] Sevalle | Aminopeptidase A contributes to the N-terminal truncation of amyloid beta-peptide[END_REF][START_REF] Castellani | Antioxidant protection and neurodegenerative disease: The role of amyloidbeta and tau[END_REF][START_REF] Brothers | The physiological roles of amyloid-beta peptide hint at new ways to treat Alzheimer's disease[END_REF]. Thus, to some extent the failure of clinical trials aimed at either inhibiting Aβ production or neutralizing Aβ once formed could be explained, at least in part, by the abolition of such Aβ-mediated physiological functions.

If considering Aβ as a physiological product, this implies that a main role of γ-secretase should be to generate bioactive molecules involved in important vital processes. Indeed, this point of view fits with genetic evidences indicating that the invalidation of γ-secretase in mice is lethal (84) and that the conditional knockout of PS1 leads to neurodegeneration at the adulthood [START_REF] Saura | Loss of presenilin function causes impairements of memory and synaptic plasticity followed by age-dependent neurodegeneration[END_REF]. Of course, these observations are probably explained by the numerous, more than 150, other γ-secretase proteolytic targets involved in physiological processes (86). Indeed, similar to APP, most of γ-secretase substrates are type I transmembrane proteins, which undergo "Regulated Intramembrane proteolysis (RIP), a proteolytic process in which γsecretase-mediated cleavage occurs inside the membrane ending up with the production of an intracellular fragment that shuttles to the nucleus and acts as a transcription factor [START_REF] Kuhnle | Intramembrane proteolysis at a glance: From signalling to protein degradation[END_REF][START_REF] Lichtenthaler | Regulated intramembrane proteolysis-lessons from amyloid precursor protein processing[END_REF]. The seminal example of RIP was described for Notch that undergoes γ-secretase-mediated cleavage, thereby generating the Notch Intracellular Domain (NICD) [START_REF] Okochi | Presenilins mediate a dual intramembranous g-secretase cleavge of Notch-1[END_REF], a key player of embryonic development [START_REF] Siebel | Notch signaling in development, tissue homeostasis, and disease[END_REF]. For instance, this lack of Notch cleavage ( 91) is believed to explain the lethal embryologic defects observed in PS-invalidated mouse embryos (84). In addition, numerous other substrates (the list is far from being exhaustive) have been reported to undergo RIP including cadherins (N and E Cadh-ICDs) [START_REF] Marambaud | A CPB binding transcriptional repressor produced by the PS1/e-cleavage of N-cadherin is inhibited by PS1 FAD mutations[END_REF], alcadeins [START_REF] Araki | Coordinated metabolism of Alcadein and amyloid beta-protein precursor regulates FE65-dependent gene transactivation[END_REF], CD44 [START_REF] Okamoto | Proteolytic release of CD44 intracellular domain and its role in the CD44 signaling pathway[END_REF], or more recently TREM2 [START_REF] Glebov | Functional involvement of gamma-secretase in signaling of the triggering receptor expressed on myeloid cells-2 (TREM2)[END_REF]. Generally, the generation of the intracellular domain of these proteins is essential in the transcriptional regulation of physiological processes such as neurite outgrowth, cell adhesion and migration, or synaptogenesis [START_REF] Jurisch-Yaksi | A fast growing spectrum of biological functions of gamma-secretase in development and disease[END_REF][START_REF] Haapasalo | The many substrates of presenilin/gamma-secretase[END_REF]. This phenomenon is also described for APP for which the intracellular domain AICD has been found to form a transcriptional active complex with Fe65 and Tip60 [START_REF] Cao | A transcriptively active complex of APP with Fe65 and histone acetyltransferase tip60[END_REF][START_REF] Von Rotz | The APP intracellular domain forms nuclear multiprotein complexes and regulates the transcription of its own precursor[END_REF] that regulates key proteins involved in the control of cell death (p53) (100), Aβ degradation (Neprilysin) (101, 102), as well as tau phosphorylation (GSK3β)( 103, 104). Thus, γ-secretase cleavage should be considered as a necessary step in the formation of both Aβ and AICD. Besides this role, γ-secretase cleavage has also been proposed to behave as a "membranous proteasome" essential for the inactivation of the buildup of toxic membranous stubs (105). It remains unclear, whether this "proteasome-like activity" is nonselective and inactivates all substrates equally, or if some substrates are cleaved by specific protease complexes. Recent evidences seem to indicate at least some substrate specificity and that not only the exact protein composition of the secretase complex, but also the cellular context, subcellular localization, and the presence of nonessential cofactors (106) can be key determinants for this control. For instance, protease heterogeneity has been shown to be related to the exact protein composition of the protease and alternative splicing of Aph1 leads to further complexity (107, 108). For instance, in mice the knockout of the 3 Aph1 subunits led to an important increase in some, but not all, membrane-bound fragments and APP-dependent neurodegeneration (107, 108). Hu and colleagues demonstrated that nicastrin is required for APP processing but not Notch processing, whereas Aph1 is necessary for the processing of both APP and Notch (109). Selectivity also seems to be determined by the subcellular localization of the protease, since, as described above, PS1-or PS2-containing complexes do not have the same cellular localization and do not cleave APP identically [START_REF] Sannerud | Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool[END_REF][START_REF] Meckler | Presenilin 1 and presenilin 2 target gamma-secretase complexes to distinct cellular compartments[END_REF]. Interestingly, mutations in PS1 change the localization toward that of PS2-containing complexes [START_REF] Sannerud | Restricted location of PSEN2/gamma-secretase determines substrate specificity and generates an intracellular Abeta pool[END_REF].

Challenges and complications of targeting γ-secretase in AD

After the discovery γ-secretase, a huge amount of research was carried out to develop potent and bioavailable γ-secretase inhibitors (GSIs), but the clinical trials based on these inhibitors all failed (Table 1). Indeed, none of them led to improvement of AD-linked cognitive decline and even sometimes they worsened them. In most of these trials, Aβ levels in plasma or cerebrospinal fluid (CSF) were significantly decreased, thus clearly indicating that a reduction in total Aβ was not sufficient to restore cognitive function. This was the case for Semagacestat, the first GSI tested in late-stage clinical trials for AD, which was reported to reduce Aβ levels by more than 60% in the plasma (110), and new Aβ synthesis was decreased by more than 80% in CSF (111). More alarming, many trials were readily stopped because of increased risk of developing skin cancer and infections, which was supposed to be tightly linked to a defective Notch signaling in the presence of the inhibitors (112). Therefore, several groups started to screen for drugs having a higher affinity for APP than toward Notch, such as Avagacestat, initially reported to be 140-fold more selective for APP than Notch (113), although its Notch sparing ability remains controversial (114,115). Avagacestat led to a 40% Aβ reduction in CSF (Table 1), but demonstrated similar side effects in clinical trials (116). Therefore, due to the huge number of different γ-secretase substrates, it has appeared difficult to target specifically and exclusively the γ-secretase-mediated APP cleavage.

Furthermore, data from preclinical trials proposed that the lack of efficacy, or even worsening, of cognitive function in these trials was also linked to the progressive accumulation of APP-CTFs in the presence of GSIs (117). To avoid such effects, therapeutic development became focused on γ-secretase modulators (GSMs) that were expected to be safer, since they interact with γ-secretase complex through the allosteric binding site, thereby modifying the enzyme activity but not blocking it (118). The concept of γ-secretase modulation was discovered with some nonsteroidal anti-inflammatory drugs (NSAIDs) such as flurbiprofen, indomethacin, and ibuprofen, which are considered as GSMs, because they induce conformational changes in PS1 and shift the cleavage of C99 toward shorter Aβ species such as Aβ37 and Aβ38 (16,119). In that way, GSMs do not lead to increased APP-CTFs levels. Indeed, in AD animal models, the chronic treatment with GSMs did not lead to the worsening of cognitive function observed with GSIs (117). Moreover, GSMs are also safer because they reduce the level of Aβ42 and increase shorter Aβ peptides without affecting Notch signaling (120, 121). Nonetheless, the first GSMs tested in clinical trials, Rofecoxib (122), Tarenflurbil (123) or Naproxen (124), did not show efficacy, but the lack of effects seemed to be related to a very poor blood-brain barrier crossing ability of these drugs (Table 1). Thus, taken together, γ-secretase-based strategies have so far been unsuccessful, even if GSMs seem to be more promising (125). Still, if considering γ-secretase as a C99inactivating enzyme, we believe that one should carefully question the validity of targeting this enzyme.

C99 is toxic and γ-secretase inhibitors potentiate pathogenic phenotypes

Emerging data propose a pathological role of the intraneuronal accumulation of C99 (Table 2). Indeed, a very recent work showed that the accumulation of C99, rather than that of Aβ, correlates with neuronal vulnerability in AD-affected patients (126). To distinguish between C99 and Aβ in situ, which can be very challenging because the two of them have common epitopes, the group of Drs Bustos and Greengard developed a sophisticated technique adapted from the proximity ligation assay (PLA) and using both N-and C-terminal directed antibodies. This link between neuronal vulnerability and C99 in human AD was an important observation previously supported by many animal models. The first evidence of C99 accumulation in AD mouse models came from studies on the widely used 3xTg-AD model (APP swe , Tau P310L , PS1 M146V ) developed by the group of La Ferla (127). In the original work, the authors used N-terminal Aβ antibodies and claimed that these mice harbor intracellular Aβ accumulation (127). However, years later our own work, using different and complementary approaches, indicated that this accumulated material corresponded to C99 and not to Aβ (128). Indeed, as stated above, it is challenging to discriminate between Aβ and C99 in situ. Hence, only a detailed characterization using N-and C-terminal antibodies, pharmacological treatments (β-and γsecretase inhibitors), and/or genetic approaches (as PS mutations) can allow this discrimination. Most and even recent studies do not provide this detailed characterization, which may explain the lack of information concerning C99 in the literature. In the 3xTgAD model, C99 accumulates early and much before Aβ can be detected (128). Of importance, C99 accumulation was found to also occur in many other transgenic models including Tg-CRND8 (129) and JA20 (130) mice carrying the APPswe mutation, known to increase C99 production, but also in mice displaying the APP E693Q Dutch mutation (131), in which a C99 overproduction is not expected to occur. Interestingly, and importantly, C99 accumulation also occurs in the knock-in models APP-NL and APP-NL-F, harboring the Swedish (KM670/671NL) mutation alone (APP-NL) or in combination with the I706F mutation (APP-NL-F), respectively (132, 133)( Table 2) indicating that C99 accumulation was not just an artificial phenotype linked to APP overexpression, as earlier described for other events taking place in transgenic mouse models that sometimes express very high levels of APP (132). The presence of the Swedish mutation could indeed, at least to some extent, explain the C99 accumulation in these knock-in mice. However, recent studies clearly showed that humanizing APP (substituting the three amino acids G676R, F681Y, and R684H) in mice or rats is sufficient to increase C99 levels (134-136). In animals displaying rodent APP, the protein was found to be mainly processed by α-secretase, thus generating almost only C83, whereas the presence of human APP increases β-secretase cleavage and leads to enhanced C99 levels (134). These data thus reveal a different APP processing of human and rodent APP, which could explain the higher levels of C99 in human as compared with rodents (137) and may be one of the reasons for the C99 accumulation observed even in SAD cases (126,137,138). Overall, these findings ruled out the possibility that it could be "model specific" but rather suggest that it should be a common and early alteration in AD.

In AD mouse models, such as in 3xTg-AD mice, C99 accumulation was described to be both the cause and the consequence of endosomal perturbations, ultimately leading to failure of lysosomal and autophagic processes (139). A similar link between C99 accumulation and lysosomal dysfunction was described in the mouse bearing the Dutch mutation APP E693Q (131). Moreover, in fibroblasts from Down syndrome patients or from the mouse model Ts65Dn, C99 were found to induce enlargement of Rab5 positive early endosomes, aberrant endocytosis, and impaired endosomal transport (140). Interestingly, these alterations were fully Aβ-independent, since they were rescued by β-secretase inhibitors or partial BACE1 genetic depletion and were enhanced by GSIs (139-142). Of utmost interest, C99 accumulation has also been associated to endolysosomal dysfunction in human cellular AD models. Indeed, the earlier mentioned transcriptomic analysis of induced pluripotent stem cells (iPSCs) harboring AD-related APP or PS1 mutations indicated similar changes in endocytosis-associated genes [START_REF] Kwart | A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta[END_REF]. Strikingly, these mutations brought divergent data concerning Aβ, but they all displayed increases in C99 levels as well as endosomal dysfunction [START_REF] Kwart | A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta[END_REF]. The use of notably a BACE inhibitor, GSMs and GSIs, showed that endosomal pathology was linked to C99 and not to the changes in Aβ [START_REF] Kwart | A large panel of isogenic APP and PSEN1 mutant human iPSC neurons reveals shared endosomal abnormalities mediated by APP beta-CTFs, not Abeta[END_REF]. Resembling data were obtained in another study using iPSCsderived neurons obtained from AD patients harboring APP or PS1 mutations, which displayed both C99 accumulation and dysfunction of the endosomal-lysosomal network (143). As a difference to the work of Kwarts and colleagues, mutations in APP but not in PS1 led to early endosomal (Rab5 associated) pathology, but all mutations led to enlarged late endosomes and defective lysosomal degradation. These effects were APPdependent, reversed by BACE inhibition, and exacerbated by GSIs. These results agree with the consistent data linking C99 to a dysfunction in the endolysosomal network occurring even in the presence of physiological APP levels (144). Whether C99 accumulation in these systems is linked to the partial loss of JBC REVIEWS: γ-Secretase: a beneficial C99-inactivating enzyme function of γ-secretase or to other PS but γ-secretase-independent mechanisms, such as impaired lysosomal-autophagic degradation, as discussed earlier in this review, remains to be established. Related to endolysosomal dysfunction, the pathogenicity of C99 could also be due to its propensity to aggregate and to spread through exosomes, small vesicles secreted from cells (145). Indeed, exosomes originate from endosomes and C99 is detected in these vesicles (145-147). Our recent work showed the possible presence of C99 existing as oligomers (C99 homomers and heteromers composed of C99/C83) in exosomes, the levels of which were drastically enhanced upon γ-secretase inhibition (145). The latter statement is of importance due to the proposed role of exosomes in prion-like transmission of neurotoxic molecules (148).

In addition to endosomal and lysosomal alterations, mitochondrial dysfunction is a key feature of AD with altered mitochondrial potential, increased levels of reactive oxygen species (ROS) (149-151), as well as a defect in clearance of abnormal mitochondria (mitophagy) (152,153). Our recent work proposed that in cellular models, C99 can trigger all these alterations (137). In agreement, mitochondrial structure alterations and mitophagy defects were observed in young preplaque 3xTg-AD mice, as well as in virus-induced C99 expressing mice (AAV10-C99 mice) and notably following GSI treatment (137). Other studies have demonstrated C99 accumulation in mitochondria-associated membranes (MAMs) (154, 155) and have proposed its contribution to neutral lipid accumulation (155) and cholesterol trafficking (156).

Neuroinflammation is also a key feature of AD, but little is known about its molecular triggers. The contribution of C99 to neuroinflammation was proposed as early as in 1998 in mice, in which the intracerebroventricular injection of recombinant C99 (CT105) was found to induce reactive gliosis and neurodegeneration (157). CT105 was also found to interact with inflammatory cytokines and produce a synergistic effect on working memory (158). Two more recent works also proposed a link between intraneuronal accumulation of C99 and astrocytic activation and astrogliosis in 3xTgAD and APP E693Q mice, respectively (128, 131). Furthermore, in preplaque TgCRND8 mice, C99 was shown to correlate with a TNFα augmentation and microglial activation (129). Does C99 accumulation also trigger functional and cognitive alterations? It is well established that AD is characterized by a hippocampal altered long-term potentiation (LTP) and depression (LTD) (159), as well as network activity dysfunctions (160). All of these electrophysiological signatures are linked to memory consolidation and learning ability and appear altered before senile plaques and neurodegeneration in human AD (161) and many mouse models. The first evidence of a neurotoxic effect of C99 on synaptic function was demonstrated years ago in a transgenic mouse model expressing directly the C99 fragment (162). These mice were found to develop both LTP and memory alterations, as well as neuroinflammation and neuronal loss. Our own recent studies in AAV10-C99 mice showed that LTP was significantly reduced in preplaque mice and that GSIs did not reverse LTP alterations (139). Synaptic alterations also seemed to be temporarily and spatially correlated to C99 accumulation in 3xTgAD mice (163) and in the Danish dementia mouse model, in which a deficiency in the BRI2 protein leads to increased APP levels and in which LTP alterations and memory defects were rescued by the pharmacological inhibition of β-secretase, but not γ-secretase (164). Hippocampal network oscillations, as well as thêta/gamma coupling alterations, were reported at early "Aβ-free" stages in the Tg-CRND8 mouse (165). In this study, these early network defects occurred in C99accumulating regions and were fully rescued by β-secretase inhibition (130). In contrast, another recent study reported an absence of rescue of network alterations by β-secretase inhibition in the J20 model, although the levels of both C99 and Aβ were clearly reduced (133). These data show that synaptic function is governed by complex mechanisms that may be difficult to rescue. First of all, in these studies β-secretase inhibition led to a decrease in C99 and Aβ, but it also led to a concomitant increase in C83 that maybe could contribute to these alterations, although this was not discussed by the authors. Second of all, and maybe more importantly, the lack of rescue by BACE1 inhibitors could be due to the suppression of important cell signaling pathways involved in synaptic function, due to the inactivation of other BACE1 substrates, such as Neuregulin-1 and Sez6 (166, 167). In the same way, studies investigating the role of C99 accumulation in AD-related memory impairment in mouse AD models have reported conflicting data. In 3xTgAD mice, C99 accumulation seems to trigger both electrophysiological hippocampal defects and mild cognitive dysfunction (163,168). Similar defects were observed in CT105-expressing mice (162). However, the studies from knock-in models seem to argue for an Aβ-rather than a C99dependent contribution to synaptic alterations. In fact, the two knock-in models APP-NL and APP-NL-F, harboring the Swedish (KM670/671NL) mutation alone (APP-NL), or in combination with the I706F mutation (APP-NL-F), respectively, both accumulate C99 (132), but only the APP-NL-F, displaying higher Aβ levels, develops late-stage cognitive dysfunction (132,169). Moreover, the knock-in mouse APP-NL-G-F harboring a third mutation, the E693G Arctic mutation, which generates a particular high level of oligomerized Aβ, displays cognitive dysfunction at a much younger age. Thus, taken together, these observations could suggest either that only Aβ is toxic or that Aβ and C99 contribute differently to the pathology. Whereas Aβ, as a soluble peptide, could be expected to act mainly outside cells, the toxic effect of membrane-embedded C99 should be intracellular. It is possible that intraneuronal C99 is a trigger of a more slowly developing pathological process that might be more prominent in transgenic mice that display higher levels of C99 than knock-in mice.

In 3xTgAD mice, these electrophysiological defects are not only accompanied by alterations in spatial learning and memory, but are also associated to a decrease in spontaneous activity that is reminiscent to apathy (168), a phenotype considered as an early neuropsychiatric symptom in AD patients (170,171). Several observations suggested that this apathy-like behavior could be linked to C99 accumulation.

JBC REVIEWS: γ-Secretase: a beneficial C99-inactivating enzyme This phenotype not only appears at a stage much before Aβ could be detected, but it similarly occurs in a 2xTg-AD model (harboring APPswe and Tau P30L), which was found to accumulate identical amounts C99 to the 3xTgAD mouse, but which display very low Aβ levels even at late stages (168).

Is β-secretase a good drug target?

BACE1 cleavage of APP represents the rate-limiting step of Aβ production, and targeting this enzyme would provide advantage to prevent production of both Aβ and C99. To date, there is no evidence of AD-linked mutations on BACE1, but a clue of an indirect importance of BACE1 in AD came from genetic studies showing that APP mutations lying close to the β-secretase cleaving site strongly affect the cleavage by this enzyme and can either cause early-onset AD (the Swedish and Leuven mutations) or be protective (the Icelandic mutation). BACE1 has also been shown to have a role in SAD as its expression was reported to be increased in AD patients. Thus, BACE1 protein was found to be increased 3-fold in cortical areas of AD patients as compared with age-matched controls (172), and these data were confirmed at the mRNA level in the frontal cortex of SAD patients (172,173). Yang and colleagues also showed a significant elevation of BACE1 in temporal cortex and hippocampal samples from a cohort of clinically diagnosed and neuropathologically confirmed AD patients (174). This study used fluorimetric analysis assays and in vitro proteolysis to confirm that this increase in BACE1 expression led to enhanced functional enzyme. At that stage, most of the In physiological conditions, γ-secretase cleavage of C99 (blue/red sticks) leads to the generation of Aβ and AICD, whereas when γ-secretase activity is impaired (inhibitors or PS mutations), C99 accumulates and aggregates. B, displays a schematic view of C99 generation and accumulation in conditions of blocked γ-secretase. APP (long blue/red bar) maturates and traffics through the Golgi to the plasma membrane (blue arrows), where a small part of it is processed by α-secretase (non-amyloidogenic pathway). APP that escapes α-secretase-mediated cleavage is endocytosed into early endosomes and some of it is recycled to the plasma membrane, either directly or through the Golgi network (open black arrows). Within early endosomes, some APP is cleaved by β-secretase generating C99 (short blue/red bar), which then can undergo γ-secretase cleavage, thereby releasing the Aβ peptide and AICD. Some C99 escapes from γ-secretase cleavage and is incorporated in ILVs (intra luminal vesicles), which are either released as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane (1) or degraded after their fusion of MVBs with lysosomes or with autophagosomes, which then fuse with lysosomes (2). The presence of APP and PS mutations or γ-secretase inhibitors and/or lowered lysosomal degradation lead to lysosomal, autolysosomal, and exosomal C99 accumulation. C99 has also been found to accumulate in MAMs and mitochondria.

studies attempted to correlate BACE1 expression to Aβ load. However, some later studies analyzed C99 levels and reported also an increase in both FAD and SAD brains (137,138,175). Of most interest, the recent work from Pulina and colleagues quantifying both C99 and Aβ in either controls, mildly or severely-affected AD brains, showed that C99 accumulates specifically in vulnerable neurons and correlated with the degree of cognitive alterations, whereas Aβ load was increased in both vulnerable and resistant brain regions in AD (126). The latter data are to be put back into context with the consistent former observations that Aβ expression and distribution within brain areas do not fully match neuronal loss, cognitive defects severity, and dementia AD progression (176-178). It should be emphasized that two recent studies documented that BACE may inactivate an intravesicular domain of APP, thereby exacerbating glutamate release (179), a process enhanced in a rat knock-in model bearing the Swedish mutation (135).

As stated above, BACE1 mainly cleaves APP at the β 0 site to generate C89 and therefore can be considered to be protective. In addition, BACE1 is also able to cleave Aβ40 and Aβ42 into the nonamyloidogenic (180) and neuroprotective (181)Aβ34. A recent study also proposed another neuroprotective role of BACE1, since the inhibition of this enzyme was found to enhance the levels of both ƞ-secretase and Aƞα, the latter was proposed to be neurotoxic (27). Thus, in AD, the shift from a beneficial to a deleterious BACE1 cleavage could be due to the age-dependent augmentation of BACE1 expression occurring in sporadic AD or to the expression of a subset of pathogenic mutations in FAD, thereby enhancing C99 levels above physiological threshold (32).

All clinical trials targeting BACE-1 that have entered in phase III have been reported to decrease significantly and dose-dependently Aβ levels in the brain, plasma, or CSF (Table 1). However, all of them have been terminated mostly due to reported toxicity. Indeed, many studies have demonstrated serious adverse effects such as an abnormal elevation in liver enzymes and clinical worsening (Table 1). The reasons for these side effects observed in human trials are not well established, but could be related to the high number of BACE1 substrates being involved in vital functions. BACE1 is known to cleave nearly 40 different neuronal membrane proteins (182) and some of these substrates, such as Sez6 or neuron myelination like neuregulin-1, are known to be essential for synaptic function (183). Indeed, several studies reported that BACE1 inhibition triggers synaptic and cognitive dysfunctions (166, 167). BACE1 inhibitors could also cross over with not only its homologue BACE2, but also with cathepsin D, another aspartyl protease (184,185). Inhibition of cathepsin D would be expected to be highly deleterious because of its role in lysosomal degradation. Of note, cathepsin D is one of the key enzymes involved in C99 degradation (139,186) and would therefore also be expected to slow down the degradation of C99 (139, 186). It is therefore of high importance to identify inhibitors that target BACE1 but no other acidic proteases. Moreover, ideally these inhibitors should specifically decrease the processing of APP but not that of other BACE1 substrates.

Until yet, one of the main obstacles of BACE1 inhibitors is linked to their limited drug availability due to their peptidic feature and high molecular size. Small molecules m a yb el e s se f ficient than APP to fill the large substrate pocket, but higher sized molecules may display reduced bioavailability. These drugs need to cross the brain barrier, but also the cell membrane to reach endosomes, which are the main sites for β-secretase (36). Great advances in the field have been the recent development of nonpeptidic inhibitors with promising pharmacological properties, but still these drugs have not demonstrated convincing effects on cognitive function. This has been the case for the clinical trial with the small-molecule β-secretase inhibitor Umibecestat, which was administrated to preasymptomatic carriers of APOE4 and led to more than 90% reduced Aβ levels C99 accumulation can be a consequence of either the presence of FAD mutations or a lowered degradation (possibly linked to risk factors). C99 firstly accumulates in endosomes and can itself be a direct cause of a dysfunction of the endosomal-lysosomal-autophagic (EAL) degradation pathway, thus leading to a vicious pathological cycle, in which more C99 accumulates, aggregates, and becomes neurotoxic by causing synaptic dysfunction, inflammation, mitochondrial dysfunction, and exosomal spread. Therapeutic strategies aimed at reducing C99 levels could be through either BACE1 inhibitors and/or BACE1 aptamers, which should be expected to reduce its production or autophagic/lysosomal activators or other drugs interfering with C99 degradation, which should increase its degradation.

in CSF but was stopped due to deleterious cognitive and side effects (187).

Alternative strategies to decrease C99 buildup

Besides being processed by γ-secretase, a part of C99 is eliminated by degradation through the autophagic/lysosomal pathway [START_REF] Marambaud | A CPB binding transcriptional repressor produced by the PS1/e-cleavage of N-cadherin is inhibited by PS1 FAD mutations[END_REF]143) and in some AD models, C99 accumulation is a consequence of a defective lysosomal clearance (139). Endosomal-lysosomal-autophagic dysfunction is indeed one of the earliest and major features of AD and has been proposed to be caused by both FAD mutations (as described above) and risk factors associated with SAD (for review see ( 188)). Thus, the activation of this degradation pathway could be expected to reduce C99 levels and to be beneficial. Rapamycin, a specific inhibitor of mTOR and a well-known inducer of autophagy, has been reported to ameliorate tau pathology, reduce amyloid-β (Aβ) deposition, and improve cognition in preclinical studies (189,190). However, rapamycin, as well as other autophagic activators, enhances autophagy without increasing lysosomal function that may be associated with an accumulation of nondigested autophagic vacuoles. Thus, a better way to enhance degradation could be to activate both autophagy and lysosomal function by overexpressing or pharmacologically activating the transcription factor EB (TFEB), the main regulator of the lysosomal/autophagic degradation pathway (191). TFEB activates the different steps of autophagy, from its initiation to the fusion of autophagosomes with lysosomes, but it also stimulates the biogenesis of lysosomal hydrolyses and regulates lysosomal acidification (191). Indeed, in animals, TFEB activation has been found to efficiently decrease C99 levels (192)(193)(194) and to ameliorate cognitive function, whereas it does not seem to produce any harmful effects in wild-type animals (143). The recent findings concerning TFEB activation by a curcumin analog are promising, because of the lack so far of specific TFEB-activating molecules. Earlier studies have proposed cyclodextrin, a modifier of cholesterol efflux used for the treatment of Niemann-Pick disease, as a TFEB activating drug. However, a recent work has demonstrated conflicting data concerning this molecule (195). Whereas short-term treatment was found to lead to activated autophagic degradation, long-term treatment had an opposite effect probably caused by the effect of cyclodextrin on membrane lipids (195). In addition, pharmacological treatments such as acidic nanoparticles, PLGA (poly lactic-coglycolic acid, called acidic nanoparticles (196) could constitute an alternative way to increase lysosomal function. These nanoparticles localize to lysosomes, where they degrade, and subsequently release their acidic components to acidify the local lysosomal environment (197). Acidic nanoparticles have notably been proven efficient in cellular and animal models of Parkinson's disease (197).

Finally, recent works have proposed novel mechanisms involved in C99 degradation and have provided new potential drug targets. For instance, it was demonstrated that the selective phosphorylation of presenilin 1 at ser367 could be a way to increase autophagic degradation of C99. Indeed, this phosphorylation of PS1 was found to facilitate autophagiclysosomal fusion, an effect that was mediated through the interaction of phosphorylated PS1 with Annexin 2 and snare proteins. These findings therefore proposed that drugs designed to selectively increase phosphorylation of presenilin1 at ser367 could be a way to circumvent C99 accumulation (198,199). Other works have shown that lysosomal/autophagic degradation of C99 can be promoted by its binding to the adipocyte differentiation protein AMAP, which could therefore also constitute a new drug target (200,201). The γ-secretase complex, often via the presenilins, binds to many endogenous proteins, which will influence γ-secretase cleavage in distinct ways (202,203). γ-Secretase has also been found to bind APP-CTFs, thereby stabilizing them for nonspecific degradation (204). A recent study proposed that ILE1, a member of the FAM3 superfamily, interferes with these chaperone properties and thereby enhances C99 degradation (204). ILE1 and molecules having similar properties may therefore also be plausible targets for future therapies.

Besides the strategies aimed at reducing C99 levels by enhancing its degradation, two other alternative strategies are possible. Firstly, the design of aptamers (so-called chemical antibodies) and the successful outcomes of their use in pathological conditions (205) allow to envisage this strategy to preclude side effects of BACE1 inhibitors on its physiological substrates. These chemical probes should be designed to prevent direct physical interaction of BACE1 with its targeted APP sequence in order to selectively hamper BACE1-mediated production of C99. Secondly, C99 levels could be reduced by repressing the transcriptional of APP. Indeed, a recent study has showed such effects of the compound Posiphen, which normalized C99 levels, but also reversed early endosome enlargement in Ts65Dn mice (206). This study not only reaffirms the link between C99 and endosomal perturbation but also indicates that the upstream regulation of its precursor APP could be a potential track to circumscribe C99 accumulation and subsequent cellular alterations.

Conclusion and perspectives

Taken together, two lines of conclusions can be drawn from the above-described observations. First, studies on a vast number of γ-secretase substrates have mostly if not always shown that their catalytic processing leads to the conversion into bioactive fragments, the generation of which governs a large spectrum of vital functions [START_REF] Jurisch-Yaksi | A fast growing spectrum of biological functions of gamma-secretase in development and disease[END_REF][START_REF] Haapasalo | The many substrates of presenilin/gamma-secretase[END_REF]. This is probably also the case of γ-secretase cleavage on APP leading to the generation of physiological levels of Aβ and AICD. Second, a network of consistent evidence suggests that, first, both FAD and SAD are associated with C99 accumulation; second, that C99, independently of Aβ, behaves as an early pathogenic trigger in AD, and third, that all C99-associated biochemical, anatomical, and cognitive alterations are drastically potentiated by γ-secretase inhibitors (Fig. 3). C99 accumulation is believed to begin mainly in endosomes and has been shown to be a direct cause of endolysosomal network dysfunction, thus leading to a vicious pathological cycle, in which C99 accumulates, aggregates and becomes neurotoxic by causing synaptic dysfunction, inflammation, mitochondrial pathology, and exosomal spread (Fig. 4). The causes of this AD-related C99 accumulation could be an increased production (presence of APP mutations in FAD or enhanced BACE expression in SAD), a loss of γ-secretase cleavage (FAD PS and APP mutations) or a decreased degradation due to a defective lysosomal-autophagic degradation (early feature linked to FAD mutations and risk factors in SAD).

Our conclusion reconciles genetic grounds indicating a strong link between βAPP and AD pathology and the systematic failure of Aβ-centric clinical assays. So far, evidence points to a particularly important role of C99 at early stages of AD, but these findings do not preclude the undoubted role of Aβ and particularly at later stages. The toxic effect of C99 could also be completely different from that of Aβ. While the membrane-embedded C99 could affect mostly intracellular pathways, Aβ could be expected to act mostly extracellularly. We propose that γ-secretase is an important C99-inactivating enzyme and that the consistent failure, and sometimes the deleterious worsening, of clinical trials based on γ-secretase inhibition could be linked, at least partly, to the impairment of C99 inactivation. GSMs are certainly more promising than GSIs, because they are not expected to increase C99 levels. However, the lack of C99 inactivation in the presence of these molecules still might be problematic. Future studies are needed to clarify this issue.

If one considers the fate of C99, secretases could overall be seen either as direct C99 inactivators, such as γ-secretase as stated above, α-secretase that cleaves C99 to generate C83 (207), and even β-secretase that mainly cleaves βAPP at the β 0 site, thereby precluding C99 production (Fig. 1B). In that sense, our point of view is that therapeutic strategies targeting secretases would not be the best way to tackle AD. Indeed, besides the complexity and the high number of substrates other than APP, inhibiting these cleavages would also impair C99 degradation.

Thus, in conclusion, we here suggest that γ-secretase is a crucial enzyme involved in not only the production of physiological bioactive fragments but also the inactivation of neurotoxic C99. We propose alternative strategies aiming to reduce the levels of C99, either by interfering with its production or by promoting its degradation (Fig. 4).

Mots clés :

 clés Maladie d'Alzheimer, ηCTF, Production Aβ, Vesicules Extracellulaires, Endosomes, Autophagie Acknowledgments First of all, I would like to express my sincere gratitude to the members of my jury, Dr. Marie-Claude POTIER, Dr. Nicolas SERGEANT, Dr. Santiago RIVERA, and Dr. Julien CHAPUIS, for accepting to evaluate my work.

  , Dad, Micha, and Paul, I consider myself extremely lucky to have family members like you. I wish I could tell you how much I love and value you. Mom and Dad, I can't thank you enough for being the most encouraging and loving parents anyone could have wished for.

GFLD

  You've blessed me more than I deserve! FAD: Familial forms of AD FDA: Food and Drug Administration FDG-PET: Fluorodeoxyglucose-Positron Emission Tomography GABABR1a: Gamma-Aminobutyric Acid type B Receptor subunit 1a Tumour Necrosis Factor-α TORC1: Target-Of-Rapamycin Complex 1 T-tau: Total Tau UPS: Ubiquitin-Proteasome System 2xTgAD: Double Transgenic mouse model of AD (APPswe, TauP301L) 3xTgAD: Triple Transgenic mouse model of AD (APPswe, TauP301L, PS1M146V) 5xFAD: Transgenic mouse model of AD with 5 familial mutations on APP and PSEN1 genes List of Tables and Figures

Figure 1 : 21 Figure 2 : 22 Figure 3 :

 1212223 Figure 1 : Projected Number of People Age 65 and Older (Total and by Age) in the U.S. Population with Alzheimer's Dementia, 2020 to 2060 ......................................................21 Figure 2: Physiological structure of the brain and neurons in healthy persons (left) and in Alzheimer's disease (right) patients. ............................................................................22 Figure 3 : An overview of genes which have been implicated in Alzheimer's disease. 31

Figure 4 : 35 Figure 5 : 37 Figure 6 44 Figure 7 :

 4355376447 Figure 4 : Domain architecture, functional segments and conservation of APP-family proteins. ..............................................................................................................................35 Figure 5 : Non-amyloidogenic and amyloidogenic APP-pathways. ................................37 Figure 6 : β or β'/γ-secretase pathway. .............................................................................44 Figure 7 : Progressive cleavages of the APP transmembrane domain by the Presenilin/-secretase complex. ........................................................................................46

Figure 8 : 49 Figure 9 : 51 Figure 10 : 55 Figure 11 : 58 Figure 12 : 60 Figure 13 : 74 Figure 14 : 77 Figure 15 : 79 Figure 16

 84995110551158126013741477157916 Figure 8 : Subunits of γ-secretase and substrate recognition. .......................................49 Figure 9: Schematic representation of APP trafficking and its convergence with canonical secretases in the endo-lysosomal network. ....................................................51 Figure 10 : The ubiquitin proteasome system. .................................................................55 Figure 11: Turnover of APP and CTFs via Endosomal Exosomal-Lysosomal-Autophagic network. ..........................................................................................................58 Figure 12: Scheme of the increased endocytic production of Aβ due to LOAD genetic risk factors. .........................................................................................................................60 Figure 13: Non-canonical APP-cleavages. .......................................................................74 Figure 14: The ƞ-secretase pathway. ................................................................................77 Figure 15: Metzincins superfamily members and classification of MMPs based on their domain arrangement. .........................................................................................................79 Figure 16 Domain organization in MT5-MMP. ...................................................................83

Figure 17 :Figure 18 :Figure 19 :

 171819 Figure 17: ηCTF-Nter immunostaining in human AD hippocampi. ............................... 122 Figure 18 : The detection of human APP-FL in different cell and mouse models, using ηCTF-Nter antibody. ......................................................................................................... 123 Figure 19 : ηCTF is not detected in AD brains. ............................................................. 124

Figure 20 :

 20 Figure 20: Aηα and Aηβ impair hippocampal LTP. ........................................................ 125

Figure 1 :

 1 Figure 1 : Projected Number of People Age 65 and Older (Total and by Age) in the U.S. Population with Alzheimer's Dementia, 2020 to 2060 (https://www.alz.org/media/Documents/alzheimers-facts-and-figures)

Figure 2 :

 2 Figure 2: Physiological structure of the brain and neurons in healthy persons (left) and in Alzheimer's disease (right) patients. Macroscopic brain atrophy in AD with the two histopathological markers: extracellular amyloid plaques and intraneuronal neurofibrillary tangles (NFTs). Created with BioRender.com free software.

  speaking or writing: People living with Alzheimer's may have trouble following or joining a conversation. They may stop in the middle of a conversation and have no idea how to continue or they may repeat themselves. They may struggle with vocabulary, have trouble naming a familiar object or use the wrong name (e.g., calling a watch a "hand clock"). Sometimes having trouble finding the right word. Misplacing things and losing the ability to retrace steps: People living with Alzheimer's may put things in unusual places. They may lose things and be unable to go back over their steps to find them. They may accuse others of stealing, especially as the disease progresses. Misplacing things from time to time and retracing steps to find them. Decreased or poor judgment: Individuals may experience changes in judgment or decisionmaking. For example, they may use poor judgment when dealing with money or pay less attention to grooming or keeping themselves clean. Making a bad decision or mistake once in a while, such as neglecting to schedule an oil change for a car. Withdrawal from work or social activities: People living with Alzheimer's disease may experience changes in the ability to hold or follow a conversation. As a result, they may withdraw from hobbies, social activities or other engagements. They may have trouble keeping up with a favorite sports team or activity. Sometimes feeling uninterested in family and social obligations. Changes in mood, personality and behavior: The mood and personalities of people living with Alzheimer's can change. They can become confused, suspicious, depressed, fearful or anxious.

Figure 3 :

 3 Figure 3 : An overview of genes which have been implicated in Alzheimer's disease.The internal color corresponds to their understood function; where there are two internal colors, the gene has been implicated in more than one pathway. Genes circled in yellow are also thought to influence amyloid precursor protein metabolism; genes circled in red are thought to influence tau metabolism (Lane,Hardy and Schott, 2018).

Figure 4 :

 4 Figure 4 : Domain architecture, functional segments and conservation of APP-family proteins.APP and its mammalian homologues APLP1 and APLP2 share on overall a similar domain architecture. The Aβ-peptide and the extension domain (ED) are only present in all isoforms of APP, the KPI-insertion only in APP751, the KPI/OX2-insertion only in APP770 and APLP2 and the E1domain is less conserved in APLP1 as indicated by its light green color. Additional functional units in APP isoforms are indicated by brackets [heparin binding domain (HBD); growth factor like domain (GFLD)]. Adapted from[START_REF] Coburger | The structural biology of the amyloid precursor protein APPa complex puzzle reveals its multi-domain architecture[END_REF].

Figure 5 :

 5 Figure 5 : Non-amyloidogenic and amyloidogenic APP-pathways. A) represents the non-amyloidogenic pathway in which APP is first cleaved by the α-secretase producing the soluble fragment sAPPα and the membrane-embedded Cterminal fragment, C83 that is then cleaved by γ-secretase to release the cytosolic

Figure 6 :

 6 Figure 6 : β or β'/γ-secretase pathway. APP is first cleaved by the β-secretase at β or β' site to liberate the soluble fragments sAPPβ or sAPPβ' and the membrane-embedded C99 or C89 fragments, respectively. Then γ-secretase cleavage releases the cytosolic AICD fragment and Aβ peptides (Aβ1-40/42) or Aβ11-40/42, respectively. (Checler et al., 2021).

Figure 7 :

 7 Figure 7 : Progressive cleavages of the APP transmembrane domain by the Presenilin/-secretase complex. Following β-secretase cleavage, the resultant C99 goes through a sequence of consecutive cleavages catalyzed by γ-secretase to generate Aβ. C99 first undergoes ε-cleavage, releasing Aβ48 or Aβ49, and a further ζ-cleavage, producing Aβ45 or Aβ46. Subsequently, at γ-site, Aβ45 and Aβ46 are processed into Aβ42 and Aβ43, respectively. The resulting Aβ43 then faces additional processing at γ-site to produce Aβ40. (Selkoe and Hardy, 2016).

Figure 9 :

 9 Figure 9: Schematic representation of APP trafficking and its convergence with canonical secretases in the endolysosomal network.After synthesis, APP transits along the secretory pathway through the ER to the TGN, then from the TGN to the plasma membrane where it is further metabolized by α-secretase(1). APP can be also endocytosed into early endosomes vesicles(2) or directly transported from the TGN to early endosomes(4). Early endosomes can be recycled back to the plasma membrane(3) or to the Golgi compartment(5). The maturation of early endosomes generates multivesicular bodies (MVB)/late endosomes (6) containing multiple vesicles. Once in the MVB/endosomes, APP can be degraded in lysosomes(7) or released through exosomes(8). Non-amyloidogenic processing mainly occurs at the cell surface where α-secretases are present(1). Amyloidogenic processing involves transit through the endocytic organelles where APP encounters βand γ-secretases (6 to 8). Figure created with BioRender.com free software.

Figure 11 :

 11 Figure 11: Turnover of APP and CTFs via Endosomal Exosomal-Lysosomal-Autophagic network. APP, C99, and C83 can reach early endosome either via trans-golgi network (TGN), or via cell surface.Early endosome matures to late endosome, also called MVB. APP, C99, and C83 are incorporated into ILVs of MVBs. In parallel, phagophore matures to form autophagosome, which can fuse with endolysosomal compartments, such as MVB. Fusion of autophagosome with MVB results in formation of hybrid organelle called amphisome. Amphisome can fuse with lysosome, promoting final degradation of APP, C99, and C83. Autophagosome can also fuse with lysosome resulting in formation of autolysosomes. Adapted from[START_REF] Gonzalez | Autophagosomes cooperate in the degradation of intracellular C-terminal fragments of the amyloid precursor protein via the MVB/lysosomal pathway[END_REF].

Figure 12 :

 12 Figure 12: Scheme of the increased endocytic production of Aβ due to LOAD genetic risk factors. ApoE4-mediated increase in APP endocytosis and/or via increased APP in the secretory pathway due to increased APP transcription; Bin1 loss-of-function impedes BACE1 to recycle out of sorting endosomes; Sorla loss-of-function decreases APP recycling out of endosomes to the TGN; CALM loss-of-function increases APP and γ-secretase endocytosis and delivery to sorting endosomes. The red arrows indicate the alterations (increase or decrease) of the subcellular mechanisms associated with each predisposition gene. Adapted from (Guimas Almeida et al., 2018).

Figure 13 :

 13 Figure 13: Non-canonical APP-cleavages. δ-secretase cleaves in two sites of extracellular part of APP. Meprin-β cleaves APP in six sites: 3 on the N-terminal part of APP, 1 on β-site and 2 within Aβ sequence. Cathepsins can cleave on βsite of APP and caspases within the intracytoplasmic domain. Cleavage by η-secretase occurs 92 residues above the β-site. All the residues are termed using APP695 numbering. Figure created with BioRender.com free software.

  MMPs and ADAMs, which play multiple roles in pathophysiology of AD, along with ADAMs with thrombospondin motifs (ADAMTSs) are all members of this metzincin subfamily. In humans, twenty-four MMPs have been identified and divided into five groups based on structure and substrate specificity: gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), collagenases (MMP-1, -8 and -13), membrane-type MMPs (MT-MMPs, MMP-14 or MT1-MMP, MMP-15, MMP-16 or MT3-MMP, MMP -17, MMP -24 or MT5-MMP and MMP-25) and the other MMPs (e.g., MMP-7, MMP-12 or MMP-28) (Rivera et al., 2019; Rivera et al., 2010)( Figure 15). MMPs were formerly thought to only degrade ECM proteins, but this belief is now changing as non-matrix substrates of MMPs, such as signaling molecules, trophic factors, receptors, cell adhesion molecules, and even nuclear proteins, have been discovered. Numerous studies have highlighted MMPs' important roles in development, post-injury plasticity, learning, and memory. The functional diversity of MMPs and, more crucially, their role in neuroinflammation, and in neurodegenerative diseases such as AD were reviewed by Santiago

Figure 15 :

 15 Figure 15: Metzincins superfamily members and classification of MMPs based on their domain arrangement.Matrix metalloproteinases (MMPs) share a common domain structure: the pre-domain that contains a signal peptide responsible for secretion; the pro-domain that keeps the enzyme inactive by an interaction between a cysteine residue and the Zn2+ ion group from the catalytic domain; and the haemopexin-like carboxy-terminal domain, which is linked to the catalytic domain by a flexible hinge region. Membrane-type MMPs (MT-MMPs) have a transmembrane domain or a glycosylphospatidylinositol (GPI) anchor at the C terminus. ADAM, a disintegrin and metalloproteinase; ADAMTS, ADAM with thrombospondin motifs; BMP1, bone morphogenetic protein 1.[START_REF] Vandenbroucke | Is there new hope for therapeutic matrix metalloproteinase inhibition?[END_REF].

Figure 16

 16 Figure 16 Domain organization in MT5-MMP. MT5-MM's structure contains a signal peptide, a prodomain, a RXKR activation motif, a catalytic domain, a hinge region hemopexin-like domain a transmembrane domain and a short cytosolic tail. Figure created with BioRender.com free software.

First,

  Sekine-Aizawa and colleagues found MT5-MMP colocalized with amyloid plaques in post-mortem brain tissue from AD patients (Sekine-Aizawa et al., 2001). To further understand the role of MT5-MMP in AD, Rivera's group developed the 5xFAD/MT5-/-model, which was created by crossing 5xFAD mice with familial AD mutations in the human APP and Psen1

Fig 2 )

 2 Fig 2) validating the specificity of this band as well as the in vitro usefulness of CTF-Nter. Interestingly, C83 and C99 fragments are not recognized by ηCTF-Nter (Fig.4b) allowing us to get rid of these CTFs and to analyze specifically the subcellular localization of ηCTF fragment in HeLa cells transiently transfected with ηCTF cDNA.

Fig. 1 :

 1 Fig. 1: Expression and detection of ηCTF in SH-SY5Y cells. a. Schematic illustration of APP-derived -secretase-mediated production of ηCTF and its subsequent cleavages by indicated secretases. Stars indicate the sites of recognition of proteolytic fragments or peptides by WO2 and APP-Cter antibodies. The η-secretase cleaves APP protein to produce a transmembrane fragment called ηCTF that undergoes subsequent cleavages by  and/or -secretases yielding Aη and Aη respectively. The WO2 antibody targets the amino acid residues 4-10 of Aβ peptide and recognizes full-length APP, ηCTF, C99, Aη but neither C83 nor Aη. The APP-Cter antibody is directed toward the last residues of APP protein and recognizes fulllength APP, ηCTF, C99, C83 but neither Aη nor Aη. b-c. SH-SY5Y cells were transiently transfected with C99-, ηCTF-, Aη-bearing vectors or empty pcDNA3 vector and analyzed by western-blot using APP-Cter (b) or WO2 (c) antibodies. A specific band corresponding to ηCTF is detected around 30kDa by both APP-Cter and WO2 antibodies. Recombinant ηCTF protein (ηCTFrec) is used as molecular weight control and GAPDH as loading control. Fig. 2: ηCTF fragment is degraded by both proteasome and autophagic pathways. a-d. SH-SY5Y cells were transiently transfected with ηCTF or pcDNA3 vectors and treated for 24h with proteasome inhibitors (a-b, lactacystine (Lact, 5µM), epoxomicin (Epox, 1µM), MG132 (5µM)) or with bafilomycin A1 (BafA1, 100nM) or Smer28 (50µM) that blocks or activates autophagy respectively (c-d) then analyzed by western-blot using APP-Cter antibody. Histograms in b and d correspond to the quantification of ηCTF immunoreactivity obtained in a and c and are expressed as percent of controls (H2O or DMSO-treated cells) taken as 100. Bars are the means +/-SEM of 5 to 9 independent determinations. ****p<0.0001 according to Mann Whitney test.

Fig. 3 :

 3 Fig. 3: ηCTF fragment yields both Aη and Aβ peptides. a. Wild-type (MEF APPwt) and APP/APLPs-deficient mouse embryonic fibroblasts (MEF APPKO) were transiently transfected with ηCTF or pcDNA3 vectors and analyzed by western-blot using APP-Cter and WO2 antibodies. GAPDH is used as loading control. b-d. ηCTF transfected MEF APPKO cells were treated for 24h with -or -secretase inhibitors (Gi:10µM, Bi:30µM, D6:1µM) then analyzed by western-blot using WO2 antibody (b). GAPDH is used as loading control. Bars correspond to the quantification of ηCTF immunoreactivity expressed as percent of controls (DMSO-treated cells) taken as 100 and are the means +/-S.E.M of 6 independent determinations. Ns, not statistically significant according to the Tukey one-way ANOVA test (b). Aη peptides were immunoprecipitated (IP) using WO2 antibody from conditioned medium of MEF APP KO cells expressing or not ηCTF and treated with -or secretase inhibitors. Note that A was not detectable in secretates before immunoprecipitation (Input) (c). A40 levels were measured by ELISA in the conditioned medium of MEF APPKO cells expressing or not ηCTF and treated with -or -secretase inhibitors. Bars indicate the concentration of A in pg/ml and are the means +/-S.E.M of 17 independent determinations. ****p<0.0001, * p<0.05, ns: not statistically significant according to the Tukey one-way ANOVA test (d).

Fig. 4 :

 4 Fig. 4: Characterization of a new η-CTF-Nter antibody.

dFig. 5 :Fig. 6 :

 56 Fig. 5: ηCTF fragment is localized in Golgi and endosomes. a-d. Hela cells were transiently transfected with ηCTF and immunostained with ηCTF-Nter or WO2 (green) antibodies for ηCTF detection and antibodies directed towards TGN-46 (trans-Golgi apparatus, red, a), EEA1 (early endosomes, red, b), CD63 (late endosomes, red, c) or lamp2 (lysosomes, red, d). Note that staining corresponding to ηCTF colocalized mostly with TGN-46 and partially with EEA1 and CD63 antibodies (yellow in merge, d). Nuclei were stained with DAPI. Scale bar are 10µm Fig. 6: CTF expression and in situ localization in AAV-ηCTF mouse brains. a-c. Wild-type newborn mice were infected with adeno-associated virus expressing ηCTF (AAV-η-CTF) or control empty vector (AAV-free) by intra-cerebro-ventricular (ICV) injection then sacrificed at 3-month-old.

Fig. 7 :

 7 Fig. 7: ηCTF fragment is detected in sEVs purified from cells and mouse brains.

Fig. 8 :

 8 Fig. 8: ηCTF fragment accumulates in 3xTgAD brains.

Figure 17 :

 17 Figure 17: ηCTF-Nter immunostaining in human AD hippocampi. The hippocampi of control and AD patients at different braak NFT stages (patients' information in Table2) were analyzed by DAB-immunohistochemical staining and obtained using ηCTF-Nter antibody to detect ηCTF, or WO2 for the detection of amyloid plaques.

Figure 18 :

 18 Figure 18 : The detection of human APP-FL in different cell and mouse models, using ηCTF-Nter antibody.Brains of wild-type (WT), and 3xTg mice were homogenized for membrane protein preparation then analyzed in western-blot along with SH-SY5Y, SH APPwt, MEF APPwt and MEF APP KO cells for the detection of APP full length (APP-FL), using APP-Cter and ηCTF-Nter antibodies.

Figure 19 :

 19 Figure 19: ηCTF is not detected in AD brains. Brains of control and AD patients at different Braak NFT stages (patients' information in TABLE 2) were analyzed by western blot using APP-Cter (a,b), WO2 (c,d), or ηCTF-Nter (e,f) antibodies. GAPDH is used as loading control.

Figure 20 :

 20 Figure 20: Aηα and Aηβ impair hippocampal LTP. LTP was analyzed ex vivo in hippocampal slices of wild-type mice. fEPSP, field excitatory postsynaptic potential. a,b, fEPSP slope (% baseline) pre-and post-LTP induction (time 0) in control (N=6, n=8) or in presence of a, 100nM Aηα (N=4, n=9), or b, 100 nM Aηβ (N=4, n=8). c, fEPSP magnitude 60 min after LTP induction from graphs in a,b, with statistical analysis *p< 0.05; one-way analysis of variance (ANOVA); Bars are the means +/-SEM

  Brains were quickly removed and placed in ice-cold artificial cerebrospinal fluid containing (in mM) 124NaCl, 3.75 KCl, 1.25 NaPO4, 2 MgSO4, 2 CaCl2, 26.5 NaHCO3, 10 glucose (pH, ~ 7.4, gassed with 95% O2-5% CO2). Coronal slices (350 μm thick) were cut with a vibratome (7000 smz2; Campden Instruments) and transferred for recovery to a holding chamber containing oxygenated artificial cerebrospinal fluid as described above (gassed with 95% O2-5% CO2, pH ~ 7.4) at 28-30 °C for 30min before adding of 10µM picrotoxin. Hippocampal slices recovered at least for 60 min before recording. For recording process, individual slices were transferred to a submerged recording chamber, where they were still maintained at 29-30 °C and perfused with artificial cerebrospinal fluid containing or not Aηα or Aηβ recombinant fragments (100nM), at a rate of 2 ml per min. To record fEPSPs, a monopolar electrode was placed in the Schaffer collaterals, and stimulation was applied at 0.066 Hz (every 20 s) with stimulus intensity ranging from 10µA to 100μA. The recording electrode was placed in the stratum radiatum and fEPSPs recorded with a borosilicate micropipette filled with artificial cerebrospinal fluid. Baseline was recorded for a minimum of 10 min or until stable. LTP was induced by stimulation with 100Hz with three trains of a 1s tetanus separated by 20s. Signals were amplified with an Axopatch 200B amplifier (Molecular Devices, Union City, CA, USA) digitized by a Digidata 1550B interface (Axon Instruments, Molecular Devices) and sampled at 10 kHz. Recordings were acquired using Clampex 11.0 software (Molecular Devices) and analyzed with Clampfit 11.0 sofware (Molecular Devices). The initial fESPS slope in mV per ms was determined by the software.

1 )

 1 Can ηCTF be produced under pathophysiological conditions?

  Throughout many years, AD was believed to be exclusively caused by the toxicity of Aβ. Yet, the etiology of the disease has been reevaluated in light of accumulating evidence of clinical trial failures targeting this peptide, leading scientists to search for additional causative factors as well as some other targets.

Frédéric

  Checler*, Elissa Afram, Rapha€ elle Pardossi-Piquard, and Inger Lauritzen From the Université Côte d'Azur, Institut de Pharmacologie Moléculaire et Cellulaire UMR7275 CNRS, Laboratoire d'Excellence Distalz, Sophia-Antipolis, Valbonne, France Edited by Paul Fraser

Figure 1 .

 1 Figure 1. APP metabolism. A, represents the α/γ-secretase pathway in which APP is first cleaved by the α-secretase producing the soluble fragment sAPPα and the membrane-embedded C-terminal fragment, C83 that is then cleaved by γ-secretase to release the cytosolic AICD fragment and p3 peptide, respectively. B, represents the β or β 0 /γ-secretase pathway in which APP is first cleaved by the β-secretase at β or β 0 site to liberate the soluble fragments sAPPβ or sAPPβ' and the membrane-embedded C99 or C89 fragments, respectively. Then γ-secretase cleavage releases the cytosolic AICD fragment and Aβ peptides (Aβ1-40 or Aβ1-42) or Aβ11-40, respectively. Note that in physiological conditions, β-secretase mainly cleaves at the β' site, but some FAD mutations shift the cleavage to the β-site to produce more C99. C, represents the ƞ/γ-secretase pathway in which ƞ-secretase cleavage releases the soluble fragment sAPPƞ and the membrane-embedded ƞCTF, which is processed by α-o rβ-secretase, thus generating Aƞα or Aƞβ peptides, respectively.

Figure 2 .

 2 Figure 2. γ-secretase. A, displays a schematic representation of presenilins (PS1 or PS2) composed of nine transmembrane domains and harboring the catalytic core of the complex with two aspartyl residues in the transmembrane domains TM-6 and TM-7. During maturation, presenilin undergoes endoproteolysis, and the resulting N-terminal fragment (PS-NTF) and C-terminal fragment (PS-CTF) remain associated. B, represents the multimeric γsecretase complex composed of four membrane proteins: presenilin (green), nicastrin (blue) composed of a single transmembrane and a large glycosylated N-terminal ectodomain, Aph-1 (purple) composed of seven transmembrane domains, and Pen-2 (pink) composed of two transmembrane domains. The substrate C99 (yellow) interacts with γ-secretase to form an enzyme substrate complex. C, represents the successive proteolysis of C99 transmembrane domain by γ-secretase. First, an endoproteolytic cleavage occurs close to the membrane-cytosol interface at two possible ε sites yielding long Aβ peptides Aβ48 or Aβ49, respectively. Then a carboxypeptidase activity of γ-secretase trims Aβ48 and Aβ49 each 3 or 4 amino acid at successive ζ-, γ, and γ 0 -sites leading to two Aβ product lines: Aβ49→Aβ46→Aβ43→Aβ40 and Aβ48→Aβ45→Aβ42→ Aβ38, respectively.

Figure 3 .

 3 Figure 3. Schematic models for C99 accumulation. A, displays a schematic model of C99 fate in the absence or presence of γ-secretase cleavage. In physiological conditions, γ-secretase cleavage of C99 (blue/red sticks) leads to the generation of Aβ and AICD, whereas when γ-secretase activity is impaired (inhibitors or PS mutations), C99 accumulates and aggregates. B, displays a schematic view of C99 generation and accumulation in conditions of blocked γ-secretase. APP (long blue/red bar) maturates and traffics through the Golgi to the plasma membrane (blue arrows), where a small part of it is processed by α-secretase (non-amyloidogenic pathway). APP that escapes α-secretase-mediated cleavage is endocytosed into early endosomes and some of it is recycled to the plasma membrane, either directly or through the Golgi network (open black arrows). Within early endosomes, some APP is cleaved by β-secretase generating C99 (short blue/red bar), which then can undergo γ-secretase cleavage, thereby releasing the Aβ peptide and AICD. Some C99 escapes from γ-secretase cleavage and is incorporated in ILVs (intra luminal vesicles), which are either released as exosomes after the fusion of multivesicular bodies (MVBs) with the plasma membrane(1) or degraded after their fusion of MVBs with lysosomes or with autophagosomes, which then fuse with lysosomes (2). The presence of APP and PS mutations or γ-secretase inhibitors and/or lowered lysosomal degradation lead to lysosomal, autolysosomal, and exosomal C99 accumulation. C99 has also been found to accumulate in MAMs and mitochondria.

Figure 4 .

 4 Figure 4. C99 toxicity. C99 accumulation can be a consequence of either the presence of FAD mutations or a lowered degradation (possibly linked to risk factors). C99 firstly accumulates in endosomes and can itself be a direct cause of a dysfunction of the endosomal-lysosomal-autophagic (EAL) degradation pathway, thus leading to a vicious pathological cycle, in which more C99 accumulates, aggregates, and becomes neurotoxic by causing synaptic dysfunction, inflammation, mitochondrial dysfunction, and exosomal spread. Therapeutic strategies aimed at reducing C99 levels could be through either BACE1 inhibitors and/or BACE1 aptamers, which should be expected to reduce its production or autophagic/lysosomal activators or other drugs interfering with C99 degradation, which should increase its degradation.

Table 1 : Signs of Alzheimer's dementia compared with typical age-related changes

 1 . ..26

Table 2 :

 2 Demographic

  3.1. Secretion of exosomesAs described previously (see chapter Endo-Lysosomal Network (ELN) -Generalities) ILVs and their cargos that are not destined for degradation can be released extracellularly as exosomes following MVBs' fusion with the plasma membrane. Although the exact mechanisms governing whether MVBs are directed towards the plasma membrane or lysosomes have not yet been identified, studies have observed that MVBs with high cholesterol levels are more likely to fuse with the plasma membrane, releasing exosomes

  1.1.1. Active immunotherapyThe first vaccine to undergo clinical testing was AN-1792, which consists of a synthetic Aβ1-42 with QS-21 adjuvant and conducted in 2002. Despite the promising results in amyloid plaque clearance, it was discovered that Aβ42 of AN-1792 contains a domain responsible for T-cell activation, which is why a percentage of the treated patients developed T-cell-mediated meningoencephalitis. Other Aβ vaccines that target more specific epitopes and lack the portion responsible for inflammatory T-cell activation were developed to avoid T-cell activation, such as CAD106, which is composed of multiple copies of Aβ1-6 and failed due to unexpected changes in cognitive function, brain volume loss, and body weight loss. Another Aβ-vaccine that was created, ACC-001, which contained Aβ1-7, similarly failed due to statistically insignificant results. There were several further active vaccines that were investigated afterwards and failed, including ACI-24, affitope AD02, V950, and UB-311. ABvac40 was one of the first vaccines to target the Aβ40 peptide, and it consists of numerous repeats of Aβ33-

	Aducanumab was approved by the FDA in United States with much controversy, after
	demonstrating slowing of cognitive and functional decline in patients receiving treatment,
	however, phase III clinical trials will continue until 2023. The FDA is currently evaluating
	donanemab, an antibody that targets Aβ(p3-42), the pyroglutamate form of Aβ. Finally, after
	significantly slowing the progression of cognitive decline in patients with very mild to mild

40.

In clinical trials, this vaccine displayed excellent safety and elicited a significant immune response characterized by an increase in anti-Aβ40 antibody levels; however, secondary exploratory efficacy endpoints are still being investigated

[START_REF] Se Thoe | A review on advances of treatment modalities for Alzheimer's disease[END_REF][START_REF] Song | Immunotherapy for Alzheimer's disease: targeting beta-amyloid and beyond[END_REF]

.

1.1.2. Passive immunotherapy

Although monoclonal antibodies have shown a lack of clinical efficacy, passive immunotherapy has been the most investigated in clinical trials to date. Five antibodies: bapineuzumab, solanezumab, crenezumab, gantenerumab, and BAN2401, reached phase III clinical trials and showed considerable promise in lowering CSF biomarker levels and Aβ plaques. Unfortunately, none of these antibodies have exhibited clinical benefits and failed to slow cognitive or functional impairment in patients. Aducanumab, one of the most potent medications for AD ever developed, had its phase III clinical trials discontinued for ineffectiveness in March 2019 following an interim futility analysis. However, in early 2020, AD, the FDA is reviewing Lecanemab, an antibody binding to soluble Aβ protofibrils, for accelerated approval

[START_REF] Se Thoe | A review on advances of treatment modalities for Alzheimer's disease[END_REF][START_REF] Song | Immunotherapy for Alzheimer's disease: targeting beta-amyloid and beyond[END_REF]

.

secretase-derived APP fragment ηCTF is localized in Golgi, endosomes and extracellular vesicles and contributes to Aβ production » Elissa Afram,

  This study gave rise to my first-author article currently under review to the journal, Cellular and Molecular Life Sciences (CMLS), and the preprint is available online on Research Square Inger Lauritzen, Alexandre Bourgeois, Wejdane El Manaa, Eric Duplan, Mounia Chami, Audrey Valverde, Bauer Charlotte, Raphaëlle Pardossi-Piquard and Frederic Checler.

	(https://www.researchsquare.com/article/rs-2285279/v1).

« The η-

  . Quantitative data are represented as means  SEM and subjected to non-parametric tests such as the Mann Whitney test for single comparisons and the Tukey one-way ANOVA test for multiple comparisons. Statistical significance code is: ****p<0.0001 and * p<0.05.

derived slices used in immunohistochemistry analyses

			Gender	PMD (h)	Braak's
		(years)			NFT stage#
		Brain samples used in SDS-PAGE analyses
	Control	52	Female	29	-
	Control	71	Male	26	-
	Control	84	Male	32	-
	Control	71	Female	15	-
	Control	60	Female	28	-
	AD	68	Female	51	I-II
	AD	68	Female	10	I-II
	AD	70	Male	31	III
	AD	78	Female	18	III
	AD	92	Female	NA	III
	AD	81	Male	19	VI
	AD	91	Female	34	VI
	AD	81	Female	NA	VI
	AD	82	Female	NA	VI
	Brain-Control	55	Male	NA	-
	AD	65	Male	NA	V
	AD	86	Male	NA	VI
	AD	55	Male	NA	NA

Table 2 : Demographic data and neuropathological findings related to human brain samples used in SDS-PAGE analyses, and brain-derived slices used in immunohistochemistry analyses.

 2

PMD: Post mortem delay, h: hours, NA: not available. Controls are brain samples isolated from post-mortem patients diagnosed as negative for several neuropathologies. # Braak and Braak's NFT (neurofibrillary tangle: tau-related pathology) stage, (-) means no NFT detection.

Table 1

 1 Principal antiamyloid clinical drugs and strategies

			Aβ modulation in treated AD
	Strategy	Drug/specific target	patients	FDA statute and participants	Side effects/cognitive readout	Reference
	Active immunotherapy	AN-1792 (synthetic Aβ42,	≥ 60-70% Aβ load reduction	Discontinued in 2002 (mild to	Meningoencephalitis	(208)
		Janssen)	in the brain (post-mortem	moderate AD patients)		(209)
			immonustaining)			(210)
		CAD106 (multiple copies of	1.3% Aβ reduction in brain	Discontinued in 2019 (asymp-	Worsens cognition, head-	(211)
		Aβ1-6 peptide, Novartis)	PET scan (florbetapir)	tomatic carriers of APOE-4)	ache, nasopharyngitis, py-
			2-3-fold increase in		rexia, hypertension, back
			plasma Aβ40 at 450 mg		pain…
	Passive immunotherapy	Crenezumab (monomers,	≥ 70% Aβ42 increase in CSF	Phase II ongoing in asymp-	Lack of efficacy	(212)
		oligomers, and fibrils of Aβ,	at 15 mg/kg	tomatic carriers of PS	in mild to moderate AD
		Roche)		mutations
		Solanezumab (monomeric	170-and 18-fold increase in	Phase III ongoing in asymp-	Lack of efficacy	(213)
		and soluble forms of Aβ Eli	plasma Aβ40 and Aβ42	tomatic people who have	in mild to moderate AD
		Lilly)	respectively no Aβ modu-	biomarker evidence of brain	and in asymptomatic car-
			lation in brain PET scan	amyloid deposition	riers of APP and PS
			(florbetapir) at 400 mg		mutations
		Aducanumab (oligomers, and	80% Aβ reduction in brain	Phase III ongoing in an open-	One of the two trials	www.alzforum.org/
		fibrils of Aβ, Biogen)	PET scan (florbetapir) at	label extension study in mild	(EMERGE) was positive	therapeutics/aducanumab
			10 mg/kg	to moderate AD patients	with a significant reduction
					in cognitive decline in mild
					to moderate AD a biologics
					license application was sub-
					mitted to the FDA for
					approval on July 2020
		Gantenerumab (oligomers,	≥ 15%, 35% and 78% Aβ	Phase III ongoing in an open-	A directional trend for	(214)
		and fibrils of Aβ, Roche)	reduction in brain PET	label extension study in mild	slower clinical decline in	(215)
			scan (florbetapir) at 60, 200	to moderate AD patients	mild to moderate AD
			and 1200 mg respectively	(SCarlet RoAD, Marguerite
				RoAD, and GRADUATE)
				and in asymptomatic car-
				riers of APP and PS muta-
				tions (Dian-Tu)
		BAN2401 (soluble Aβ proto-	≥ 120% Aβ40 increase in	Phase III ongoing in early	47% and 30% reduction in	(216)
		fibrils, Biogen)	plasma	symptomatic AD patients	cognitive decline as judged	www.alzforum.org/
			300 fold Aβ42 increase in	(Clarity AD) and in asymp-	by the ADAS-Cog and the	therapeutics/ban2401
			CSF	tomatic people who have	ADCOMS respetively
			93% Aβ reduction in brain	biomarker evidence of brain
			PET scan (florbetapir) at	amyloid deposition
			10 mg/kg	(AHEAD 3-45)
	β-secretase inhibitors	Verubecestat (MK-	≥ 57-84% Aβ reduction in	Discontinued in 2018 (pro-	Worsens cognition, anxiety,	(217)
		8931Merck)	CSF at 12-60 mg	dromal, mild to moderate	depression, and sleep	(218)
				AD patients)	problems
		Atabecestat (Janssen)	≥ 67-90% Aβ reduction in	Discontinued in 2018 (asymp-	Worsens cognition, elevated	(219)
			CSF at 10-50 mg	tomatic people)	liver enzymes, depression,	(220)
					anxiety, and sleep problems
		Lanabecestat (AZD3293,Eli	≥64% to 78% Aβ reduction in	Discontinued in 2018 (pro-	Lack of efficacy on cognition,	(221)
		Lilly)	plasma at 15-50 mg	dromal and mild AD	neuropsychiatric adverse	(222)
			≥51% to 76% Aβ reduction	patients)	events, weight loss, hair co-
			in CSF at 15-50 mg		lor changes
		Umibecestat (Novartis)	≥ 90% Aβ reduction in CSF at	Discontinued in 2019 (asymp-	Worsens cognition, brain at-	(223)
			85 mg	tomatic carriers of APOE-4)	rophy and weight loss	(187) www.alzforum.org/
						therapeutics/umibecestat
		Elenbecestat (Biogen)	≥5.8% and ≥13.6% Aβ	Discontinued in 2019 (pro-	Weight loss, skin rashes and	(224)
			reduction in brain PET	dromal, mild to moderate	neuropsychiatric adverse	(225)
			scan (florbetaben and flor-	AD patients)	events
			betapir respectively) at
			50 mg

Table 1 -

 1 Continued

		Reference
			(110)	(111)	(112)			(116)			(122)		(226)	(123)	(124)
		Side effects/cognitive readout	Worsens cognition, skin	cancer and infections.				Worsens cognition, skin	cancers, diarrhea, nausea,	vomiting, and rash	Lack of efficacy, cardiovascu-	lar damage	Worsens cognition, dizziness,	upper respiratory tract	infection and constipation.	Lack of efficacy, hypertension,	gastrointestinal, and	vascular or cardiac problems
		FDA statute and participants	Discontinued in 2011 (AD	patients)				Discontinued in 2012 (pro-	dromal AD)		Discontinued in 2004 (mild to	moderate AD patients)	Discontinued in 2009 (mild	AD patients)	Discontinued in 2019 (asymp-	tomatic carriers of APP and	PS mutations)
	Aβ modulation in treated AD	patients	≥58.2% to 64.6% Aβ reduc-	tion in plasma at	100-140 mg	≥47-84% newly Aβ reduc-	tion in CSF at 100-280 mg	≥40% Aβ reduction in CSF at	125 mg		Not determined		No Aβ42 modulation in	plasma and CSF	No Aβ42 modulations in CSF
		Drug/specific target	Semagacestat (Eli Lilly)					Avagacestat (Bristol-Myers	Squibb)		Rofecoxib (Merck)		Tarenflurbil (Myriad	Genetics)	Naproxen (Procter & Gamble)
		Strategy	γ-Secretase inhibitors								γ-Secretase modulators

ADAS-Cog, Alzheimer's Disease Assessment Scale-Cognitive Subscale; ADCOMS, Alzheimer's Disease Composite Score; CSF, cerebrospinal fluid; FDA, US Food and Drug Administration; MCI, mild cognitive impairment.

Table 2

 2 C99-associated toxicity in AD mice models and human brain

	References	(128, 137, 139, 163, 168)			(224)	(155)	(131)	(129, 227)	(130)	(132-134)		(143)		(140, 228-230)		(126, 137, 138, 175)
	C99 accumulation-associated pathology	Endolysosomal and autophagic dysfunction	Inflammation, LTP alterations	Mitochondrial pathology	Mitochondrial pathology	Accumulation in MAMs	Lysosomal dysfunction, inflammation	Brain network alterations	Brain network alterations	Nd	Nd	Lysosomal dysfunction	Endosomal pathology	Endosomal pathology lysosomal dysfunction	Axonal transport defects	Nd	Correlation with neuronal vulnerability
	Mutations	APP K670N,M671L , PS1 M146V , MAPT P301L APP K670N,M671L , MAPT P301L	APP KM670/671NL, APP I716V, APP V717I , PS1 M146L,I286V APP751 K670 N,M671L , PS1 G384A	APP E663Q APP KM670/671NL , APP V717F APP KM670/671NL , APP V717F APP KM670/671NL , APP KM670/671NL,I716F APP KM670/671NL,EI716F,E693G hAβ sequence (G676R,F681Y,R684H)	Various APP and PS mutants		Ts65DN mice or human patients		FAD	SAD
	Mouse models	3xTgAD	2xTgAD		5xFAD	APP/PS1	APPE663Q	TgCRND8	J20 Knock-in APP NL APP NL-F and APP NL-G-F APP Hm/Hm	Human IPSCs		Down syndrome fibroblasts		Human brain

APP, amyloid precursor protein; FAD, familial Alzheimer disease; IPSC, induced pluripotent stem cells; LTP, long-term potentiation; MAPT, microtubule-associated protein tau; nd, not determined; PS, presenilin; SAD, sporadic Alzheimers disease.

J. Biol. Chem. (2021) 296 100489 3

J. Biol. Chem. (2021) 296 100489 5

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J. Biol. Chem. (2021) 296 100489

Acknowledgements:

We would like to thank Dr Frank LaFerla for providing the 3xTgAD mice, Paul Fraser for the APP-Ct antibody and Mathilde Cohen-Tannoudji Maria-Grazia Biferi and Martine Barkats for their help in producing AAVs.

Acknowledgments-This work was partly supported by the "Fondation Alzheimer," the "Fondation pour la Recherche Médicale" and the French National Foundation for Alzheimer's Disease and Related Disorders. This work has been developed and supported through the LABEX (excellence laboratory, program investment for the future) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to ALZheimer's disease) and the University hospital Federation (FHU) OncoAge.

Fundings

This work was supported by University hospital Federation (FHU) OncoAge and the LabEx (excellence laboratory) DISTALZ (Development of Innovative Strategies for a Transdisciplinary approach to Alzheimer's disease) which fully provided the PhD funding for EA.

Availability of data and materials

Not applicable. All full gels are provided in Supplementary figure 5

Abbreviations

II. O B J E C T I V E S III.

R E S U L T S

All animal experimental procedures were in accordance with the ethical standards of the European Communities Council Directive of 24 November 1986 and approved by the local Institutional Ethics Committee for the Laboratory Animal (CIEPAL-azur) from Nice, the National Council on animal care of the Ministry of Health (APAFIS#9766-201704261624789v3) and the local committee for animal welfare of IPMC institute.

Consent for publication

All authors read the final version of the article and consent to its publication

Competing Interests:

The authors have nothing to disclose and declare no financial conflict of interest.

Author's contributions

Supplementary Materials and Methods

Organotypic hippocampal slices preparation, culture and immunostaining

Organotypic hippocampal slices were obtained from postnatal day 5-7 C57BL6JRj mice as previously described (1). Briefly, hippocampi were sliced into 400 µm sections and carefully transferred on sterile hydrophilic membrane millicell discs (Millipore, FHLC01300) placed in semiporous cell culture inserts (Millipore, 0.4 µm) within a 6-well plate, balanced with the warm culture medium (Minimum Essential Medium Eagle (MEM)+Glutamax-1 (50%), EBSS (18%), EBSS (13%)/D-glucose (5%), penicillin-streptomycin (5000U/ml, 1%), Horse serum (25%) and Nystatin 10000U/ml (0.06%). Slices were infected 2 h after plating with adenoassociated viruses AAV-free or AAV-ηCTF: 5 x 10 12 vg/ml). Slices were incubated at 37°C, 5% CO2 and half of the medium was replaced every 3 days. After 9 days in culture, slices were fixed for 20 min in paraformaldehyde 4% then stored at 4°C in PBS and kept for immunostaining.

For immunostaining, organotypic hippocampal slices were permeabilized overnight in PBS Triton (0.4%), blocked with BSA (5%)/Tween20 (0.1%), then incubated with primary antibodies: α-APPct, mouse monoclonal (1:1000, Biolegend) or ηCTF-Nter (1:800) for ηCTF detection or/and α-EEA1 goat polyclonal (1:1000) for early endosomes labelling. After washes, coverslips were incubated with Alexa Fluor-488, Alexa Fluor-590 and Alexa Fluor-647 conjugated antibodies (1:1000) and DAPI (1: 20,000) staining. Finally, the sections were washed with PBS, then mounted onto glass slides and cover-slipped. The stained slices were kept at 4•C before analysis with confocal microscopy (Zeiss LSM 780 with 63X Objective).

Supplementary legends to figures

Sup