Baranger Kévin

Van Gijsel-Bonnello Manuel

Stephan Delphine
email: delphine.stephan@univ-amu.fr

Carpentier Wassila

Rivera Santiago
email: santiago.rivera@univ-amu.fr

Khrestchatisky Michel
email: michel.khrestchatisky@univ-amu.fr

Gharib Bouchra
email: bouchra.gharib@univ-amu.fr

Reggi De

Benech Max

Philippe

Kevin Baranger
email: kevin.baranger@univ-amu.fr

Manuel Van

Delphine Stephan

Wassila Carpentier

Long-term pantethine treatment counteracts pathologic gene dysregulation and decreases Alzheimer's disease pathogenesis in a transgenic mouse model

Keywords: Alzheimer'disease, pantethine, gene expression profiles, neuroprotection, phagocytosis, 5xFAD mice

Background:

The low-molecular weight thiol pantethine, known as a hypolipidemic and hypocholesterolemic agent, is the major precursor of co-enzyme A. We have previously shown that pantethine treatment reduces amyloid- (A)-induced IL-1 release and alleviates pathological metabolic changes in primary astrocyte cultures. These properties of pantethine prompted us to investigate its potential benefits in vivo in the 5XFAD (Tg) mouse model of Alzheimer's disease (AD).

Methods: 1.5-month-old Tg and wild type (WT) male mice were submitted to intraperitoneal administration of pantethine or saline control solution for 5.5 months. The effects of such treatments were investigated by performing behavioral tests and evaluating astrogliosis, microgliosis,  deposition and whole genome expression arrays, using RNAs extracted from the mice hippocampi.

Results:

We observed that long-term pantethine treatment significantly reduced glial reactivity and  deposition, and abrogated behavioral alteration in Tg mice. Moreover, the transcriptomic profiles revealed that after pantethine treatment, the expression of genes differentially expressed in Tg mice, and in particular those known to be related to AD, were significantly alleviated. Most of the genes overexpressed in Tg compared to WT were involved in inflammation, complement activation and phagocytosis, were found repressed upon pantethine treatment. In contrast, pantethine restored the expression of a significant number of genes involved in the regulation of  processing and synaptic activities, which were down-regulated in Tg mice.

Conclusions: Altogether, our data support a beneficial role for long-term pantethine treatment in preserving CNS crucial functions altered by A pathogenesis in Tg mice, and highlight the potential efficiency of pantethine to alleviate AD pathology.

Background

Alzheimer's disease (AD) is the most common cause of dementia worldwide. It is characterized by a progressive cognitive impairment thought to be associated with the accumulation of extracellular amyloid plaques, composed of amyloid- peptide (A) and intracellular neurofibrillary tangles (NFTs). NFTs result from an alteration of microtubule stabilization following the abnormal hyperphosphorylation of Tau, a microtubule-associated protein. The production of A is triggered by a successive proteolytic cleavage of the amyloid precursor protein (APP) by the -secretase, BACE-1 (beta-site APP cleaving enzyme 1) and the -secretase complex. Among the different A peptides generated from APP, A42 is believed to play a primary role in the development of AD [START_REF] Selkoe | The amyloid hypothesis of Alzheimer's disease at 25 years[END_REF]. The deposition of extracellular A is associated with a neuroinflammatory response and it is suggested that upon sustained A production, the resulting chronic inflammation contributes to AD pathology [START_REF] Tuppo | The role of inflammation in Alzheimer's disease[END_REF][START_REF] Ardura-Fabregat | Targeting Neuroinflammation to Treat Alzheimer's Disease[END_REF]. Therapeutic strategies based on the reduction of Tau hyperphosphorylation or A are under evaluation in addition to those developed to improve cognitive function (acetylcholinesterase inhibitors) and mood disturbance or to provide neuroprotection (memantine). However, to date, available treatments show only modest symptomatic effects.

Moreover, epidemiological studies have suggested that longstanding use of anti-inflammatory drugs such as NSAIDs (non-steroidal anti-inflammatory drugs) can prevent or delay the development of AD [START_REF] Stewart | Risk of Alzheimer's disease and duration of NSAID use[END_REF][START_REF] Etminan | Effect of non-steroidal anti-inflammatory drugs on risk of Alzheimer's disease: systematic review and meta-analysis of observational studies[END_REF]. Unfortunately, treatment of AD patients with anti-inflammatory drugs such as celecoxib or naproxen failed to demonstrate clinical benefits [START_REF] Soininen | Long-term efficacy and safety of celecoxib in Alzheimer's disease. Dementia and geriatric cognitive disorders[END_REF][START_REF] Group | Follow-up evaluation of cognitive function in the randomized Alzheimer's Disease Anti-inflammatory Prevention Trial and its Follow-up Study. Alzheimer's & dementia : the journal of the Alzheimer[END_REF]. These failures illustrate our poor understanding of the sequential events of neuroinflammation in AD that, depending possibly on timing and context, can either promote protective effects or exacerbate the disease [START_REF] Wyss-Coray | Inflammation in Alzheimer disease: driving force, bystander or beneficial response?[END_REF]. Thus, in addition to their potential therapeutic efficacy, search for new modulators of inflammation can be useful in determining whether inflammation is a driving force or a bystander effect in AD.

Pantethine, a dietary low-molecular-weight thiol widely distributed in the living world and synthesized from pantothenic acid (vitamin B5) and cysteamine, is an intermediate in the production of Co-enzyme A (CoA). This activity is thought to be related to its well-known effect on lowering cholesterol synthesis [START_REF] Chen | Efficacy and tolerability of coenzyme A vs pantethine for the treatment of patients with hyperlipidemia: A randomized, double-blind, multicenter study[END_REF]. However, pantethine can be rapidly hydrolyzed in vivo to pantothenic acid and cysteamine by pantetheinase encoded by the vanin gene, and it cannot be excluded that its observed activities might result from cysteamine or pantothenic acid [START_REF] Vecsei | Effects of pantethine, cysteamine and pantothenic acid on open-field behavior and brain catecholamines in rats[END_REF][START_REF] Kaskow | Diverse biological activities of the vascular non-inflammatory molecules -the Vanin pantetheinases[END_REF]. Pantethine elicits broad physiological activities involving multiple cellular pathways. It has been shown to exert neuroprotective effects and to reduce metabolic dysfunctions [START_REF] Brunetti | Pantethine treatment is effective in recovering the disease phenotype induced by ketogenic diet in a pantothenate kinase-associated neurodegeneration mouse model[END_REF][START_REF] Cornille | Enhancement of L-3-hydroxybutyryl-CoA dehydrogenase activity and circulating ketone body levels by pantethine. Relevance to dopaminergic injury[END_REF][START_REF] Rana | Pantethine rescues a Drosophila model for pantothenate kinase-associated neurodegeneration[END_REF], but also to decrease inflammation and mediate immune responses [START_REF] Kavian | Pantethine Prevents Murine Systemic Sclerosis Through the Inhibition of Microparticle Shedding[END_REF][START_REF] Penet | Protection against cerebral malaria by the low-molecular-weight thiol pantethine[END_REF]. We have previously shown that pantethine is able to protect mice against cerebral malaria by preserving blood brain barrier integrity and by lowering TNF- levels [START_REF] Penet | Protection against cerebral malaria by the low-molecular-weight thiol pantethine[END_REF]. More recently, we demonstrated that pantethine alleviates metabolic dysfunctions, reduces astrogliosis and IL-1 production in primary cultured astrocytes of 5XFAD mice [START_REF] Van Gijsel-Bonnello | Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine[END_REF]. Such beneficial effects were also observed in WT astrocytes treated with A oligomers. These data suggest that pantethine could help maintain brain homeostasis and function during AD pathogenesis.

Such observations prompted us to explore pantethine activity in vivo using 5XFAD mice. This animal model bears five mutations linked to familial forms of AD and recapitulates in a few months the main features of AD. These mutations act in an additive manner to boost the production of Aβ peptides, particularly Aβ 42 . Though they do not present tau pathology, these mice develop cerebral amyloid plaques and gliosis as early as two months [START_REF] Oakley | Intraneuronal betaamyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation[END_REF], along with increased levels of pro-inflammatory mediators Il-1 and Ccl2 mRNAs [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].

Electrophysiological studies have detected hippocampal synaptic dysfunctions in 4-to 6month-old 5XFAD animals, concomitant with memory deficits [START_REF] Oakley | Intraneuronal betaamyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation[END_REF][START_REF] Kimura | Impairments in remote memory stabilization precede hippocampal synaptic and cognitive failures in 5XFAD Alzheimer mouse model[END_REF][START_REF] Crouzin | Area-specific alterations of synaptic plasticity in the 5XFAD mouse model of Alzheimer's disease: dissociation between somatosensory cortex and hippocampus[END_REF][START_REF] Girard | Evidence for early cognitive impairment related to frontal cortex in the 5XFAD mouse model of Alzheimer's disease[END_REF][START_REF] Girard | Onset of hippocampus-dependent memory impairments in 5XFAD transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Baranger | MT5-MMP is a new pro-amyloidogenic proteinase that promotes amyloid pathology and cognitive decline in a transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Baranger | Chronic treatments with a 5-HT4 receptor agonist decrease amyloid pathology in the entorhinal cortex and learning and memory deficits in the 5xFAD mouse model of Alzheimer's disease[END_REF]. Neuronal death, which is absent in most AD mouse models, has been described in the cortex and subiculum in 9-month-old 5XFAD mice [START_REF] Oakley | Intraneuronal betaamyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation[END_REF][START_REF] Eimer | Neuron loss in the 5XFAD mouse model of Alzheimer's disease correlates with intraneuronal Abeta42 accumulation and Caspase-3 activation[END_REF]. In a previous longitudinal transcriptomic study, we showed that the main processes altered in 5xFAD mice, from 4 months onwards, were related to microglial activation and inflammation [START_REF] Landel | Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer's disease[END_REF].

To investigate the effects of pantethine on AD pathology, 1.5 month-old 5xFAD transgenic (Tg thereafter) and wild type (WT thereafter) mice were treated with pantethine for 5.5 months (see materials and methods). In line with our previous data [START_REF] Van Gijsel-Bonnello | Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine[END_REF], a significant decrease in gliosis and A plaque deposits was observed in treated Tg mice compared to untreated.

Treatment also improved behavior with a significant decrease of aggressiveness.

Transcriptomic analyses showed that pantethine was indeed able to tune down the modulations in gene expression characteristic of the Tg mice. We present for the first time an array of evidences supporting a beneficial effect of pantethine in AD. Thus, its current use and the possibility of chronic treatment in the absence of known side effects provide the grounds to test the efficacy of pantethine in AD as a new therapeutic agent in preclinical and clinical studies.

Materials and methods

Chemical

Pantethine was purchased from Sigma-Aldrich (Saint Quentin-Fallavier, France). Purity of the preparation was ascertained using high performance liquid chromatography-coupled mass spectrometry (HPLC-LC-UV-MSD) and the purity of the compound reached 99%. Sodium Chloride (NaCl) and Phosphate Buffer Saline (PBS) were purchased from Euromedex (Souffelweyersheim, France). Proteinase inhibitors cocktail was from Millipore (Molsheim, France). All the chemical powders were of analytical grade and purchased from Sigma-Aldrich.

Animals and pantethine treatment

We used 5XFAD male transgenic mice, which overexpress two human transgenes bearing five mutations linked to familial AD: App (Swedish mutation K670N, M671L; Florida mutation I716V; London mutation V717I) and Psen1 (M146L; L286V), under transcriptional control of the mouse Thy1 promoter. These mice exhibit AD-related symptoms earlier than other AD animal models and amyloid deposition starts in the cortex and subiculum at two months of age [START_REF] Oakley | Intraneuronal betaamyloid aggregates, neurodegeneration, and neuron loss in transgenic mice with five familial Alzheimer's disease mutations: potential factors in amyloid plaque formation[END_REF]. Heterozygous male 5XFAD transgenic animals (B6/SJL background) and WT controls were obtained after breeding in our animal house facility [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] Landel | Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer's disease[END_REF][START_REF] Landel | Vitamin D interacts with Esr1 and Igf1 to regulate molecular pathways relevant to Alzheimer's disease[END_REF]. Animals were housed in cages in a controlled environment (22-25°C, 50% humidity and a 12hour light/dark cycle) with free access to standard laboratory diet and water. At 1.5 month, Tg and WT mice were each separated into treated and vehicle groups (n=10 per group). Based on a previous study [START_REF] Cornille | Enhancement of L-3-hydroxybutyryl-CoA dehydrogenase activity and circulating ketone body levels by pantethine. Relevance to dopaminergic injury[END_REF], Tg and WT treated mice were given each an intraperitoneal injection of 15 mg of pantethine diluted in saline, three times a week for 5.5 months; vehicle animals received saline injections as control. At the age of 7 months, the animals were submitted to the intrudal aggressivity test (see below) and then sacrificed. Mice were anesthetized with pentobarbital (0.36 g per kg) and transcardially perfused with 50 ml of NaCl 0.9%. Brains were microdissected and hippocampi collected for the assays. All experimental procedures were approved by the Ethics Committee of the Medical Faculty of Marseille and were carried out in accordance with the guidelines published in the European Communities Council Directive of November 24, 1986 (86/609/EEC). All efforts were made to reduce animal suffering and the number of mice.

Total RNA isolation and extraction

Hippocampi from each experimental group (n=3 per group) were quickly snap-frozen in liquid nitrogen and stored at -80°C until use. Total RNA was extracted using the RNeasy Mini kit (Qiagen, Courtaboeuf, France) according to the manufacturer's instruction. RNA concentration was determined using a nanodrop 2000 spectrophotometer (ThermoFisher Scientific, Villebon sur Yvette, France) and RNA integrity assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Les Ulis, France).

Microarray assay

The microarray assay included four different experimental conditions: hippocampi from WT and Tg mice vehicle or treated with pantethine (n=3 per group). A total of 12 biological samples were therefore processed for the microarray experiment. The mouse WG-6 expression BeadChip (Illumina Inc., Cambridge, UK), comprising 47,232 transcripts was used to generate gene expression profiles and hybridized to cRNAs, which were synthesized, amplified and purified using the Illumina TotalPrep RNA Amplification Kit (Ambion, Foster City, CA, USA) following manufacturer's recommendations. Briefly, 200 ng of RNA were reverse transcribed. After second strand synthesis, the cDNA was transcribed in vitro and cRNA labelled with biotin-16-UTP. Labelled probe hybridization to the Beadchips was carried out using Illumina's protocol. The Beadchips were scanned on the Illumina IScan using Illumina IScan image data acquisition software. Illumina GenomeStudio software and LumiR (www.bioconductor.org) were used for preliminary data analysis, data normalization and quality controls. Microarray statistical analyses were performed using the Biometric Research Branch array tools (http://linus.nci.nih.gov/BRB-Array Tools.html) and the Multi Experiment Viewer software (MeV4.0; [START_REF] Saeed | TM4: a free, opensource system for microarray data management and analysis[END_REF]). Expression values were filtered for a p value ≤ 0.05. Functional analysis was performed using the text-mining based software, PredictSearch [START_REF] Landel | Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer's disease[END_REF]

Real-time quantitative PCR (qPCR)

Following microarray analysis, the expression levels of some selected genes were further validated using qPCR technique. Total RNA samples (1 μg) extracted from the hippocampi of all animals (n=3) in each group were subjected to reverse-transcription reactions to synthesize cDNA using oligodT, RNase-out and M-MLV RT enzyme (ThermoFisher Scientific) according to the manufacturer's instructions. Mm99999915_g1. We used 7.5 ng of previously prepared cDNA and samples were run in triplicates. Relative expression levels were determined according to the Ct method where the expression level of the mRNA of interest is given by 2 -CT where CT = CT target mRNA -CT reference mRNA (Gapdh) in the same sample [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].

ELISA assay

The cortices of mice brains (n=3 for each condition) were homogenized in 100 mM PBS containing 2 mM NaF, 2.5 mM Na 4 P 2 O 7 , 1 mM Na 3 VO 4 , 1% Triton X-100, pH 7.2, supplemented with 1% protease inhibitor cocktail and centrifuged at 12,000 x g for 20 min at 4°C. Protein concentration was quantified by using the Bio-Rad DC™ protein assay kit following the manufacturer's instructions (Bio-Rad, Marnes-La-Coquette, France). The levels of IL-1β were analyzed by ELISA assay (Peprotech, Neuilly-sur-Seine, France) according to the manufacturer's instructions.

Immunostaining, image analysis and quantification

Anesthetized mice were transcardially perfused with 50 ml of NaCl 0.9%, followed by 50 ml of AntigenFix solution (Diapath, MM France, Brignais, France). Coronal sections of 30 μm were obtained using a vibratome (Microm HM650V, Thermo Fisher Scientific), and stored at -20°C in cryoprotectant containing 30% glycerol, 30% ethylene glycol and 40% in phosphate buffer 0.5 M, pH 7.4. Free-floating sections were first permeabilized and blocked for 1 h at room temperature using a solution of PBS 1X, 0.1% Triton X-100 and 3% BSA. Sections were then incubated overnight at 4°C with primary antibodies: mouse monoclonal anti-Aβ 6E10 (1/300; Covance, Ozyme, Saint-Quentin en Yvelines, France), rabbit polyclonal anti-IBA1 (1/300; Wako, Sobioda, Montbonnot, France), Rabbit polyclonal anti-GFAP (1/300, Dakocytomation, France) followed by an incubation with appropriate secondary antibodies:

AlexaFluor488-goat anti-rabbit (1/800) or AlexaFluor568-goat anti-mouse (1/800) (Thermo Fisher Scientific) for 1 h at room temperature. Nuclei were stained with Hoechst (0.5 µg/mL, Thermo Fisher Scientific). Omission of the primary antibody was used as control.

Sections were mounted using Prolong Gold Antifading reagent on Superfrost glass slides (Thermo Fisher Scientific). Images were taken and processed using a confocal microscope (LSM700) and Zen software (Zeiss, Jena, Germany), as previously described [START_REF] Baranger | MT5-MMP is a new pro-amyloidogenic proteinase that promotes amyloid pathology and cognitive decline in a transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Baranger | MT5-MMP Promotes Alzheimer's Pathogenesis in the Frontal Cortex of 5xFAD Mice and APP Trafficking in vitro[END_REF].

Densitometric analysis of immunostained sections was performed using ImageJ software on 3 hippocampal sections from 3 animals for each experimental group. The area of GFAP + astrocytes and IBA1 + microglia was determined. The same threshold of fluorescence was applied across the experimental groups and data were normalized to the values in the WT group. Extracellular amyloid deposits were blindly scored in the three brain sections per animal and expressed as number of plaques per mm 2 of brain surface.

Intrudal aggressivity test

The effect of pantethine on Tg male mice aggressiveness was evaluated with the residentintruder paradigm according to a previously published method [START_REF] Koolhaas | The organization of intraspecific agonistic behaviour in the rat[END_REF], and modified according to our experimental situation. The animals to be tested, treated or not with pantethine (n=10 per group) were maintained in their individual resident cage in order to mimic the natural pattern of wild animals that defend their territory. The intruders were WT male mice (n=10) from the same genetic background, but not included in this protocol. For the test, a WT animal was introduced in the cage of a Tg and their behavior were monitored with a video camera for 180 sec. The following behavioral elements were scored: 1) latency: the time between the introduction of the intruder and the first threat or attack; 2) number of threats that are generally in the form of frenetic tail agitation; 3) number of assaults. Threats and assaults were exclusively facts of the Tg resident as the WT intruder always behaved passively. In the case of violent assault, the animals were immediately separated and the test ended. The offense intensity was estimated on a scale of 0 to 5. The latency time was set at 180 sec when no reaction, threat or attack was noticed. The tests were done 3 times on 3 consecutive days under normal light, starting at 10 am. From one test to another, the intruders were circularly permuted. Intrudal aggressivity experiments were performed at least three times for each animal.

Statistical analysis

All

Results

Pantethine reduces gliosis and amyloid plaque number

The 5XFAD mouse model of AD is characterized by an increase in glial reactivity and amyloid plaque number as aging progresses. At first, immunohistological analyses of brain sections at the hippocampal level in 7-month-old Tg and WT mice (Fig. 1A) supported our previous results on cultured astrocytes [START_REF] Van Gijsel-Bonnello | Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine[END_REF]. Indeed, in sections immunostained with anti-GFAP and anti-IBA1 antibodies, Tg mice consistently exhibited a higher signal (10-fold and 20-fold increase, respectively) than WT mice (Fig. 1A,B). Pantethine treatment reduced this signal significantly in Tg animals, with an 80% and 40% reduction for GFAP and IBA1, respectively. Interestingly, pantethine reduced GFAP staining nearly to WT levels (Fig. 1B).

Similarly, staining with A-specific antibodies showed that the numerous amyloid deposits observed in Tg mice were dramatically reduced by 85% upon pantethine treatment (Fig. 1C).

These results indicated that pantethine was able to reduce significantly glial reactivity as well as the number of amyloid plaques in Tg mice.

Pantethine reduces 5XFAD aggressive behavior

AD pathology leads to cognitive impairment and behavioral alterations linked to amyloidmediated neuroinflammation and neurodegeneration. Although altered cognition has been well characterized in a variety of transgenic models, it has been reported that disturbance in hippocampal learning performance can be detected using the Morris water maze (MWM) in male 5XFAD not before 6 to 9 months of age [START_REF] Girard | Onset of hippocampus-dependent memory impairments in 5XFAD transgenic mouse model of Alzheimer's disease[END_REF][START_REF] Schneider | Behavioral and EEG changes in male 5xFAD mice[END_REF]. Alternatively, social withdrawal and aggressiveness have been clearly documented not only in AD patients [START_REF] Bidzan | Aggressive and impulsive behavior in Alzheimer's disease and progression of dementia[END_REF][START_REF] Rouch | Personality, Alzheimer's disease and behavioural and cognitive symptoms of dementia: the PACO prospective cohort study protocol[END_REF][START_REF] Ballard | Agitation and aggression in people with Alzheimer's disease[END_REF], but also in different AD mouse models [START_REF] Rouch | Personality, Alzheimer's disease and behavioural and cognitive symptoms of dementia: the PACO prospective cohort study protocol[END_REF][START_REF] Pugh | Non-cognitive behaviours in an APP/PS1 transgenic model of Alzheimer's disease[END_REF][START_REF] Vloeberghs | Aggressive male APP23 mice modeling behavioral alterations in dementia[END_REF][START_REF] Yan | Age-related neuropsychiatric symptoms in presenilins conditional double knockout mice[END_REF], and was proposed to be useful in screening putative drug treatments for AD [START_REF] Pugh | Non-cognitive behaviours in an APP/PS1 transgenic model of Alzheimer's disease[END_REF]. For these reasons, we chose to evaluate the potential benefit of pantethine on the behavior of 7 month-old AD mice rather than on cognitive performances.

Thus, we performed an intrudal aggressive test with treated and untreated Tg mice in the presence of WT animals. When comparing untreated and treated 5XFAD mice (Fig. 2), we observed a significant difference between these two groups, not only in offense intensity (Fig. 2A), but also in attack latency (Fig. 2B). Most (8 out of 10) vehicle-treated Tg animals react aggressively to the presence of WT mice, while only 4 out of 10 treated animals exhibit such a behavior (Fig. 2A). Moreover, pantethine treated animals present a longer latency period (150s) between the attacks (Fig. 2B), confirming that pantethine treatment is able to modulate the aggressive attitude of Tg mice.

Panthetine moderates the dysregulation of gene expression observed in 5XFAD mice

cDNA microarrays were performed to investigate whether the reduced astroglial and microglial activation, as well as the positive impact on behavior observed after pantethine treatment, could be related to a modulation of the expression of genes involved in these processes. The selection of genes in Tg animals presenting a reduced expression upon pantethine treatment (filtered for a threshold ≤0.6) led to the identification of 47 specific genes (Fig. 3, left panel). Interestingly, 91% of these genes (gene set 1) were expressed at higher levels in Tg mice compared to WT. Similarly, 89% of the top 47 genes (gene set 2) upregulated in pantethine-treated Tg animals when compared to untreated Tg mice (filtered for a threshold ≥1.5), were down-regulated in Tg when compared to WT (Fig. 3, right panel).

Noteworthy, pantethine had very little if no effect at all on the expression of most of these genes in WT animals (Fig. 3). It must be noted that none of the genes encoding proteins directly associated with the APP/A metabolism (e.g., Bace-1, App, or -secretase members)

were modulated between vehicle and pantethine-treated Tg mice (data not shown).

A similar tendency was confirmed when we analyzed the genes modulated in Tg mice after pantethine treatment in relation to all genes differentially expressed in Tg compared to WT (Fig. 4). Among the 154 genes down-regulated in Tg, 80% (124 genes) were up-regulated by pantethine in Tg while none were further repressed. On the other hand, out of the 362 genes whose expression was upregulated in Tg versus WT, only 43 genes (12%) showed a reduced expression upon pantethine treatment (Fig. 4). These observations suggested that pantethine was able to counteract the modulation of gene expression in 5XFAD mice and this inhibitory effect could account for the preservation by pantethine of physiological functions normally altered in these mice.

Inhibitory effect of pantethine on the expression of 5XFAD dysregulated genes in inflammation and related activities

At first, to investigate which processes could be targeted by pantethine, we focused our analysis on genes whose up-regulation was statistically significant in Tg compared to WT and that presented a lower expression in pantethine-treated Tg animals. A significant number of these genes (Table 1) were reported in our previous temporal gene profiling study [START_REF] Landel | Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer's disease[END_REF] and are known to be related to glial activation and neuroinflammation in an AD context. Figure 5 shows the relationships between these genes and related AD activities identified through a bibliographic search using PredictSearch software [START_REF] Landel | Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer's disease[END_REF][START_REF] Molino | Gene expression comparison reveals distinct basal expression of HOX members and differential TNF-induced response between brain-and spinal cord-derived microvascular endothelial cells[END_REF]. Indeed, most of these activities highlight the role of the selected genes in astroglial activation (Gfap, S100a6), microglial activation (Aif1/Iba1, CD68, Hexb2) and phagocytosis (Cd14, Trem2, Tyrobp, Csf1r, C1qa, C1qb, C1qc, C4a, and C4b).

Similarly to allograft inflammatory factor 1/Iba1 (AIF1), CD68 is considered a marker of phagocytic microglia. Cd63, encoding a marker of exosomes, and Lgals3 (galectin 3), were found to be co-expressed in 5XFAD mice in A plaque-associated microglia [START_REF] Yin | Immune hyperreactivity of Abeta plaque-associated microglia in Alzheimer's disease[END_REF], cells that are considered as primary contributors to neuroinflammation in AD pathology.

Furthermore, the reduced expression of Gfap, a marker of astroglial activation, suggested that the effects of pantethine were not restricted to microglia. Indeed, S100a6, that encodes a Ca 2+ /Zn 2+ binding protein, was reported to be overexpressed in astrocytes of AD patients and AD mouse models [START_REF] Boom | Astrocytic calcium/zinc binding protein S100A6 over expression in Alzheimer's disease and in PS1/APP transgenic mice models[END_REF]. In contrast to astrocytes of white matter where all S100A6 proteins were expressed homogeneously, almost all S100A6 immunoreactive cells within the gray matter were concentrated in astrocytes surrounding the A deposits.

Genes encoding TLR2, TLR7, and CD14 were upregulated in the cortex of distinct transgenic murine models of various neuroinflammatory diseases, including AD [START_REF] Letiembre | Screening of innate immune receptors in neurodegenerative diseases: a similar pattern[END_REF]. It must be noted that the TLR-mediated microglial response has both beneficial roles in stimulating phagocytosis as well as detrimental roles in the Aβ-stimulated release of neurotoxic products [START_REF] Landreth | Toll-like receptors in Alzheimer's disease[END_REF]. TLR2 is a known primary receptor for A peptide to trigger neuroinflammation [START_REF] Hoffmann | TLR2 mediates neuroinflammation and neuronal damage[END_REF][START_REF] Liu | TLR2 is a primary receptor for Alzheimer's amyloid beta peptide to trigger neuroinflammatory activation[END_REF].

Its co-receptor, CD14, is a critical regulator of the microglial inflammatory response that modulates A deposition [START_REF] Reed-Geaghan | Deletion of CD14 attenuates Alzheimer's disease pathology by influencing the brain's inflammatory milieu[END_REF].

In addition to other inflammatory markers, complement activation products are known to be markedly upregulated in AD brains in response to soluble and insoluble amyloid peptides [START_REF] Yasojima | Up-regulated production and activation of the complement system in Alzheimer's disease brain[END_REF][START_REF] Kolev | Implication of complement system and its regulators in Alzheimer's disease[END_REF]. Their activation, detected at the early stages of amyloid deposition, coincides with the clinical expression of AD [START_REF] Akiyama | Inflammation and Alzheimer's disease[END_REF][START_REF] Bradt | Complement-dependent proinflammatory properties of the Alzheimer's disease beta-peptide[END_REF]. The encoded products are components of both the alternative phagocytic pathway (AP), triggered by bacterial membranes, and the classical phagocytic pathway (CP), initiated when C1Q binds to immune complexes [START_REF] Kolev | Implication of complement system and its regulators in Alzheimer's disease[END_REF][START_REF] Gasque | Complement expression in human brain. Biosynthesis of terminal pathway components and regulators in human glial cells and cell lines[END_REF][START_REF] Morgan | Extrahepatic complement biosynthesis: where, when and why? Clinical and experimental immunology[END_REF]. The expression of these components in transgenic mice might also be considered as part of a protective response. For instance, C1Q was shown to protect cultured primary neurons against A [START_REF] Pisalyaput | Complement component C1q inhibits beta-amyloid-and serum amyloid P-induced neurotoxicity via caspase-and calpain-independent mechanisms[END_REF], and C3 deficiency in AD transgenic mice was reported to elicit an acceleration of plaque deposition and neurodegeneration compared to WT [START_REF] Maier | Complement C3 deficiency leads to accelerated amyloid beta plaque deposition and neurodegeneration and modulation of the microglia/macrophage phenotype in amyloid precursor protein transgenic mice[END_REF].

Other genes revealed by our analyses such as Csf1r, Trem2 and Tyrobp (Table 2) have been reported to be involved in phagocytosis (Fig. 5). In addition to the classical phagocytic receptors FCGR3 and CD68, CSF1R (colony stimulating factor 1 receptor) as well as CD14, contribute to A phagocytosis [START_REF] Liu | LPS receptor (CD14): a receptor for phagocytosis of Alzheimer's amyloid peptide[END_REF][START_REF] Smith | M-CSF increases proliferation and phagocytosis while modulating receptor and transcription factor expression in adult human microglia[END_REF]. In a recent report [START_REF] Sosna | Early long-term administration of the CSF1R inhibitor PLX3397 ablates microglia and reduces accumulation of intraneuronal amyloid, neuritic plaque deposition and pre-fibrillar oligomers in 5XFAD mouse model of Alzheimer's disease[END_REF], the role of CSF1R was determined as being crucial for the accumulation of intraneuronal amyloid peptides and the consequent formation of neuritic plaques. Here, we confirmed that the expression of Csf1r was increased in Tg compared to WT and significantly decreased upon pantethine treatment (Table 2). Furthermore, pantethine reduced the expression of Trem2, which presented the highest fold change (10-fold) within the set of Tg overexpressed genes (Table 1), along with the gene encoding its interactor, Tyrobp. Trem2 is highly expressed in microglia and the Trem2/Tyrobp pathway activates phagocytosis of apoptotic neurons as well as A in cultured cells [START_REF] Lue | What happens to microglial TREM2 in Alzheimer's disease: Immunoregulatory turned into immunopathogenic?[END_REF]. While this pathway inhibits TLR-induced inflammation [START_REF] Painter | TREM2 in CNS homeostasis and neurodegenerative disease[END_REF], it was reported that, in a mouse model of AD, Trem2 deficiency reduced pro-inflammatory cytokines (IL-1 and IL-6) as well as astrogliosis [START_REF] Jay | TREM2 deficiency eliminates TREM2+ inflammatory macrophages and ameliorates pathology in Alzheimer's disease mouse models[END_REF]. According to this observation, in AD mice, the reduced expression of Trem2 in response to pantethine at a particular inflammatory stage might contribute to hamper the production of cytokines in Trem2 expressing cells.

AIF1 binds to and polymerizes F-actin, and regulates RAC1 activity [START_REF] Autieri | AIF-1 is an actin-polymerizing and Rac1activating protein that promotes vascular smooth muscle cell migration[END_REF]. RAC1 participates in the activation of the NADPH oxidase (NOX) complex leading to production of superoxide (Fig. 5). Among the genes encoding the different subunits of this complex, only the overexpression of Cyba in Tg (cytochrome b-245 alpha gene/p22phox) was drastically reduced in pantethine-treated Tg as shown in Table 1. CYBA is considered as a critical component of the membrane-bound oxidase of phagocytes that generates superoxide. A is known to act on NOX in both hippocampal microglial and astrocyte cells to produce neurotoxic superoxide [START_REF] Utomo | Vav proteins in neutrophils are required for FcgammaR-mediated signaling to Rac GTPases and nicotinamide adenine dinucleotide phosphate oxidase component p40(phox)[END_REF][START_REF] Shimohama | Activation of NADPH oxidase in Alzheimer's disease brains[END_REF], through the interaction of the NOX complex with phosphatidylinositol-3-phosphate (PI3P). Production of PI3P requires activation of RAC2, a member of the Ras superfamily of small guanosine triphosphate (GTP)-metabolizing proteins, VAV (vav guanine exchange factor) and FCG receptor ligation [START_REF] Niedergang | Signaling and membrane dynamics during phagocytosis: many roads lead to the phagos(R)ome. Current opinion in cell biology[END_REF]. Among these factors, Tg overexpression of Vav1 was indeed lowered by pantethine (Table 1). Thus, the inhibitory effect of pantethine on the up-regulation of Cyba, and Vav1 genes in transgenic mice supported a reduced activity of the NOX complex and therefore an ongoing decreased production of superoxide.

On the other hand, we found that some of the genes up-regulated in Tg and lowered by pantethine (Table 1) are known to be involved in neuroprotection, such as Lyz and Hexb2

(Fig. 5). For instance, rescuing Hexb expression in Hexb -/-mice, which was detected exclusively in microglia [START_REF] Hickman | The microglial sensome revealed by direct RNA sequencing[END_REF], reduces neuroinflammation and can attenuate behavioral deficits [START_REF] Kyrkanides | The trigeminal retrograde transfer pathway in the treatment of neurodegeneration[END_REF].

As previously reported [START_REF] Landel | Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer's disease[END_REF], we also observed an increased expression of Grn (granulin) in Tg that was reduced by pantethine (Table 1). GRNs are a family of secreted peptides generated by proteolytic release from progranulin. Interestingly, mutations in the Grn gene cause frontotemporal lobar degeneration and may contribute to AD [START_REF] Ghidoni | Circulating progranulin as a biomarker for neurodegenerative diseases[END_REF].

Regarding genes of the TGF family, such as Tgfb1 and Tgfbr2, studies in transgenic animal models suggest that TGF-β might primarily contribute to AD pathogenesis by influencing Aβ production and deposition, resulting in brain microvasculature damage [START_REF] Masliah | Functional role of TGF beta in Alzheimer's disease microvascular injury: lessons from transgenic mice[END_REF]. Moreover, TGFβ was shown to act in synergy with IL-6 to turn on the expression of Gfap [START_REF] Kasten-Jolly | Central nervous system cytokine gene expression: modulation by lead[END_REF]. In contrast, several studies report evidence for neuroprotective features of TGF-1 [START_REF] Caraci | Dysfunction of TGF-beta1 signaling in Alzheimer's disease: perspectives for neuroprotection[END_REF][START_REF] Huang | TGF-beta1 blockade of microglial chemotaxis toward Abeta aggregates involves SMAD signaling and down-regulation of CCL5[END_REF]. Indeed, it was reported that reduced TGF- signaling leads to neurodegeneration and results in increased levels of secreted A [START_REF] Tesseur | Deficiency in neuronal TGF-beta signaling promotes neurodegeneration and Alzheimer's pathology[END_REF].

Overall, these data confirmed an inhibitory effect of pantethine on genes involved in inflammation. Thus, reducing inflammation might in turn abrogate its downstream induced activities including NO production, phagocytosis and as a consequence the need of neuroprotection. However, we cannot exclude that pantethine acts further upstream by interfering with A production, which, in the long term, will tune down inflammatory processes. Of note, despite the overexpression of inflammatory markers in the 5XFAD mice, no changes were observed in our arrays for the expression of pro-inflammatory genes such as

Il-1 or Tnf-. To validate the transcriptional data, RT-qPCR analyses were performed on genes differentially expressed in Tg when compared to WT and the effect of pantethine was evaluated. As shown in Fig. 6A, expression of Gfap, Aif1 and Tlr2 was increased in Tg compared to WT and this increase was significantly reduced by pantethine treatment in Tg but not in WT. Furthermore, while the absence of modulated expression was confirmed for Tnf- (Fig. 6A), we observed a significantly higher expression of Il-1 in Tg when compared to WT that was decreased by pantethine (Fig. 6B). Such a change in Il-1 expression was also confirmed at the protein level (Fig. 6C) with a two-fold increase in Tg compared to WT, an increase that was completely abrogated by pantethine.

Pantethine reduces the expression of underexpressed genes in 5XFAD mice involved in neurotransmission and A production

For this analysis, we only considered genes filtered for a threshold ≤0.6 in Tg compared to WT animals (Table 2) that were underexpressed in our previous transcriptomic study on hippocampi of either 6-or 9-month-old 5XFAD mice [START_REF] Landel | Temporal gene profiling of the 5XFAD transgenic mouse model highlights the importance of microglial activation in Alzheimer's disease[END_REF]. Decreased expression of Syn2 (synapsin 2) was indeed observed at the earliest clinically detectable stage of AD ( 73) and significantly down-regulated in the hippocampus of the 3xTg-AD mice compared to the WT mice [START_REF] Nie | Ginsenoside Rg1 Ameliorates Behavioral Abnormalities and Modulates the Hippocampal Proteomic Change in Triple Transgenic Mice of Alzheimer's Disease. Oxidative medicine and cellular longevity[END_REF]. It encodes a neuron-specific phosphoprotein involved in the regulation of neurotransmitter release and synaptogenesis (Fig. 7) that binds to small synaptic vesicles in the presynaptic nerve terminal [START_REF] Wang | alpha-synuclein multimers cluster synaptic vesicles and attenuate recycling[END_REF]. Noteworthy, as pantethine, ginsenoside Rg1, a drug shown to improve behavioral abnormalities in AD, restores its expression in the hippocampus of 3xTg-AD mice [START_REF] Nie | Ginsenoside Rg1 Ameliorates Behavioral Abnormalities and Modulates the Hippocampal Proteomic Change in Triple Transgenic Mice of Alzheimer's Disease. Oxidative medicine and cellular longevity[END_REF].

Reduced Gria2 (glutamate ionotropic receptor AMPA type subunit 2) expression was observed also at 12 months in the hippocampus in 3xTg-AD mice [START_REF] Saura | Gene expression parallels synaptic excitability and plasticity changes in Alzheimer's disease[END_REF]. Its encoded product belongs to a family of glutamate receptors, the main excitatory neurotransmitter at many synapses in the CNS, and functions as ligand-activated cation channels. It was found that

Gria2 is subjected to RNA editing, a process shown to be essential for brain function [START_REF] Satterlee | Novel RNA modifications in the nervous system: form and function[END_REF], and defective Gria2 editing was observed in the hippocampus of AD patients [START_REF] Gaisler-Salomon | Hippocampus-specific deficiency in RNA editing of GluA2 in Alzheimer's disease[END_REF].

Several other selected genes encoded proteins involved in neurotransmission (Fig. 7). DGKG, a member of the diacylglycerol kinase family belongs to a class of enzymes that catalyze the ATP-dependent conversion of diacylglycerol (DAG1) to phosphatidic acid (PtdOH). It is well documented that the coordinated regulation of these two lipid second messengers is particularly important in the nervous system where it modulates a variety of neurological functions. Indeed, several reports support roles for these enzymes in neuronal spine density, myelination, synaptic activity, neuronal plasticity, epileptogenesis and neurotransmitter release (reviewed in [START_REF] Tu-Sekine | Regulation and roles of neuronal diacylglycerol kinases: a lipid perspective[END_REF]). Kcnma1 (potassium calcium-activated channel subfamily M alpha 1) encodes the pore-forming alpha subunit of the MaxiK channels that play a role in neuronal excitability [START_REF] Hu | Presynaptic Ca2+activated K+ channels in glutamatergic hippocampal terminals and their role in spike repolarization and regulation of transmitter release[END_REF]. NRXN1 (neurexin 1) is a single-pass type 1 membrane protein that binds neuroligins to form a Ca 2+ -dependent complex at synapses in the CNS required for neurotransmission [START_REF] Paatero | Crystal Structure of an Engineered LRRTM2 Synaptic Adhesion Molecule and a Model for Neurexin Binding[END_REF]. Heterozygous mutations in NRXN1 were found to impair synaptic functions in human neurons [START_REF] Pak | Human Neuropsychiatric Disease Modeling using Conditional Deletion Reveals Synaptic Transmission Defects Caused by Heterozygous Mutations in NRXN1[END_REF]. Calcium voltage-gated channels such as CACNA2D1 have an important role in neurotransmission and genomic aberrations of its gene were found in patients with epilepsy and intellectual disability [START_REF] Vergult | Genomic aberrations of the CACNA2D1 gene in three patients with epilepsy and intellectual disability[END_REF].

In addition, pantethine up-regulation of several genes suggested a possible inhibitory effect of pantethine on APP processing and A production (Table 3, Fig. 7), with the exception of Grm5 (metabotropic glutamate receptor 5). Indeed, genetic deletion of this gene inhibits A production of oligomers and amyloid plaques, thus reducing cognitive impairment and pathogenesis in APP/PS1E9, another AD mice model [START_REF] Hamilton | Metabotropic glutamate receptor 5 knockout reduces cognitive impairment and pathogenesis in a mouse model of Alzheimer's disease[END_REF]. In contrast, restored expression of Apba2, Appbp2 and Gpd2 should decrease A toxicity. Apba2 (amyloid beta precursor protein family A member 2) encodes a neuronal adapter protein interacting with APP. This interaction stabilizes APP and inhibits production of proteolytic APP fragments including A [START_REF] Sakuma | Phosphorylation of the amino-terminal region of X11L regulates its interaction with APP[END_REF][START_REF] Swistowski | Novel mediators of amyloid precursor protein signaling[END_REF][START_REF] Tomita | Interaction of a neuron-specific protein containing PDZ domains with Alzheimer's amyloid precursor protein[END_REF][START_REF] Lee | The neuronal adaptor protein X11beta reduces amyloid beta-protein levels and amyloid plaque formation in the brains of transgenic mice[END_REF][START_REF] Araki | Novel cadherin-related membrane proteins, Alcadeins, enhance the X11-like protein-mediated stabilization of amyloid beta-protein precursor metabolism[END_REF]. Furthermore, it was shown that APBA2-mediated reduction in cerebral A was associated with normalization of both cognition and in vivo long-term potentiation in an AD transgenic mice model [START_REF] Mitchell | X11beta rescues memory and long-term potentiation deficits in Alzheimer's disease APPswe Tg2576 mice[END_REF]. The protein encoded by Appbp2 (Amyloid beta precursor protein binding protein 2) interacts with microtubules and is functionally associated with APP transport and/or processing [START_REF] Senol | PAT1 inversely regulates the surface Amyloid Precursor Protein level in mouse primary neurons[END_REF].

On the other hand, the restored expression of Gpd2, glycerol-3-phosphate dehydrogenase 2, can be related to the effect of pantethine on glycolysis since GPD2 is involved in maintaining a high rate of this process [START_REF] Chowdhury | High activity of mitochondrial glycerophosphate dehydrogenase and glycerophosphate-dependent ROS production in prostate cancer cell lines[END_REF]. Thus, restoring glycolysis, whose impairment was shown to promote an increase in A aggregation and internalization [START_REF] Fu | Bioenergetic mechanisms in astrocytes may contribute to amyloid plaque deposition and toxicity[END_REF], might be part of the beneficial effect of pantethine. Altogether, these data supported that pantethine maintained the constitutive expression of genes underexpressed in Tg to preserve proper CNS functions.

Discussion

Although the clinical impact of pantethine remains to be proven, we presented here some lines of evidence in favor of a beneficial effect of pantethine in AD. Our behavioral test suggested that pantethine treatment reduced aggressiveness (Fig. 2). Such an effect was supported by the decrease of astrogliosis and microgliosis as well as a reduction of the number of amyloid plaques (Fig. 1). While it is well admitted that AD starts with A deposition, the precise process that leads to neurodegeneration, is still unknown. A aggregation triggers neuroinflammation that is accompanied with an increase of ROS production resulting in an oxidative stress. Despite the ability of inflammation to increase phagocytosis for A clearance, it is thought that its chronicity can impact negatively the protective activities, and restoration of CNS functions cannot be anymore achieved.

The inhibition of pantethine on Il-1 expression seen in primary cultures of astrocytes upon treatment with A oligomers [START_REF] Van Gijsel-Bonnello | Metabolic changes and inflammation in cultured astrocytes from the 5xFAD mouse model of Alzheimer's disease: Alleviation by pantethine[END_REF] was confirmed in vivo in the present work at both mRNA and protein levels (Fig. 6B andC). Such a decrease might suggest a direct effect of the drug on the signaling pathway leading to Il-1 expression. For instance, the nuclear factor kappalight-chain-enhancer of activated B cells (NF-B) is one of the factors involved in the transcriptional induction of cytokines such as Il-1 and IL-1-induced inflammatory genes [START_REF] Warner | MyD88: a critical adaptor protein in innate immunity signal transduction[END_REF]. Reduced activity of NF-B by blocking IL-1 signaling was described to alleviate cognitive deficits, attenuate Tau pathology, and partly decrease certain fibrillar and oligomeric forms of A [START_REF] Kitazawa | Blocking IL-1 signaling rescues cognition, attenuates tau pathology, and restores neuronal beta-catenin pathway function in an Alzheimer's disease model[END_REF]. At least part of the mechanism leading to Il-1 expression requires the translocation of NF-B to the nucleus that can be achieved after binding of A 42 to TLR2. Although the mechanism of action of pantethine is not yet elucidated, it was shown that pantethine alters lipid composition and cholesterol content of membrane rafts [START_REF] Van Gijsel-Bonnello | Pantethine Alters Lipid Composition and Cholesterol Content of Membrane Rafts, With Down-Regulation of CXCL12-Induced T Cell Migration[END_REF]. Such an alteration with methyl--cyclodextrin was reported to decrease the levels of nuclear translocation of NF-B p65, phosphorylated ERK1/2 and TLR4 [START_REF] Pan | Metformin reduces morphine tolerance by inhibiting microglial-mediated neuroinflammation[END_REF][START_REF] Pan | Lipid Rafts Promote trans Fatty Acid-Induced Inflammation in Human Umbilical Vein Endothelial Cells[END_REF]. It is therefore possible that through its effects on lipid rafts as prime subcellular regions where amyloidogenic APP processing takes place (99, 100), pantethine affects NF-B activation, thus preventing the transcription of inflammatory genes. However, on one hand, neuroinflammation and IL-1 contribute to neurodegeneration, a hallmark of AD pathology, on the other hand, IL-1 overexpression in a transgenic AD model was reported to trigger beneficial effects (101). Indeed, crossing the APP/PS1 AD mouse model with transgenic mice that specifically overexpress Il-1 (APP/PS1-IL-1 in the hippocampus led to a reduction of amyloid pathology (101). It was suggested that reduced amyloid plaques and therefore the beneficial effect of IL-1 resulted from the enhancement of microglial phagocytosis.

Recently, mapping the immune cell subsets in AD onset and progression (102) led to the identification of a potential microglia protective-type associated with neurodegenerative diseases (DAM). This subset of cells, which was also detected in post-mortem human brain, is characterized by higher expression levels of genes involved in phagocytic and lipid metabolism pathways. This DAM activation requires a mechanistically coupled temporal event that is initiated in a TREM2-independent pathway, followed by activation of the TREM2-dependent program. As suggested in APP/PS1-IL-1 transgenic mice, a beneficial role of this DAM population was related to its phagocytic activity that should trigger A clearance. However, despite a reduction of plaques, it was shown that neuronal loss induced by low concentration of A (not leading to neuronal apoptosis) was mediated by primary phagocytosis of neurons by microglia (103). These observations should provide an explanation for the failure of therapeutic assays based on A vaccination that did not alleviate cognitive deficits (104). We can hypothesize that pantethine through its anti-inflammatory effect might hamper the phagocytic activity (illustrated by the reduced expression of Tyrobp and Trem2) to a level where healthy neurons are preserved, while formation of amyloid plaques is reduced.

Although pantethine treatment may be preventive, according to the lipid raft hypothesis, we cannot exclude that it acts only in an AD context. Preliminary results (data not shown) on 4month-old mice failed to detect any change following pantethine treatment in the expression of genes that were modulated in Tg compared to WT. At this time point, neither Aif1, Gfap, Cd68, Cd14, Lyz, Cyba, Grn, S100a6, Tyrobp, Trem2 nor genes encoding complement factors, whose expression was increased in Tg, were affected by pantethine. Moreover, selecting the genes modulated by pantethine in WT did not lead to the identification of processes that can be supported by a significant number of genes.

In addition, another feature that could help understand the beneficial effect of pantethine is related to its role in the production of CoA. Inborn errors of CoA biosynthesis have been implicated in neurodegeneration with brain iron accumulation (NBIA), a group of rare neurodegenerative disorders, suggesting pantethine as suitable treatment for affected individuals (105). Moreover, glucose-derived pyruvate is a principal source of acetyl-CoA in all brain cells through pyruvate dehydrogenase complex (PDHC) activity. Pantethine could therefore compensate the decrease of PDHC activity observed in AD (106) that is responsible for cholinergic deficits and loss of cognitive functions.

On the other hand, it is possible that the effects observed in the 5XFAD result from pantethine metabolites. Pantethine can be converted to its monomeric form (pantetheine), which is metabolized by pantetheinase to generate pantothenic acid (vitamin B5) and cysteamine, known to have direct anti-oxidant effects (107-109). Cysteamine can cross the blood brain barrier (110) and was shown to induce the release of BDNF (brain derived-neurotrophic factor), inhibiting neuronal loss in Huntington's disease (111). Cysteamine is highly reactive and its oxidation leads to form cystamine. It has been shown that cystamine can activate the activity of NRF2, a crucial transcriptional factor involved in the anti-oxidant response both in cell cultures and in brain tissue (112). Interestingly, although cystamine inhibits the glutamyl synthase that consequently blocks ROS scavenging, it reduces transglutaminase activity known to be associated with protein aggregates in AD brain and to trigger A oligomerization and aggregation (113). Noteworthy, no side effects of pantethine were reported (114, 115). Thus, in case of clear clinical benefits of pantethine treatment, determining whether pantethine itself or one of its metabolites is responsible should not be a barrier to its use.

Conclusions

The present study demonstrated the efficiency of pantethine to reverse AD features such as astrogliosis, microgliosis, A deposition and to AD-associated aggressive behavior. These changes were accompanied by the reversion of the overexpression of several genes in Tg mice related to inflammation/phagocytosis (summarized in figure 8). Since neuroinflammation and phagocytosis are crucial events in the course of AD, the various effects of pantethine or one of its metabolites (cysteamine, cystamine, pantothenic acid) on these processes are of importance. Blocking the signaling pathways that lead to either A deposition, inflammation or oxidative stress or the vicious cycle that amplifies these alterations will have several downstream consequences that altogether can alleviate AD pathology. It will decrease: i) APP processing that will in turn reduce A deposition; ii) the NF-B signaling pathway involved in the transcriptional activation of inflammatory genes and in particular those involved in phagocytosis such as TREM2. Ultimately, the reduced expression of these genes will impact the generation of the highly phagocytic DAM population and consequently might protect healthy neurons from phagocytosis and therefore neuronal loss.

Investigating more deeply the mechanism that sustain the effects of pantethine in 5XFAD mice overtime should determine whether pantethine treatment is preventive or curative. In addition to test depicting mood changes (i.e., aggressiveness), cognitive evaluations will have to be performed in order to provide further confirmation of the beneficial therapeutic effect of pantethine treatment. Based on our results, pantethine, provided routinely as a dietary supplement, could be considered as a serious therapeutic option for preventing, slowing or halting AD progression.

Declarations

Ethics approval and consent to participate

All experimental procedures were approved by the Ethics Committee of the Medical Faculty of Marseille and were carried out in accordance with the guidelines published in the European Communities Council Directive of November 24, 1986 (86/609/EEC). All efforts were made to reduce animal suffering and the number of mice.

Figures, tables, legends

Figure 1 :

 1 Figure 1: Effects of pantethine on astroglia and microglia reactivity in WT and Tg mice. A) Representative epifluorescence microphotographs showing changes in astroglia and microglia reactivity with GFAP and Iba1 immunostaining, respectively, and amyloid plaques (anti-A 6E10 immunostaining) in the hippocampus (Hc) of untreated or pantethine-treated WT and Tg animals, counterstained with nuclear marker Hoechst (blue). Scale bar 100 M. Quantification of changes (B) in GFAP, IBA1 immunostaining and (C) number of A plaques measured as the immunostained area/total area of the hippocampus. Values are the mean ± SEM of three brains (3 slices/brain) per group. *p<0.05; **p<0.01; ***p<0.005 when comparing WT+P, Tg and Tg+P groups vs WT, ANOVA followed by post hoc Fisher LSD test for (B) and Student t test for (C).

Figure 2 :

 2 Figure 2: Effects of pantethine on the aggressive behavior of 5XFAD mice. Quantification of the number of attacks (A) and latency to attacks (B) of untreated (Tg, black circles) and pantethine-treated (Tg+P, black squares) Tg mice towards WT male mice intruders. Values of ten animals are represented as median with interquartile range (done in triplicate with a different intruder as described in Materials and Methods). *p<0.05, Mann-Whitney test.

Figure 3 :

 3 Figure 3: Effects of pantethine on genes differentially expressed in Tg and WT mice. Expression values were filtered for p values <0.05 as provided by Illumina technology. Fold changes were deduced from the ratios (Tg+P/Tg, Tg/WT, WT+P/WT) between the four different groups and means were calculated. Selecting a fold change ≤0.6 leads to 47 genes repressed by pantethine (Gene set 1 in left panel). A similar number of the top pantethineinduced genes (Gene set 2 in right panel) selected for a fold change ≥1.5 is displayed. All the repressed or induced fold changes values are colored in white or in dark gray, respectively while unmodulated genes are indicated in light gray.

Figure 4 :

 4 Figure 4: Gene distribution in untreated and treated Tg mice. Genes were numbered and selected for a fold change ≥1.5 (up-regulated genes) and for a fold change ≤0.6 (downregulated genes) indicated by an up and a down arrow, respectively. Only genes with expression values exhibiting p values <0.05 as provided by Illumina technology were considered.

Figure 5 :

 5 Figure 5: Schematic representation of functional networks involving genes whose overexpression in Tg was repressed by pantethine.Table of legends is provided (at the top

  For the aggressiveness analysis we used a non-parametric Mann-Whitney test and results are represented as median and interquartile range. Only p values less than 0.05 were considered significant. The p levels p<0.05, p<0.01 and p<0.001 are denoted with asterisks *, ** and

the data were analyzed with Kaleida Graph or Prism softwares and the values represent the means +/-SEM of the indicated number of independent experiments/animals. ANOVA followed by a Fisher's LSD post-hoc test was used for multiple comparisons. ***, respectively.

Table 1 : Pantethine reverses the overexpression of genes involved in neuroinflammation in Tg.

 1 Table of legends is provided (at the top right of the figure). Modulated genes are within a gray box and reduced expression by pantethine is indicated by P-in superscript. Values represent the ratio of the indicated comparison between groups (three animals per group and normalized to WT). p*<0.05; **p<0.01 and ***p<0.005. ANOVA followed by post hoc Fisher LSD test.

	Genes	Tg/WT	Tg+P/Tg	Tg+P/WT	WT+P/WT
	Aif1	2.5***	0.6***	1.5	1
	Cd68	4.5**	0.5**	2.1	0.9
	Tlr2	2.7**	0.6**	1.6	1
	Tlr7	1.9**	0.7*	1.3	1
	Cd14	2.2***	0.7**	1.4	0.9
	Gfap	5.1**	0.5**	2.6*	1.3
	S100a6	2.8**	0.5**	1.3	1
	Lyz	10**	0.4**	3.7	0.9
	Hexb	3.1**	0.6*	1.9	1.1
	Grn	2.7***	0.6**	1.5	1
	Tgfb1	3**	0.6*	1.5	1
	Cd63	1.8*	0.5**	0.9	1.1
	Lgals3	1.9*	0.6*	1	1

Table 2 : Pantethine reverses the overexpression of genes involved in phagocytosis in Tg.

 2 Values represent the ratio of the indicated comparison between groups (three animals per group and normalized to WT). p*<0.05; **p<0.01 and ***p<0.005. ANOVA followed by post hoc Fisher LSD test.

	Genes	Tg/WT	Tg+P/Tg	Tg+P/WT	WT+P/WT

Acknowledgements

The authors thank Dr. Véréna Landel for critical reading of the manuscript and Dr. Marek Gierlinski for his help with statistical analysis.

Funding

This work was supported by funding from CNRS and Aix-Marseille Université; Agence Nationale pour la Recherche (MK, #ANR-11-MALZ-0007 and SR, #ANR-15-CE16-0006); Fonds Européens de Développement Régional (FEDER in PACA, MK and SR, #3013-33278); DHUNE project supported by A*MIDEX (MK and SR, #ANR-11-IDEX-0001-02) and Vaincre l'Alzheimer (MK, #13745). KB was granted a research associate fellowship (Management of Talents) by A*MIDEX and the "Fondation Plan Alzheimer". MvGB was recipient of a private doctoral fellowship.

Availability of data and materials

The microarray datasets generated in the present study are available in the Array Express database [www.ebi.ac.uk/arrayexpress] under accession number E-MTAB-6772.

Competing interests

The authors declare no financial conflict of interest that might be construed to influence the results or interpretation of the manuscript.

Author contributions


Marie-Odile Soyer-Gobillard

Laura Gaspari

Philippe Courtet

Mauricette Puillandre

Françoise Paris

Charles Sultan

Neurodevelopmental disorders in children exposed in utero to synthetic progestins: Analysis from the national cohort of the Hhorages Association*

Keywords: Adolescence, adulthood, prenatal progestin exposure, psychiatric disorders

The medical and scientific communities have not yet fully acknowledged the undesirable effects of the synthetic hormones that have been administered to pregnant women for decades. The somatic effects of in utero exposure to diethylstilbestrol (DES), such as genital malformations, infertility and cancer, have long been recognized but this has not been the case concerning psychiatric disorders. The progestins used in contraception and hormone replacement therapy are known to affect the adult brain, but no data exist on their effects due to in utero exposure of children. The Hhorages Association, a national patient support group, has assembled a cohort of 1,200 women who took synthetic hormones during pregnancy. These women had a combined 1,934 children. We obtained full questionnaire responses from 46 women treated with progestins onlyand not an oestrogenic cocktailwho gave birth to 115 children. Three groups were observed: Group 1 (n=18): firstborn unexposed children, Group 2 (n=62): children exposed in utero to synthetic progestins, and Group 3 (n=35): children born after a previous 2 pregnancy treated with progestins. No psychiatric disorders were reported in Group 1 and the incidence of psychiatric disorders was drastically elevated in Group 2. Our work shows a striking increase in psychiatric disorders among children exposed in utero to progestins and strongly suggests that prenatal exposure is associated with a high risk of psychiatric disorders in adolescence and adulthood, whether accompanied or not by disorders of sex development.

Introduction

Neuroactive steroids like progesterone play an important role in shaping the CNS structure and function (neurodevelopment, neurogenesis and cognition) throughout the lifespan.

Progesterone exerts powerful effects on the brain, such as regulation of neurogenesis and astroglial and synaptic plasticity. Moreover, it regulates the development of neuronal types like Purkinje cells and oligodendrocytes, as well as the myelinisation process. Progesterone exerts a significant influence on the activity of several neurotransmitters involved to the pathophysiology of psychosis, including the dopaminergic, glutamatergic and GABAergic systems. In addition to progesterone itself, it can be converted to dehydroprogesterone and then allopregnanolone (or iso-pregnanolone) [START_REF] Schumacher | Revisiting the role of progesterone and allopregnanolone in the nervous system : resurgence of the progesterone receptors[END_REF], potent ligands of the GABA-A receptor.

Progesterone elicits its effects by gene transcription, mediated by nuclear progesterone receptors, as well as by non-genomic mechanisms through the activation of a signal transduction pathway. Preclinical studies have provided hints that neurosteroids might be involved in the pathophysiology of psychosis.

Severe psychiatric disorders were recently documented in children exposed in utero to synthetic oestrogens like diethylstilbestrol (Distilbene®, DES) and ethinyl oestradiol (EE) [START_REF] Mo | Perturbateurs endocriniens et troubles du comportement : non, nous n'avons pas encore tiré toutes les leçons de l'histoire du DES[END_REF][START_REF] Soyer-Gobillard | Behavioral and somatic disorders in children exposed in utero to synthetic hormones: a testimony case study in a french family troop[END_REF][START_REF] Soyer-Gobillard | Association between fetal DES-exposure and psychiatric disorders in adolescence/adulthood: evidence from a French cohort of 1002 prenatally exposed children[END_REF]. The actions of these oestrogens involve epigenetic mechanisms [START_REF] Harlid | In utero exposure to diethylstilbestrol and blood DNA methylation in women ages 40-59 years from the sister study[END_REF] and especially differential specific methylations, which were found on the genes ZPF57 and ADAMTS9 in young psychotic patients exposed in utero to DES [START_REF] Rivollier | Methylomic changes in individuals with psychosis, prenatally exposed to endocrine disrupting compounds: Lessons from diethylstilbestrol[END_REF]. In this recent work, the authors suggested that in exposed individuals, ZFP57 gene methylation may be associated with psychosis. The ADAMTS9 gene is implicated in the control of organ shape, especially the development and function of the uterus and reproductive organs [START_REF] Mittaz | Adamts-1 is essential for the development and function of the urogenital system[END_REF], which are often abnormal after in utero DES exposure, as well as in the control of CNS development [START_REF] Lemarchant | ADAMTS proteoglycanases in the physiological and pathological central nervous system[END_REF] and several kinds of cancer [START_REF] Zhang | High-resolution melting analysis of ADAMTS9 methylation levels in gastric, colorectal, and pancreatic cancers[END_REF]. Synthetic progestins, widely used for contraception or hormonal replacement therapy, are known to impact the adult brain [START_REF] Dub | Hormones sexuelles et troubles psychiques[END_REF][START_REF] Zhu | How Progestin, a synthetic female hormone could affect the brain[END_REF], but no data exist on the postnatal effects after in utero exposure. The aim of this work was to determine whether prenatal exposure to synthetic progestins is a risk factor for psychiatric disorders in adolescence or adulthood.

Material and Methods

Our clinical data were collected from 1,200 families (1,934 children) involved in the French Hhorages Association (Halt to Synthetic Hormones for Pregnancies), a patient-support group, using a detailed questionnaire drawn up by practitioners and researchers and described in previous papers [START_REF] Mo | Perturbateurs endocriniens et troubles du comportement : non, nous n'avons pas encore tiré toutes les leçons de l'histoire du DES[END_REF][START_REF] Soyer-Gobillard | Behavioral and somatic disorders in children exposed in utero to synthetic hormones: a testimony case study in a french family troop[END_REF]. We included questions on patient sex, rank among siblings, exposure during gestation, and somatic and psychiatric disorders of the descendants. Most families had children exposed to either oestrogens alone or cocktails of oestrogens and progestins, but only 46 families (115 children) had at least one child exposed to one or more progestins prescribed alone, without oestrogens. In the extensive questionnaires (Registration number to French National Commission of Computer Science and Freedom, CNIL n°1006460), we obtained the family history, including the mother's hormone treatment before and during pregnancy, and the medical records of the children's health problems, including diagnoses, prescriptions and treatments and/or hospitalizations. The psychiatric disorders reported in the questionnaires were classified as: schizophrenia, bipolar disorders (maniac-depressive psychosis), severe depression, behaviour disorders, aggressiveness, and eating disorders. The diagnoses had been made by experienced psychiatrists and the patients received appropriate drugs and psychotherapy with or without hospitalization in specialized institutions. When suicides or suicide attempts were reported, a second questionnaire validated by members of the Research Group on Suicide (CHU Lapeyronie Montpellier) was sent to the families.

From the questionnaires completed by the families and returned to the Hhorages Association, it appears that one or several progestins were prescribed and administered to pregnant mothers as shown in the numerous conserved medical prescriptions. The prescribed pharmaceuticals were: 17α-hydroxyprogesterone caproate (synthetic progestin, SP): 32 (Note: withdrawn in 2000 but re-authorized in 2011), 17α-hydroxyprogesterone heptanoate (SP): 10 (Note: withdrawn in 2002), chlormadinone acetate (SP derived from hydroxyprogesterone): 11 (Note: withdrawn on 1970), dydrogesterone (6-dehydroprogesterone, synthetic isomer of progesterone): 4 (Note: against total indication for pregnant women; not withdrawn), "natural progesterone" (derived from soybean, micronized): 4 (Note: not withdrawn), Norethisterone Base (SP): 1 (Note: against total indication for pregnant women; withdrawn in 1998). Total:

62.

Results

In the entire national Hhorages cohort of 1,934 children, we detected 46 families in which at least one child had been exposed to one or several synthetic progestins.

As shown in Table 1, among the 115 children born from these 46 mothers, we observed three well-differentiated groups: Group 1: 18 firstborn unexposed children (9 boys + 9 girls) free of disorders, Group 2: 62 children exposed to synthetic progestins (40 boys + 22 girls), and Group 3: 35 children (21 boys + 14 girls) born after a previous exposure, free of disorders except 1. Group 1 + Group 3 (18 + 35 = 53 children) served as intra-familial controls. Among the 62 children exposed to synthetic progestins (see the list of medications in Material and Methods), 49 presented psychiatric disorders (79.03%), whereas only 6 (4 boys + 2 girls) presented somatic disorders alone (9.67%) and 7 (5 boys + 2 girls) presented no disorder (11.29%).

In Group 2, among the 49 patients affected by psychiatric disorders, 10 suffered from both somatic and psychiatric disorders and 39 from psychiatric disorders alone. Among the psychiatric disorders, we noted: schizophrenia: 29 (25 boys+ 4 girls); severe depression, bipolar disorder: 16 (6 boys + 10 girls); and behaviour disorders, aggressiveness, eating disorders: 4 (0 boys + 4 girls) (Table 2). It should be noted that boys mostly suffered from schizophrenic psychosis (25 boys versus 4 girls), whereas girls mostly suffered from bipolar disorder (10 girls versus 6 boys). The questionnaires completed by the families and returned to the Hhorages Association revealed that one or several progestins were prescribed and administered to the pregnant mothers, with the most frequently prescribed drugs being hydroxyprogesterone caproate, heptanoate alone, or heptanoate in a cocktail with other progestins.

In Group 3, among the 35 post-exposed childrenthat is, born after a previous exposed pregnancyonly one presented a psychiatric disorder as severe depression.

In Group 2, composed of children exposed to progestins, schizophrenia (n=29), and bipolar disorder and severe depression (n=16) made up most of the psychiatric disorders, whereas eating disorders (n=2) and behaviour disorders (n=2) were less numerous. Seven series of suicide attempts (a series comprising between 2 and 15 suicide attempts per person) were also counted and one death, as shown in Table 3, which presents the psychiatric disorders along with their prevalence in the general population [START_REF] Nicoli | Ideation et conduites suicidaires en France: prévalence sur la vie et facteurs de risques dans l'étude ESEMeD[END_REF][START_REF] Lépine | Prévalence et comorbidité des troubles psychiatriques dans la population générale française: résultats de l'étude épidémiologique ESEMeD/MHEDEA 2000/ (ESEMeD)[END_REF].

Discussion

Although selection biases (family-patient association) may somewhat affect the impact of our data, the high prevalence of psychiatric disorders in the exposed subjects is quite striking.

It should be noted that the intra-familial controls (18 firstborn unexposed versus 62 exposed) presented no disease and among the 35 post-exposed (born after a previous treated pregnancy) only one psychiatric disorder was observed. Among the 115 children born from 46 families of the Hhorages cohort, 42 unexposed children had no psychiatric disorders, while of the 62 exposed children, only 7 were without psychiatric disorders (55 with a disorder). As shown in Table 3, our data demonstrate that the prevalence of psychiatric disorders in patients who were exposed in utero to progestins was significantly different from the prevalence of the same disorders in the general population: schizophrenia: 29 = 46.77% versus 1% in the general population, behaviour disorders: 2 = 3.22% versus 3% in the general population, bipolar disorder and severe depression: 16 = 25.80% versus 6.3% in the general population, eating disorders: 2 = 3.22% versus 1.6%, suicides or suicide attempts: 7 = 11.29% versus 0.3% in the general population, and death: 1 = 1.6% versus 0.02% in the general population. Seven in utero exposed patients (11.29%) presented no disorder.

We compared our data with the recent results (4) obtained from the Hhorages cohort in which we analysed 1002 patients who were exposed in utero to the synthetic oestrogens diethylstilbestrol and ethinyl oestradiol. Of the 720 patients exposed in utero, 250 boys and 353 girls showed severe psychiatric disorders: schizophrenia, bipolar disorder, severe depression, behaviour disorders, eating disorders, suicide attempt series (612 = 85%) and death (32 = 4.4%).

Psychiatric disorders were the same in the groups exposed to oestrogens or progestins, with minor differences: for boys, the number of cases of bipolar disorder seemed less high in patients exposed to progestins than in patients exposed to synthetic oestrogens, with behaviour disorders more prevalent in the latter category. In both categories, schizophrenia was the most prevalent disorder for boys. For girls, however, the proportions of psychiatric cases were nearly the same in the two categories. Bipolar disorder and severe depression were the most abundant disorders in the two categories.

Cumulative evidence from experimental, hormonal, genetic and epidemiological studies suggests a neurodevelopmental origin of most psychiatric disorders. Environmental disturbances are known to increase brain disorder risk, interacting with a genetic predisposition.

During foetal life, neuronal differentiation, migration and interaction are managed by molecular processes, regulating the dopaminergic, glutamatergic, and the GABAergic systems. While the GABA system is the major inhibitory neurotransmitter in postnatal life, GABA-A receptor activation is excitatory during foetal life. In addition to progesterone itself, progesterone is converted to dehydroprogesterone and then to allopregnanolone (or iso-pregnanolone), potent ligands of the GABA-A receptor. These neurosteroids are known to be involved in neuronal and glial development and plasticity, and the regulation of mood and affection. Any neurodevelopmental disruption of this system can induce a dysfunction of cell migration and synaptic integration and may have marked consequences leading to chronic disability.

The data regarding the impact of synthetic progestins on the developing brain are currently conflicting. In adults, progestins have been suggested to exert neuroprotective effects in several animal models of neurological disease [START_REF] Melcangi | Levels and actions of progesterone and its metabolites in the nervous system during physiological and pathological conditions[END_REF]. After two years of treatment with progestogen via a COC combination (Oral Contraceptive Combination), impaired social behaviour was observed in female rats [START_REF] Giatti | The other side of preogestins : effects in the brain[END_REF]. This combination was also able to alter GABA receptor exposure, increasing the exposure of the gamma2-subunit in the cerebral cortex and thus affecting anxiety behaviour. According to this group, progestin may alter brain function in animal models. In 2016, Willing and Wagner [START_REF] Willing | Exposure to the Synthetic Progestin, 17α-Hydroxyprogesterone caproate during development impairs cognitive flexibility in adulthood[END_REF] showed that exposure to synthetic progestins during development could impair brain function later in life. These authors noted that many regions of the developing brain are sensitive to progestins, including the mesocortical dopamine pathway, a neural circuit important in rats for complex cognitive behaviour later in life. They found that rats exposed to synthetic progestins during development expressed impaired cognitive flexibility with increased perseveration later in life. Although this data cannot be extrapolated to humans, these authors pointed out the risk of developmental behavioural effects of synthetics progestins prescribed for pregnant women.

Negative mood symptoms have been reported by Andreen et al. [START_REF] Andreen | Sex steroid induced negative mood may be explained by the paradoxical effect mediated by GABAA modulators[END_REF] in women as a result of progesterone during the luteal phase of the menstrual cycle or progestin in COC. The symptoms are believed to be mediated via the action of allopregnanolone on the GABA-A system. In male patients with early psychosis, Belvederi Murri et al. [START_REF] Murri | Neuroactive Steroids in First-Episode Psychosis: A Role for Progesterone?[END_REF] reported a lower level of progesterone and suggested that this hormone is involved in the pathophysiology of psychotic disorders. This hypothesis is supported by the antipsychotic-like effects observed in animal models of schizophrenia. Recently Slopien et al. [START_REF] Slopien | Correlation between allopregnanolone levels and depressive symptoms during late menopausal transition and early postmenopause[END_REF] reported a reduction in circulating allopregnanolone levels that correlates with depressive symptoms. Conversely, healthy women reported increased anxiety and mood disorders after long-acting subdermal implant of progestogens.

In 2011, Guest et al. [START_REF] Guest | Altered levels of circulating insulin and other neuroendocrine hormones associated with the onset of schizophrenia[END_REF] reported an elevated concentration of progesterone in a group of 236 first and recent onset of schizophrenia patients and suggested that steroid hormones influence brain function, underlying schizophrenia and major depressive disorders. Moreover, [START_REF] Buoli | Sex Steroids and Major Psychoses: Which Role for DHEA-S and Progesterone?[END_REF] found high DHAS levels in patients with a history of psychotic symptoms, suggesting a role of steroids in the aetiology of psychosis and mood disorders [START_REF] Buoli | Sex Steroids and Major Psychoses: Which Role for DHEA-S and Progesterone?[END_REF].

Lastly, citing the French National Agency of Medicine [START_REF] Ansm | Rapport d'expertise-Mirena, Jaydess[END_REF], the independent medical journal "Prescrire", reported that 2,714 adverse effects were signalled in the last year by women using Mirena®, an intrauterine device containing levonorgestrel, a well-known progestin [START_REF]Rédaction Prescrire. Dispositif intra-utérin au lévonorgestrel: un afflux de notifications en 2017 en France[END_REF].

In 85% of the cases, the side effects were mainly related to psychiatric disorders, such as anxiety and depression.

[UMO1]
Although a direct relationship cannot be formulated, a link between progestin treatment during foetal life and later psychiatric disorders in offspring should be considered. Since progestins are known to induce GABA receptor activity/neural activation before birth, it is likely that the GABAergic system contributes to schizophrenia, anxiety, depression, panic disorders, epilepsy, autism and other disorders [START_REF] Schmidt | Neurodevelopment, GABA System dysfunction and schizophrenia[END_REF]. Disruption of GABA signalling in early development alters cell migration and cortical architecture, which then may lead to chronic disability in postnatal life. Our results point out the postnatal consequences of synthetic progestin treatment during foetal life and highlight the potential impact of synthetic progestin.

They highlight the need for reevaluation of the potential outcome of progestin administration during gestation.

Tables

Table 1 . Classification of the three groups of Hhorages children born from 46 families in which at less one child per family has been exposed in utero to progestins. _____________________________________________________________________ 115 children Group 1 Group 2 Group 3

 1

	Firstborn unexposed	Exposed to synthetic progestins	Post-exposed
	(n=18)	(n=62)	n=35
		Psychiatric disorders
	0 disorder	n=49 (79.03%)	n=1
		0 disorder n=7 (11.29%)
		Somatic disorders only:
		n=6 (9.67%)
	___________________________________________________________________________________

Table 2 . Total number of psychiatric disorders among the 62 children exposed to progestins.

 2

	_________________________________________________________________________________
	Schizophrenia (25 boys, 4 girls)	29
	Maniac-depressive psychosis (bipolar disorder), severe depression (6 boys, 10 girls)	16
	Behaviour disorders, aggressiveness, eating disorders (0 boys, 4 girls)	4
	Somatic disorders only (4 boys, 2 girls)	6
	With no disorder (5 boys, 2 girls)	7
	Total number of exposed children	62
	___________________________________________________________________

Table 3 . Prevalence of psychiatric disorders and comparison with the general population.

 3

	___________________________________________________________________________
		Group 1	Group 2	Group 3
	_______________	_____________ ___________
	Firstborn unexposed	Progestin exposed	Post-exposure General population
		(n=18)	(n=62)	(n=35)
	___________________________________________________________________________
	Schizophrenia	(0%)	(n= 29) (46.77%)	(0%)	(1%)
	Behavioural disorders	(0%)	(n= 2) (3.22%)	(0%)	(3%)
	Bipolar disorder, depression	(0%)	(n= 16) (25.80%)	(n=1) (2.8%)	(6.3%)
	Eating disorders	(0%)	(n= 2) (3.22%)	(0%)	(1.6%)
	Suicides	(0%)
	Attempts (n=7)	(0%)	(n= 7) (11.29%)	(0%)	(0.3%)
	Death (n= 1)	(0%)	(n= 1) (1.6%)	(0%)	(0.02%)
	With no disorder		(n=7) (11.29%)
	___________________________________________________________________________

Acknowledgements

The authors acknowledge Professor M. Dolan (University of Massachusetts, Amherst, USA) for critical reading of the manuscript as well as the Hhorages board for its ongoing support and precious collaboration concerning the files and relations with families. The authors also warmly acknowledge the patient families of the Hhorages Association for their ongoing support and participation via their testimonies.

Declaration of interest

The authors declare that they have no conflict of interest concerning this work, the Hhorages Association being exclusively financed by memberships and donations.