Geoffrey Canet

Fanny Pineau

Charleine Zussy

Célia Hernandez

Hazel Hunt

Nathalie Chevallier

Véronique Perrier

Joan Torrent

Joseph K Belanoff

Onno C Meijer

Catherine Desrumaux

Dr Laurent Givalois
email: laurent.givalois@umontpellier.fr

Glucocorticoid receptors signaling impairment potentiates amyloid-β oligomers-induced pathology in an acute model of Alzheimer's disease AUTHORS

Keywords: Selective GR modulator, GSK-3β, Cdk5, ROCK, PDK1

Aβ: amyloid-β peptide ADAM10: A disintegrin and metalloproteinase Domain-containing protein 10 (α-secretase) APP: amyloid precursor protein AR: androgen receptors BACE1: β-APP cleaving enzyme type 1 (β-secretase

INTRODUCTION

Alzheimer's disease (AD), the most common cause of dementia in the elderly, is characterized by a progressive impairment of cognitive functions and the presence of senile plaques and neurofibrillary tangles (NFT) throughout the brain, including areas particularly involved in memory formation and emotional regulation. Plaques are composed of insoluble extracellular aggregates consisting mainly of amyloid-β (Aβ) peptides, while NFT result from hyper-and abnormal phosphorylation of the microtubulestabilizing protein Tau [START_REF] Selkoe | Alzheimer's disease: Genes, proteins, and therapy[END_REF]. There are several forms of AD. Familial forms with known mutations of specific genes represent less than 5% of cases, whereas 95% of patients develop sporadic forms, with unknown mechanisms, but with identified risk factors. The principal risk factor for sporadic AD is aging. But, there is also growing evidence that stressful lifetime events may increase the probability of developing AD (2). This view is particularly supported by the fact that in AD patients, cognitive and psychological symptoms are associated with an early deregulation of the hypothalamic-pituitary-adrenal (HPA) axis, as well as elevated levels of glucocorticoids (GC) in plasma and CSF [START_REF] Hartmann | Twenty-four hour cortisol release profiles in patients with Alzheimer's and Parkinson's disease compared to normal controls: ultradian secretory pulsatility and diurnal variation[END_REF][START_REF] Hoogendijk | Increased cerebrospinal fluid cortisol level in Alzheimer's disease is not related to depression[END_REF].

The HPA axis, highly involved in the stress response, triggers the adrenal cortex to release GC. These steroid hormones readily cross the BBB and bind to low affinity glucocorticoid receptors (GR) and high affinity mineralocorticoid receptors (MR) [START_REF] Reul | Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation[END_REF]. GC are necessary for normal cellular activity and fundamental for many CNS functions, including learning and memory [START_REF] Roozendaal | Glucocorticoids and the regulation of memory consolidation[END_REF]. While MR are localized mainly in the hippocampus, GR are more ubiquitous and are particularly found in several structures of the limbic system (prefrontal cortex (PFC), hippocampus and amygdala), which are strongly involved in cognitive and psychological functions, but also are important components of the neural circuitry modulating HPA axis activity [START_REF] Jankord | Limbic regulation of hypothalamo-pituitary-adrenocortical function during acute and chronic stress[END_REF].

GC act synergistically with excitatory amino acids (like glutamate) in neurotoxicity.

Hence, a deregulation of the HPA axis activity or a modification of GR functioning could be extremely toxic, especially in limbic structures [START_REF] Mcewen | Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators[END_REF], and thus could contribute to the cognitive decline and psychological symptoms that occur in AD. In chronic animal models of AD (transgenic mice), stress and GC administration affect the course of the pathology. Chronic stress accelerates the onset of cognitive deficits, triggers amyloid precursor protein (APP) misprocessing, enhances plaque pathology, reduces Aβ clearance, increases Aβ levels, stimulates Tau hyperphosphorylation and its neuronal accumulation [START_REF] Green | Glucocorticoids increase amyloid-beta and tau pathology in a mouse model of Alzheimer's disease[END_REF][START_REF] Rothman | 3xTgAD mice exhibit altered behavior and elevated Aβ after chronic mild social stress[END_REF]. In the same line of evidence, a recent study showed that early life stress in APP/PS1 mice induced elevated corticosterone levels, associated with enhanced hippocampal Aβ 1-40/42 and BACE1 levels [START_REF] Lesuis | Targeting glucocorticoid receptors prevents the effects of early life stress on amyloid pathology and cognitive performance in APP/PS1 mice[END_REF]. In an acute pathomimetic model of AD obtained after a single intracerebroventricular (icv) injection of an oligomeric solution of Aβ (oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] ) (12-14), we demonstrated a strong, long-lasting activation of the HPA axis, associated with a modification of GR and MR expression in brain regions involved in the control of GC secretion (hippocampus, amygdala and hypothalamus) [START_REF] Brureau | Deregulation of HPA axis functions in an Alzheimer's disease rat model[END_REF], supporting its involvement in the etiology of AD [START_REF] Green | Glucocorticoids increase amyloid-beta and tau pathology in a mouse model of Alzheimer's disease[END_REF][START_REF] Catania | The amyloidogenic potential and behavioral correlates of stress[END_REF][START_REF] Baglietto-Vargas | Mifepristone alters amyloid precursor protein processing to preclude amyloid beta and also reduces tau pathology[END_REF][START_REF] Notarianni | Hypercortisolemia and glucocorticoid receptor-signaling insufficiency in Alzheimer's disease initiation and development[END_REF][START_REF] Lanté | Subchronic glucocorticoid receptor inhibition rescues early episodic memory and synaptic plasticity deficits in a mouse model of Alzheimer's disease[END_REF]. We also observed that an antagonist and selective modulators of the GR could potently counteract the effects of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] injection in the hippocampus, arguing strongly for a therapeutic potential of modulating GR activity (14).

In the present preclinical study, we focused our attention on the PFC for several reasons. [START_REF] Selkoe | Alzheimer's disease: Genes, proteins, and therapy[END_REF] It is a cerebral region highly involved in the control of the HPA axis. Indeed, the PFC is involved in both driving the stress-induced activation of the HPA axis, and in mediating negative feedback regulation in times of stress [START_REF] Sullivan | Prefrontal cortical regulation of hypothalamic-pituitaryadrenal function in the rat and implications for psychopathology: side matters[END_REF]. [START_REF] Querfurth | Alzheimer's disease[END_REF] The different subdivisions of the PFC are particularly involved in cognitive and emotional processing [START_REF] Davidson | Anxiety and affective style: role of prefrontal cortex and amygdala[END_REF]. [START_REF] Hartmann | Twenty-four hour cortisol release profiles in patients with Alzheimer's and Parkinson's disease compared to normal controls: ultradian secretory pulsatility and diurnal variation[END_REF] In AD, the PFC is rapidly affected (22). ( 4) GR levels in the PFC are five-fold higher than MR levels [START_REF] Diorio | The role of the medial prefrontal cortex (cingulate cortex) in the regulation of hypothalamic-pituitary-adrenal responses to stress[END_REF], suggesting that the PFC could be particularly sensitive to a dysregulation of the HPA axis activity. Thus, on the basis of our previous findings (12-14) we took advantage of specific properties of a new selective GR modulator (sGRm) CORT113176 to decipher the role of GR in AD. CORT113176 is representative of a series of novel, selective non-steroidal GR ligands (1H-pyrazolo(3,4-g)hexahydro-isoquinoline sulfonamides) developed by Corcept Therapeutics (Fig. 1B). It exhibits excellent affinity for GR with no measurable affinity for the other nuclear hormone receptors (progesterone, androgen, mineralocorticoid and estrogen) (14,24-26). CORT113176 demonstrated only partial antagonism and also some agonism in reaction to a viral protein in rat hepatocytes (14). Hence, we refer to it as a sGRm, with the implication of the advantages linked to selective receptor modulation that we previously reported in detail (14). In fact, this family of molecules has the potential to more selectively abrogate pathogenic GR-dependent processes in the brain (as antagonist), while retaining beneficial aspects of GR signaling (as agonist) (14, [START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF].

MATERIALS AND METHODS

Animals

Adult male Sprague-Dawley rats (Janvier Lab., Le Genest-Saint-Isle, France) weighing 260-280g (8 weeks) at the beginning of the experiments, were housed 1 week before experiments in a standard animal facility (12H/12H light/dark cycle with lights on at 07H00; 21 ± 1°C, food and water ad libitum). All experiments, including sacrifices, were performed in conscious rats between 09H00 and 14H00, during the diurnal trough of the HPA axis circadian rhythm.

Ethical considerations

Animal procedures were conducted in strict adherence to the European Union Directive of 2010 (2010/63/EU). The National French Animal Welfare Committee and the local committee at the University of Montpellier approved all protocols (authorization:

CEEA-LR-12160). All efforts were made to minimize the number of animals used, potential pain, suffering and distress.

Amyloid-β peptide

In patients, soluble Aβ oligomers contains mainly the sequences Aβ 1-40 and Aβ 1-42 [START_REF] Selkoe | Alzheimer's disease: Genes, proteins, and therapy[END_REF]. However, they also contain peptides with shorter sequences such as Aβ 25-35 or Aβ 25- 35/40 [START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF], identical between human and rodent [START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF]. It can be produced in AD patients by enzymatic cleavage of Aβ 1-40 [START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF]. This Aβ peptide includes extracellular and transmembrane residues that have been reported to represent a biologically active region of Aβ [START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF][START_REF] Pike | Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25-35 region to aggregation and neurotoxicity[END_REF][START_REF] Yamada | Animal models of Alzheimer's disease and evaluation of anti-dementia drugs[END_REF] and to contain the highly hydrophobic region forming stable aggregates [START_REF] Pike | Structure-activity analyses of beta-amyloid peptides: contributions of the beta 25-35 region to aggregation and neurotoxicity[END_REF].

Interest in this undecapeptide, which itself shows a β-sheet structure (12,36), has grown over the last decade, mainly because it induces neurite atrophy, neuronal cell death, synaptic loss, as well as synaptic plasticity and memory deficits in a similar way to Aβ 1-40 and Aβ 1-42 [START_REF] Yamada | Animal models of Alzheimer's disease and evaluation of anti-dementia drugs[END_REF], but with better solubility and efficiency [START_REF] Yankner | Neurotrophic and neurotoxic effects of amyloid beta protein: reversal by tachykinin neuropeptides[END_REF][START_REF] Varadarajan | Different mechanisms of oxidative stress and neurotoxicity for Alzheimer's A beta(1--42) and A beta(25--35)[END_REF]. Aβ 25-35 and scrambled Aβ 25-35 peptides (PolyPeptide, Strasbourg, France) were dissolved in sterile water (1 µg/µl) and stored at -20°C. Since soluble Aβ oligomers correlate better with the progression of the disease [START_REF] Selkoe | The amyloid hypothesis of Alzheimer's disease at 25 years[END_REF], Aβ 25-35 and scrambled peptides were aggregated by in vitro incubation at 37°C (4 days) to obtain a solution mainly composed (more than 95%) of a mixture of soluble oligomer species (oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] ), as previously characterized [START_REF] Zussy | Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-β peptide injection in rats. PlosOne 8[END_REF].

Experimental procedures

To evaluate the impact of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] (acute model of AD), animals were divided into three groups. One group had no surgery (control rats), a second received an icv injection of incubated scrambled peptide (10 µg/rat) and a third received an icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] (10 µg/rat). The animals were anesthetized with an intraperitoneal (ip) injection of 1 ml of a mixture of Ketamine and Xylazine (80 and 10 mg/kg b.w., respectively). oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] was injected directly into the lateral ventricles using a David-Kopf stereotaxic apparatus (Phymep, Paris, France), (coordinates: AP -1 mm, L ±1.5 mm, DV -3.5 mm) (Fig. 1A) [START_REF] Paxinos | The rat brain in stereotaxic coordinates[END_REF].

Based on a previous study (14) and in order to decipher the role of GR in oAβ toxicity, treatment with sGRm was conducted 1 week after the icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] .

CORT113176 (10 mg/kg b.w. per injection) (Corcept Therapeutics, Menlo Park, CA, USA) (Fig. 1B) was injected ip twice a day (09H00 and 18H00) for 1 week. The short-term memory or anxiety state of different groups of rats were tested (day 14) in a T-maze or in an elevated plus maze test (EPM), respectively. The following day (day 15) and 30 min after the last ip injection, the unanesthetized animals were sacrificed by decapitation.

Blood samples and the PFC were rapidly collected for corticosterone assay and WB analysis. Naive rats received no treatment but were manipulated in the same manner as treated rats. Vehicle rats received only ip injections of sesame oil and served as negative controls for pharmacological treatments.

Spatial short-term memory

T-maze test was used to rapidly assess the delayed alternation of rats. This memory behavioral test was used as a non-invasive recurrent readout that we usually perform to make sure we have an appropriate toxicity (12-15). The T-maze consisted of two short arms (A and B), extending from a longer alley and enclosed with high walls. The test involved two trials separated by 1 h. During the training session, one short arm (B) was closed. Rats were placed at the end of the long alley, allowed to visit the maze for 10 min and then returned into their home cage. During the test session, animals were placed in the maze for 2 min, with free access to all arms. The number of visits and time spent in each arm were measured. The results were expressed as ratio of the time spent in the initially closed novel arm, over the time spent in the previous arm and as a ratio of the number of entries into the novel arm over the familiar one. The apparatus was cleaned with diluted ethanol (50%) between animals.

Anxiety behavior

The anxiety state of rats was measured using their ability to explore open and enclosed arms of an EPM, as previously described [START_REF] Brureau | Deregulation of HPA axis functions in an Alzheimer's disease rat model[END_REF]. The clear plexiglass apparatus consisted of two open arms (50 x 10 cm) and two enclosed arms (50 x 10 x 45 cm high), extending from a central platform and placed 60 cm above the floor. Each rat was placed at the center of the plus-maze facing the closed arm and its exploration behavior was recorded for 10 min. The results were expressed as total time spent in the open arms and the total number of entries was counted to verify general motor activity. An entry into an arm was recorded if the animal crossed the line that connected that arm with the central platform with all four legs. The apparatus was cleaned with diluted ethanol (50%) between animals.

Corticosterone assay

Blood samples were collected at the time of sacrifice (day 15), on 1 mg/ml EDTA (Sigma-Aldrich, Saint Quentin Fallavier, France), centrifuged at 4°C, and plasma stored at -20°C until assayed for corticosterone (14). Plasma corticosterone concentrations were assayed using a conventional ELISA kit (Enzo-Life Sciences, Farmingdale, NY, USA) in a 10-µl plasma sample diluted (1:40) with the assay buffer. The assay sensitivity was 27 pg/ml. The intra-and inter-assay coefficients were 6.6 and 7.8%, respectively.

Aβ 1-42 assay

Rats were sacrificed by decapitation 15 days after oAβ 25-35 injection and brains were rapidly removed, PFC dissected out, weighted, frozen in liquid nitrogen and stored at -80°C until assayed. After thawing, PFC were sonicated (VibraCell; Sonics & Materials, Newtown, CT, USA) for 20s in a lysis buffer [START_REF] Meunier | The γ-secretase inhibitor2-[(1R)-1-((4-chlorophenyl)sulfonyl) (2,5-difluorophenyl) amino)ethyl-5-fluorobenzenebutanoic acid (BMS-299897] alleviates Aβ1-42 seeding and short-term memory deficits in the Aβ25-35 mouse model of Alzheimer's disease[END_REF]. After centrifugation (14000 rpm for 25 min, 4°C), supernatants were used for Aβ 1-42 ELISA assay (Anaspec, Fremont, CA, USA), according to the manufacturer's instructions. Absorbance was read at 450 nm (Tecan icontrol, ThermoFisher Scientific, Illkirch , France) and sample concentration was calculated using the standard curve (SFig.3A). Results were then expressed in pg of Aβ 1- 42 /g of tissue. The assay sensitivity was 3.91 pg/ml. The intra-and inter-assay coefficients were 4.3 and 6.4%, respectively.

WB analysis

WB were performed as previously described (14) in the whole PFC. All antibodies used are detailed in the Table 1. Briefly, after sacrifice, the PFC was micro-dissected, weighed, immediately frozen on liquid nitrogen and stored at -20°C. Tissues were sonicated (VibraCell; Sonics & Materials, Newtown, CT, USA) in a lysis buffer (12) and centrifuged (4°C). Supernatants were collected and the protein concentration was measured using a BCA kit (ThermoFisher Scientific, Illkirch , France). Sixty µg from each sample were taken for WB analysis. Samples were separated in SDS-polyacrylamide gel (12%) and transferred to a PVDF membrane (Merck-Millipore, Dachstein, France). The membrane was incubated overnight (4°C) with the primary antibody, rinsed and then incubated for 2h with the appropriate horseradish peroxidase-conjugated secondary antibody. Peroxidase activity was revealed by using enhanced-chemiluminescence (ECL) reagents (Luminata-Crescendo, Merck-Millipore). The intensity of peroxidase activity was quantified using Image-J software (NIH, Bethesda, MA, USA). β-tubulin (β-Tub) was used as a loading control for all immunoblotting experiments.

Statistical analysis

Data are presented as mean ± SEM and analyzed using two-way ANOVA followed by a Tukey's multiple comparison test (GraphPad-Prism 5.0). P 0.05 was considered significant. The number of animals in each group is indicated within the columns. Before each analysis of variance, the Gaussian distribution was evaluated and validated by a Kolmogorov-Smirnov test (GraphPad-Prism 5.0).

RESULTS

To characterize the impact of oAβ 25-35 , we previously tested over time (after 1 and 2 weeks) two different doses (5 and 10 µg/rat) on several parameters previously characterized in this acute model of AD (12) (SupFig.1A). While the scrambled peptide induced no modification in comparison with control naive rats, the dose of 10 µg of oAβ 25- 35 was more efficient than the dose of 5 µg. Indeed, after 10 µg short-term memory deficit (T-maze) was observed up to 2 weeks post-injection (SupFig.1B) and plasma levels of corticosterone were increased from 1 to 2 weeks (SupFig.1C).

We next evaluated (in the PFC) the role of GR in the oAβ toxicity using a sGRm (CORT113176). Animals were treated one week after the icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] with CORT113176 according to a protocol and a dose established in a previous study (14).

Control scrambled peptide and vehicle treatment induced no changes in any of the readouts relative to untreated animals (Fig. 123456). All blots of control conditions were presented in a supplementary document (SFig.2) to improve the clarity of figures and to highlight key effects. Two weeks after oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] , animals presented anxious behavior (Fig. 1C), short-term memory deficits (Fig. 1D), and high plasma concentrations of corticosterone (Fig. 1E). Treatment with CORT113176 reversed all of these parameters (Fig. 1).

Behavioral deficits observed two weeks after oAβ 25-35 , were associated with pre-(SYN) and post-synaptic (PSD95) deficits, increased apoptotic marker expression (Fig. 2A-D) and a marked neuroinflammation characterized by the activation of astrocytes (GFAP) (Fig. 2A,E) and microglial cells (Iba1) (Fig. 2A,F). Treatment with the sGRm normalized Caspase 3 expression, the pre-and post-synaptic deficits (Fig. 2A,C,D) and blocked the neuroinflammatory processes (Fig. 2A,F), as previously reported at the hippocampus level (14) or at the spinal cord level in an experimental model of amyotrophic lateral sclerosis [START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF].

In order to determine in the PFC the effects of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] (and of CORT113176) on glucocorticoid receptor signaling, as part of a potential feed-forward or feedback process, animals were treated as previously detailed (Fig. 1A). The icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] increased the expression of MR and GR (Fig. 3A-C). These effects were associated with an over-activation of GR, as characterized by an increase of the phosphorylated form of GR (Ser 211 ) (Fig. 3A,D). There were opposite changes in the expression of HSP90 and HSP70, the two main chaperones involved in the activity of GR [START_REF] Kirschke | Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles[END_REF], but also involved in the control of Aβ and Tau aggregation [START_REF] Dou | Chaperones increase association of tau protein with microtubules[END_REF][START_REF] Chen | Hsp90 chaperone inhibitor 17-AAG attenuates Aβ-induced synaptic toxicity and memory impairment[END_REF][START_REF] Lackie | The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases[END_REF]. The HSP90/HSP70 ratio (which reflects GR activation) [START_REF] Kirschke | Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles[END_REF] accordingly was substantially increased (Fig 3A,E). Treatment with CORT113176 reversed the increase of GR and MR, decreased the phosphorylation of GR and normalized the HSP90/HSP70 ratio (Fig. 3).

The GR phosphorylation status (47) may constitute an important link between AD and GC. Indeed, GR can be phosphorylated on several serine and threonine residues. Thus, we characterized the impact of oAβ 25-35 on GSK-3β and Cdk5, the two main enzymes involved in both the phosphorylation of GR [START_REF] Galliher-Beckley | Emerging roles of glucocorticoid receptor phosphorylation in modulating glucocorticoid hormone action in health and disease[END_REF], and the hyperphosphorylation of Tau [START_REF] Beurel | Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases[END_REF][START_REF] Sun | Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation[END_REF]. We first confirmed changed expression ratios of p(Ser9)GSK-3β/GSK-3β and p(Tyr216)GSK-3β/GSK-3β (51) (Fig. 4A-C), reflecting an increase in GSK-3β activation.

Second we observed increased levels of Cdk5 (Fig. 4A,D), in association with those of p35 and p25, which are involved in Cdk5 activation (52) (Fig. 4A,E). We also measured increased levels of Calpain 1, a member of cysteine proteases family regulated by intracellular calcium and showing aberrant activity in AD [START_REF] Kurbatskaya | Upregulation of calpain activity precedes tau phosphorylation and loss of synaptic proteins in Alzheimer's disease brain[END_REF]. Calpain 1 is particularly involved in the activation of GSK-3β [START_REF] Jin | Truncation and activation of GSK-3β by calpain 1: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease[END_REF], and in the maturation of p35 in p25 [START_REF] Lee | Neurotoxicity induces cleavage of p35 to p25 by calpain[END_REF] (Fig. 4A,F). Lastly, we observed increases in the levels of Fyn (Fig. 4A,G), a Src kinase associated with non-genomic effects of GR [START_REF] Oakley | The biology of the glucocorticoid receptor: New signaling mechanisms in health and disease[END_REF], involved in the activation of GSK-3β and the phosphorylation of Tau [START_REF] Beurel | Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases[END_REF][START_REF] Lesort | Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase[END_REF]. This family of enzymes is bound to the inactive form of GR as chaperone, and released when GC bind to their receptors [START_REF] Oakley | The biology of the glucocorticoid receptor: New signaling mechanisms in health and disease[END_REF]. Under physiological conditions, GC inhibit Fyn activity and phosphorylation [START_REF] Löwenberg | Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn[END_REF], but under chronic stress, with high levels of GC, Fyn is upregulated especially in the hippocampus of adult rats [START_REF] Li | Effects of chronic multiple stress on learning and memory and the expression of Fyn, BDNF, TrkB in the hippocampus of rats[END_REF]. To summarize, the icv injection of oAβ 25-35 activated both GSK-3β and Cdk5 pathways. This activation was associated with an increase of p25, Calpain 1 and Fyn levels in the PFC (Fig. 4). Treatment with CORT113176 inhibited the activation of GSK-3β and Cdk5, the maturation of p35 into p25 and the increase of Calpain 1 and Fyn (Fig. 4).

In the next part of this study, we characterized the different pathways of APP maturation, through the assessment of the different cellular elements involved in the mutually exclusive processing pathways of APP, the amyloidogenic and nonamyloidogenic pathways (Figs 5 & 6). APP processing and induction of the amyloidogenic pathway (Fig. 5) were evaluated by measuring PFC levels of full-length APP (precursor of amyloid proteins) (Fig. 5A,B), C99 (precursor of Aβ peptides) (Fig. 5A,C), BACE1 (β-APP cleaving enzyme) (Fig. 5A,D), PS1 (presenilin-1, a subunit of the γ-secretase) (Fig. 5A,E) and IDE (insulin degrading enzyme, involved in the clearance of Aβ) (Fig. 5A,F). Two weeks after the injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] , APP levels were increased and amyloidogenic processing was enhanced. This activation was associated with an increased formation (BACE1 and PS1), and a decreased clearance (IDE). One week of treatment with CORT113176 inhibited the activation of the amyloidogenic pathway. The increase in levels of APP, C99, PS1 and BACE1, but also the IDE down-regulation were fully reversed (Fig. 5). To characterize the non-amyloidogenic pathway in the PFC, we evaluated by western blot the levels of the α-secretase-cleaved soluble APP ectodomain (sAPPα) and ADAM10 (A disintegrin and metalloproteinase Domain-containing protein 10, a component of α-secretase) (Fig. 6A-C). Two weeks after the injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] , sAPPα and ADAM10 were decreased. This inhibition of the non-amyloidogenic processing of APP was totally reversed by one week of treatment with CORT113176 (Fig. 6A-C). Finally, in order to confirm the induction of the amyloidogenic pathway, we assayed the endogenous levels of Aβ 1-42 in the PFC (SFig.3). As expected, two weeks after the icv injection of oAβ 25-35 , Aβ 1-42 levels were increased by 18% in the PFC. This upregulation was totally reversed by the treatment with CORT113176 (SFig.3B). Even if the levels of Aβ 1-42 assayed in the PFC were relatively low, they are consistent with some previous studies in rats [START_REF] García-Rojo | In Vivo Sub-chronic Treatment with Dichlorvos in Young Rats Promotes Synaptic Plasticity and Learning by a Mechanism that Involves Acylpeptide Hydrolase Instead of Acetylcholinesterase Inhibition. Correlation with Endogenous β-Amyloid Levels[END_REF][START_REF] Lin | Lidocaine attenuates cognitive impairment after isoflurane anesthesia in old rats[END_REF].

To understand by which mechanisms non-amyloidogenic pathways could be inhibited, we evaluated the involvement of the Rho-kinases system. These Ser/Thr kinases are involved in cell motility, cell proliferation, autophagy and apoptosis [START_REF] Chang | Activation of Rho-associated coiled-coil protein kinase 1 (ROCK-1) by caspase-3 cleavage plays an essential role in cardiac myocyte apoptosis[END_REF][START_REF] Loirand | Rho Kinases in Health and Disease: From Basic Science to Translational Research[END_REF][START_REF] Swanger | Double-edge sword of sustained ROCK activation in prion diseases through neuritogenesis defects and prion accumulation[END_REF]. They have been suggested as potential therapeutic targets for neurodegenerative diseases, including AD (66-68). This effect seems to be mediated by Rho-associated coiled-coil kinases (ROCK)-induced overactivation of the 3-phosphoinositide-dependent kinase 1 (PDK1) activity (65,68). In fact, exacerbated ROCK activity seems to increase the pool of PDK1 molecules physically interacting with and phosphorylated by ROCK (65,68). The overactivation of this system inhibits the non-amyloidogenic pathway (65,68) and affects Tau phosphorylation [START_REF] Amano | Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase[END_REF][START_REF] Gentry | Rho kinase inhibition as a therapeutic for progressive supranuclear palsy and corticobasal degeneration[END_REF]. They seem particularly involved in the inhibition of sAPPα synthesis [START_REF] Pietri | PDK1 decreases TACE-mediated α-secretase activity and promotes disease progression in prion and Alzheimer's diseases[END_REF] and in the phosphorylation of Tau [START_REF] Amano | Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase[END_REF][START_REF] Castro-Alvarez | ROCK inhibition prevents tau hyperphosphorylation and p25/CDK5 increase after global cerebral ischemia[END_REF]. Their activity is modulated by GC (72) via GR activation [START_REF] Rubenstein | Selective glucocorticoid control of Rho kinase isoforms regulate cell-cell interactions[END_REF]. To characterize the Rho-kinase system, we measured the PFC levels of Rho-associated coiled-coil kinases (ROCK1 & ROCK2) (Fig. 6A,D,E) and the 3phosphoinositide-dependent kinase (PDK1) (Fig. 6A,F). Here, two weeks after injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] , we observed in the PFC an increase of ROCK1, ROCK2 and PDK1, which was normalized by one week of treatment with the sGRm (Fig. 6A, D-F).

DISCUSSION

In previous studies, we provided evidence for a vicious cycle between AD and the HPA axis. We showed that the pathology, and especially the amyloid toxicity, rapidly increases GC secretion that in turn, modulates APP processing (14,[START_REF] Brureau | Deregulation of HPA axis functions in an Alzheimer's disease rat model[END_REF]. This dysregulation seems to be under the control of GR, since treatment with a new class of selective GR ligands blocks the installation of this cycle in the hippocampus and re-establishes all parameters analyzed and disturbed by the amyloid toxicity (memory and synaptic deficits, neuroinflammation, apoptosis, APP processing and high levels of GC) (14). This recent study (14), allowed us to design and validate the experimental protocol of treatment with sGRm. Briefly, we determined treatment timing and duration, doses and specificities of the two sGRm (CORT108297 & CORT113167) tested, in comparison to the non-selective antagonist of reference, Mifepristone. It appeared that one week of treatment (two ip injection per day) with CORT113176 (10 mg/kg per injection) displayed the most effective therapeutic potential against toxicity induced by oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] . Owing to its efficacy and selectivity, this sGRm was selected in the present mechanistic study.

Here, on the basis of our precedent finding (14), we aimed to decipher the role of GR in AD and to characterize associated underlying mechanisms. For this purpose, we evaluated the impact of oAβ 25-35 on several intracellular pathways involved in the activation of GR, but also in the pathophysiology of AD. We show the establishment of several intracellular vicious cycles involving GC and GR, providing mechanistic insight to a central role of these receptors in the etiology of AD (Fig. 7). The notion of a vicious cycle between GC signaling and pathogenesis is reinforced by the fact that modulation of GR activity with CORT113176 normalized all changes induced by the amyloid toxicity.

In the first part of our study, in order to validate our protocols in comparison with the previous study (14), we confirmed that one week of treatment with a sGRm (CORT113176) at the dose of 10 mg/kg is sufficient to reverse short-term memory deficits and to reestablish plasma concentrations of corticosterone disrupted by the icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] . In addition, and for the first time, we show that treatment with sGRm is able to reverse anxious behavior induced by the amyloid toxicity [START_REF] Brureau | Deregulation of HPA axis functions in an Alzheimer's disease rat model[END_REF]. We equally demonstrate that sGRm treatment is able to reverse synaptic deficits, neuroinflammation and apoptosis processes induced by oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] in the PFC, as previously observed in the hippocampus (14).

Then, we showed that the icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] increased GR phosphorylation on Ser 211 , a site involved in the activation of GR in rats [START_REF] Galliher-Beckley | Emerging roles of glucocorticoid receptor phosphorylation in modulating glucocorticoid hormone action in health and disease[END_REF]. This activation was associated with an increase and activation of GSK-3β and Cdk5, with a substantial increase of the HSP90/HSP70 ratio (important for GR activity [START_REF] Kirschke | Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles[END_REF]), but also, importantly, in the control of Aβ and Tau aggregation [START_REF] Dou | Chaperones increase association of tau protein with microtubules[END_REF][START_REF] Chen | Hsp90 chaperone inhibitor 17-AAG attenuates Aβ-induced synaptic toxicity and memory impairment[END_REF][START_REF] Lackie | The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases[END_REF]. The activation of GSK-3β and Cdk5 is under the control of several enzymes [START_REF] Beurel | Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases[END_REF][START_REF] Sun | Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation[END_REF][START_REF] Jin | Truncation and activation of GSK-3β by calpain 1: a molecular mechanism links to tau hyperphosphorylation in Alzheimer's disease[END_REF][START_REF] Lee | Neurotoxicity induces cleavage of p35 to p25 by calpain[END_REF][START_REF] Lesort | Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase[END_REF], including Fyn and Calpain 1 that are regulated by GC [START_REF] Löwenberg | Rapid immunosuppressive effects of glucocorticoids mediated through Lck and Fyn[END_REF][START_REF] Li | Effects of chronic multiple stress on learning and memory and the expression of Fyn, BDNF, TrkB in the hippocampus of rats[END_REF], induced by the amyloid toxicity, and increased in AD patients [START_REF] Ho | Altered p59Fyn kinase expression accompanies disease progression in Alzheimer's disease: implications for its functional role[END_REF]. Our data show that the over-activation of GR induced by oAβ 25-35 coincided with an increase of these two enzymes (Fig. 7).

We also observed that oAβ 25-35 induced a strong increase of Calpain 1. This augmentation could be the reflection of its proteolytic activity, given that it was concomitant with an activation of two of its substrates, GSK-3β and an increase of p25/p35 ratio. These effects were reversed by treatment with the sGRm (CORT113176), providing evidence of an intracellular loop by which pathology increases the activation of GR (directly via GSK-3β and Cdk5 and indirectly via Fyn and Calpain 1), which in turn increases the activation of these key enzymes and the HSP90/HSP70 ratio, worsening the AD pathogenesis (44-46) (Fig. 7).

Our current data show that the seeding and the accumulation of endogenous Aβ induced by the icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] [START_REF] Kirschke | Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles[END_REF] (SFig.3B), not only results from the activation of amyloid-pathways, but also from the inhibition of non-amyloid-pathways. Indeed, the icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] provoked the activation of the amyloidogenic pathway in the PFC, through an increase of Aβ synthesis (APP/C99, Aβ 1-42 , BACE1 and PS1 up-regulation) and a decrease of Aβ clearance (IDE down-regulation), as previously reported for the hippocampus (14). Besides, Aβ oligomers also inhibit the non-amyloidogenic pathway (sAPPα and ADAM 10 down-regulation) and that this effect could be controlled by the activation of ROCK1 and ROCK2 (Fig. 7). We showed that the oAβ 25-35 injection-induced inhibition of sAPPα and ADAM10 is associated with an activation of ROCK/PDK1 pathways. These results are consistent with several studies showing that ROCKs modulate the shedding of sAPPα through an inhibition of tumor necrosis factor-αconverting enzyme (TACE or ADAM) activity, and that ROCKs depletion reduces Aβ levels [START_REF] Pietri | PDK1 decreases TACE-mediated α-secretase activity and promotes disease progression in prion and Alzheimer's diseases[END_REF][START_REF] Herskowitz | Pharmacologic inhibition of ROCK2 suppresses amyloid-β production in an Alzheimer's disease mouse model[END_REF][START_REF] Henderson | Rho-associated protein kinase 1 (ROCK1) is increased in Alzheimer's disease and ROCK1 depletion reduces amyloid-β levels in brain[END_REF]. ROCK activity seems to be directly up-regulated by GC [START_REF] Huang | Up-regulation of Rho-associated kinase 1/2 by glucocorticoids promotes migration, invasion and metastasis of melanoma[END_REF][START_REF] Rubenstein | Selective glucocorticoid control of Rho kinase isoforms regulate cell-cell interactions[END_REF][START_REF] Liu | The glucocorticoid dexamethasone inhibits U937 cell adhesion and neutrophil release via RhoA/ROCK1-dependent and independent pathways[END_REF], and ROCK/PDK1 activation after oAβ 25-35 is reversed by treatment with CORT113176, again constituting a vicious cycle based on feedforward effects on GR signaling (Fig. 7).

ROCK also affect Tau hyperphosphorylation [START_REF] Amano | Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase[END_REF][START_REF] Gentry | Rho kinase inhibition as a therapeutic for progressive supranuclear palsy and corticobasal degeneration[END_REF]. They activate the two principal enzymes involved in Tau phosphorylation, Cdk5 and GSK-3β [START_REF] Castro-Alvarez | ROCK inhibition prevents tau hyperphosphorylation and p25/CDK5 increase after global cerebral ischemia[END_REF][START_REF] Hamano | Pitavastatin decreases tau levels via the inactivation of Rho/ROCK[END_REF]. In addition, ROCK/PDK1 inhibition of sAPPα may affect Tau, since it was shown that sAPPα reduces GSK-3β-mediated Tau phosphorylation [START_REF] Deng | Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3β signaling pathway[END_REF]. These two mechanisms again link GR and GC to the pathophysiology of AD (Fig. 7). Likewise, Tau phosphorylation may also be directly impacted by the other GR-related enzymes we described. Indeed, Fyn can directly phosphorylate Tau on tyrosine residues [START_REF] Scales | Tyrosine Phosphorylation of Tau by the Src Family Kinases Lck and Fyn[END_REF]. Fyn can also directly activate GSK-3β to rapidly induce Tau phosphorylation in human neuroblastoma cells [START_REF] Lesort | Insulin transiently increases tau phosphorylation: involvement of glycogen synthase kinase-3beta and Fyn tyrosine kinase[END_REF]. In the same line of evidence, it appears that Fyn controls the activity of PDK1 through an up-regulation of ROCK [START_REF] Liu | Inhibition of SRC family kinases protects hippocampal neurons and improves cognitive function after traumatic brain injury[END_REF][START_REF] Lv | Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro[END_REF], and thus could also participate in the inhibition of the non-amyloidogenic pathway. Calpain 1 also activates GSK-3β and Cdk5, promoting Tau phosphorylation and Tau-associated neurodegeneration [START_REF] Beurel | Glycogen synthase kinase-3 (GSK3): regulation, actions, and diseases[END_REF][START_REF] Sun | Activation of Cdk5/p25 and tau phosphorylation following chronic brain hypoperfusion in rats involves microRNA-195 down-regulation[END_REF]. Calpain 1 can cleave the neuron-specific Cdk5 activator p35 to produces p25, which accumulates in the brains of AD patients [START_REF] Patrick | Conversion of p35 to p25 deregulates Cdk5 activity and promotes neurodegeneration[END_REF].

In fact, it was shown that induction of p25 by Calpain 1 causes prolonged activation and mislocalization of Cdk5 and that the p25/Cdk5 kinase hyperphosphorylates Tau, disrupts the cytoskeleton and promotes the apoptotic death of primary cortical neurons [START_REF] Lee | Neurotoxicity induces cleavage of p35 to p25 by calpain[END_REF]. Thus Fyn and Calpain 1 upregulations may be involved in both the increased Tau phosphorylation after oAβ 25-35 injection [START_REF] Zussy | Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-β peptide injection in rats. PlosOne 8[END_REF], and in the activity of GSK-3β, Cdk5, and the processing of APP. This highlights the potential link that GR activation could play between Aβ and Tau (Fig. 7).

The mechanisms by which GR affects the multiple enzymes identified here is unclear. GR are nuclear receptors that directly interact with specific genes via binding to glucocorticoid response element (GRE), interactions with other transcription factors, or via non-genomic mechanisms, such as epigenetic modifications [START_REF] Reul | Two receptor systems for corticosterone in rat brain: microdistribution and differential occupation[END_REF][START_REF] Lv | Fyn Mediates High Glucose-Induced Actin Cytoskeleton Reorganization of Podocytes via Promoting ROCK Activation In Vitro[END_REF]. A GRE has been described in the promoter regions of APP and BACE1 [START_REF] Lahiri | Functional characterization of amyloid beta precursor protein regulatory elements: rationale for the identification of genetic polymorphism[END_REF][START_REF] Sambamurti | Gene structure and organization of the human beta-secretase (BACE) promoter[END_REF][START_REF] Laping | Transcriptional control of glial fibrillary acidic protein and glutamine synthetase in vivo shows opposite responses to corticosterone in the hippocampus[END_REF]. For the other proteins no mechanism of transcriptional regulation via GR has been identified. A recent study on hippocampal slices pre-treated with an inhibitor of transcription activity, showed a nongenomic activation of GSK-3β by GC [START_REF] Dey | Glucocorticoid-mediated activation of GSK3β promotes tau phosphorylation and impairs memory in type 2 diabetes[END_REF]. The efficacy of the sGRm suggests that it is able to antagonize also non-genomic GR signaling. Of note, involvement of membranelocalized GR was linked to AD not only for the regulation of GSK-3β ( 88), but alsosurprisingly -in the regulation of BACE1 [START_REF] Choi | Membrane-Associated Effects of Glucocorticoid on BACE1 Upregulation and Aβ Generation: Involvement of Lipid Raft-Mediated CREB Activation[END_REF]. For the other proteins, further investigations will be needed to decipher which mechanisms are involved in GR regulatory activity. There is also the potential for more indirect mechanisms, since membrane-localized GR can facilitate glutamatergic transmission [START_REF] Groeneweg | Rapid non-genomic effects of corticosteroids and their role in the central stress response[END_REF][START_REF] Groeneweg | Mineralocorticoid and glucocorticoid receptors at the neuronal membrane, regulators of nongenomic corticosteroid signalling[END_REF][START_REF] Popoli | The stressed synapse: the impact of stress and glucocorticoids on glutamate transmission[END_REF], affect some of the factors that we identified here via stimulation of excitotoxicity pathways [START_REF] Mcewen | Central effects of stress hormones in health and disease: Understanding the protective and damaging effects of stress and stress mediators[END_REF].

This new study provides new arguments supporting the development of a vicious cycle based on GR activation in AD, here based on analysis of changes in the PFC. In this preclinical study, the new sGRm (CORT113176) blocked this cycle and normalized all AD processes analyzed, including extracellular (ADAM10, BACE1 and IDE), intramembrane (PS1) and intracellular enzymes (Cdk5/p25, GSK-3β, Fyn, Calpain 1, ROCKs and PDK1).

In addition, we cannot exclude, as previously mentioned in the hippocampus (14), the involvement of MR in the effects observed after sGRm treatment. Indeed, several studies suggested a neuroprotective role of these receptors in a context of GR blockade (93-95).

Thus, further investigations are needed to decipher the precise role of MR in the pathophysiology of AD. We moreover demonstrated that the accumulation of endogenous Aβ, induced by the amyloid toxicity and the concomitant dysregulation of HPA axis, resulted from the activation of amyloidogenic and the inhibition of the non-amyloidogenic pathways (Fig. 7). All of these data place HPA axis dysregulation and GR in a central and crucial position in the pathophysiology of AD, linking amyloid toxicity and Tau deregulation. This work also highlights the therapeutic potential of sGRm to counteract negative effects induced by the amyloid toxicity and to reestablish the functionality of GR and a fortiori to reestablish the primal role of GC in the maintenance of homeostasis.

AUTHOR'S CONTRIBUTIONS

GC, FP, CH and CZ performed experiments. HH and JKB provided glucocorticoid receptor ligand tested in this study. GC, FP, CZ, CH, CD, NC, VP, JT, HH, OM and JKB corrected the manuscript. LG designed the study, wrote the protocol (with the help of GC), performed part of experiments, analyzed the data (with the help of CD, NC, and OM) and wrote the manuscript with GC. All authors read and approved the final manuscript. All funders had no role in data collection, analysis or in the writing of the manuscript.

LEGENDS Figure 1

Effects of selective GR modulator on, anxious behavior, spatial short-term memory deficits and high levels of corticosterone induced by the icv injection of oAβ [START_REF] Beaudry | Effects of selective and non-selective glucocorticoid receptor II antagonists on rapidonset diabetes in young rats[END_REF][26][START_REF] Zalachoras | Differential targeting of brain stress circuits with a selective glucocorticoid receptor modulator[END_REF][START_REF] Meijer | Glucocorticoid receptor modulators[END_REF][START_REF] Solomon | The selective glucocorticoid receptor antagonist CORT 108297 decreases neuroendocrine stress responses and immobility in the forced swim test[END_REF][START_REF] Meyer | The selective glucocorticoid receptor modulator CORT113176 reduces neurodegeneration and neuroinflammation in Wobbler mice spinal cord[END_REF][START_REF] Kaneko | Drastic neuronal loss in vivo by beta-amyloid racemized at Ser(26) residue: conversion of non-toxic [D-Ser(26)]beta-amyloid 1-40 to toxic and proteinase-resistant fragments[END_REF][START_REF] Kubo | In vivo conversion of racemized beta-amyloid ([D-Ser 26]A beta 1-40) to truncated and toxic fragments ([D-Ser 26]A beta 25-35/40) and fragment presence in the brains of Alzheimer's patients[END_REF][START_REF] Gruden | Differential neuroimmune markers to the onset of Alzheimer's disease neurodegeneration and dementia: autoantibodies to Abeta((25-35)) oligomers, S100b and neurotransmitters[END_REF][START_REF] Fung | Interaction of human and mouse Abeta peptides[END_REF][START_REF] Pike | Neurodegeneration induced by beta-amyloid peptides in vitro: the role of peptide assembly state[END_REF] . Panel A:

Experimental protocol -At T0, adult male rats (Sprague Dawley) were injected directly into the lateral ventricles using a stereotaxic apparatus (coordinates: AP -1 mm, L ±1.5 mm, DV -3.5 mm). One group has not undergone surgery (control rats, white column), a second group received an icv injection of scrambled Aβ 25-35 peptide (negative control -10 µg/ratorange column) and a third group received an icv injection of oAβ 25-35 peptide (Aβ -10 µg/rat -red column). Animals were weighted daily and one week after the icv injection (at Week 1), animals were treated with vehicle or CORT113176 (10 mg/kg per injection) through two intraperitoneal (ip) injections per day during 7 days. At day 14 (Week 2), the anxious behavior or the spatial short-term memory of each rat were tested in an elevated plus maze (EPM) or in a T-maze, respectively. The following day (day 15), 30 min after the last ip injection, the animals were sacrificed, blood samples and PFC were rapidly PS1 and BACE-1 contents, concomitantly with a decrease of IDE, evidencing the induction of the amyloidogenic pathway and, as previously observed, Aβ 1-42 production [START_REF] Meunier | The γ-secretase inhibitor2-[(1R)-1-((4-chlorophenyl)sulfonyl) (2,5-difluorophenyl) amino)ethyl-5-fluorobenzenebutanoic acid (BMS-299897] alleviates Aβ1-42 seeding and short-term memory deficits in the Aβ25-35 mouse model of Alzheimer's disease[END_REF] and Tau hyperphosphorylation [START_REF] Zussy | Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-β peptide injection in rats. PlosOne 8[END_REF]. In parallel, the icv injection of Aβ oligomers inhibits sAPPα and ADAM10, evidencing the inhibition of non amyloidogenic pathway. Aβ oligomers induce, in addition to an excess of circulating GC, an overactivation of GR, which is associated with an increase of the two main chaperones (HSP90 & HSP70) particularly involved in the activity of GR [START_REF] Kirschke | Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles[END_REF], but also in the control of Aβ and Tau aggregation [START_REF] Dou | Chaperones increase association of tau protein with microtubules[END_REF][START_REF] Chen | Hsp90 chaperone inhibitor 17-AAG attenuates Aβ-induced synaptic toxicity and memory impairment[END_REF][START_REF] Lackie | The Hsp70/Hsp90 Chaperone Machinery in Neurodegenerative Diseases[END_REF]. Thus, active GR translocate to the nucleus where they exert their genomic effects through GRE, inducing APP and BACE-1 gene transcription, and potentiating Aβ oligomers production.

Therefore, it evidences a first intracellular vicious cycle by which pathology increases circulating GC, which in turn increase pathology. Aβ oligomers also enhance contents and activity of key enzymes involved directly (Cdk5 and GSK-3β) or indirectly (Calpain 1 and Fyn) in the activation of GR, but also in the hyperphosphorylation of Tau. Enzyme inductions which are regulated by GR, as evidenced after treatment with the selective GR modulator (CORT113176), demonstrate non-genomic effects of GR and thus a second intracellular vicious cycle. Indeed, pathology increases the activation of GR via several keys enzymes (Cdk5, GSK-3β, Calpain 1 and Fyn), which in turn increase the activation of these enzymes involved in the pathophysiology of AD. Finally, it appears that amyloid toxicity inhibits also the non-amyloidogenic pathways, reinforcing the displacement of the equilibrium in favor of endogenous amyloid seeding and evidencing another intracellular loop by which pathology increases the activation of GR, which in turn increases the activation of ROCKs/PDK1 pathways, as evidenced after treatment with the selective GR modulator (CORT113176). This activation accentuates the pathology through the inhibition of α-secretase (ADAM10) and sAPPα synthesis, as previously reported [START_REF] Deng | Soluble amyloid precursor protein alpha inhibits tau phosphorylation through modulation of GSK3β signaling pathway[END_REF], but also by increasing the phosphorylation of Tau [START_REF] Amano | Identification of Tau and MAP2 as novel substrates of Rho-kinase and myosin phosphatase[END_REF][START_REF] Gentry | Rho kinase inhibition as a therapeutic for progressive supranuclear palsy and corticobasal degeneration[END_REF][START_REF] Castro-Alvarez | ROCK inhibition prevents tau hyperphosphorylation and p25/CDK5 increase after global cerebral ischemia[END_REF][START_REF] Hamano | Pitavastatin decreases tau levels via the inactivation of Rho/ROCK[END_REF].

Red arrow: showed in this study. Purple arrow: showed in our previous studies [START_REF] Zussy | Alzheimer's disease related markers, cellular toxicity and behavioral deficits induced six weeks after oligomeric amyloid-β peptide injection in rats. PlosOne 8[END_REF]65). Black arrow: known in the literature (See discussion for references).

  collected for corticosterone assay and Western blot analysis, respectively. Panel B: Chemical structure of the selective GR modulator tested in this study, CORT113176 (reproduction with the permission of Corcept Therapeutics). Panel C: Anxious behavior was determined in the EPM paradigm. Each animal was placed at the center of the EPM and allowed to freely explore for 10 min. Data were expressed as time spent in the open arms (OA) in % of total time. Two-way ANOVA: F 2,59 = 7.90 for group, p< 0.001, F 2,59 = 1.54 for treatment, ns; and F 4,59 = 2.95 for interaction, p< 0.05. Panel D: Spatial short-term memory performance was determined in a T-maze test and was expressed as the ratio of the time spent in the initially closed arm (B) over the time spent in the previous arm (A). Two-way ANOVA: F 2,48 = 9.10 for group, p< 0.001; F 2,48 = 8.51 for treatment, p< 0.001; and F 4,48 = 2.36 for interaction, ns. Panel E: Plasma concentrations of corticosterone (CORT) were determined by Elisa and expressed as ng/ml. Two-way ANOVA: F 2,45 = 39.0 for group, p< 0.0001; F 2,45 = 15.3 for treatment, p< 0.0001; and F 4,45 = 10.8 for interaction, p< 0.0001. * p< 0.05 and ** p< 0.01 vs. respective control (C) group. + p< 0.05 and ++ p< 0.01 vs. respective scrambled (S) group. x p< 0.05 and xx p< 0.01 vs. respective naive rat in each group (C, S or oAβ). The number of animals in each group is indicated within the columns.

Figure 2

 2 Figure 2

Figure 3

 3 Figure 3 The effects in the PFC of CORT113176 on the activation of GR induced by the icv injection of oAβ 25-35 , were evaluated by Western blot. Variations of the expression of MR (100 kDa) (Panels A,B) and GR (95 kDa) (Panels A,C), the phosphorylation of GR (p[Ser211]GR, 95 kDA) (Panels A,D) and the expression ratio of HSP90/HSP70 (90 kDa / 70 kDa) (Panels A,E) were evaluated in control (C -white column) and in icv injected rats with 10 µg/rat of scrambled peptide (S -orange column) or oAβ 25-35 (Aβ -red column), treated or not with vehicle (sesame oil) or the selective GR modulator CORT113176 (10 mg/kg per ip injection). For experimental protocol see Fig.1A. The variations of all proteins in the PFC were normalized with the variations of β-tubulin (β-tub, 50 kDa) and compared with non-injected rats (control group: C). Two-way ANOVA: MR: F 2,54 = 11.0 for group, p< 0.0001; F 2,54 = 2.96 for treatment, p< 0.05; and F 4,54 = 3.06 for interaction, p< 0.05; GR: F 2,51 = 22.1 for group, p< 0.0001; F 2,51 = 8.45 for treatment, p< 0.001; and F 4,51 = 5.97 for

Figure 4

 4 Figure 4 The effects in the PFC of CORT113176 on the activation of GSK-3β and Cdk5 pathways induced by the icv injection of oAβ 25-35 , were evaluated by Western blot. The activation of GSK-3β (ratio of p[Tyr216]GSK-3β/GSK-3β total & ratio of p[Ser9]GSK-3β/GSK-3β total, 46kDa each) (Panels A-C) and Cdk5 (30 kDa) (Panels A,D) pathways, the expression ratio of p25/p35 (25 & 35 kDa) (Panels A,E), Calpain 1 (80 kDa) (Panels A,F) and FYN (59 kDa) (Panels A,G) were evaluated in control (C -white column) and in icv injected rats with 10 µg/rat of scrambled peptide (S -orange column) or oAβ 25-35 (Aβred column), treated or not with vehicle (sesame oil) or the selective GR modulator CORT113176 (10 mg/kg per ip injection). For experimental protocol see Fig.1A. The variations of all proteins in the PFC were normalized with the variations of β-tubulin (β-tub, 50 kDa) and compared with non-injected rats (control group: C). Two-way ANOVA: p[Tyr216]GSK-3β/GSK-3β: F 2,51 = 17.0 for group, p< 0.0001; F 2,51 = 6.07 for treatment, p< 0.01; and F 4,51 = 2.49 for interaction, p< 0.05; p[Ser9]GSK-3β/GSK-3β: F 2,51 = 11.9 for group, p< 0.0001; F 2,51 = 6.99 for treatment, p< 0.01; and F 4,51 = 2.81 for interaction, p< 0.05; Cdk5: F 2,48 = 12.1 for group, p< 0.0001; F 2,48 = 0.87 for treatment, ns; and F 4,48 = 3.73 for interaction, p< 0.01; p25/p35: F 2,47 = 13. 2 for group, p< 0.0001; F 2,47 = 12.1 for treatment, p< 0.0001; and F 4,47 = 16.5 for interaction, p< 0.0001; Calpain 1: F 2,49 = 15.5 for group, p< 0.0001; F 2,49 = 5.54 for treatment, p< 0.01; and F 4,49 = 4.10 for interaction, p< 0.01; Fyn: F 2,50 = 16.1 for group, p< 0.0001; F 2,50 = 2.82 for treatment, p< 0.05; and F 4,50 = 4.70 for interaction, p< 0.01. The variations are expressed as means ± SEM in % of control values. * p< 0.05 and ** p< 0.01 vs. respective control (C) group. + p< 0.05 and ++ p< 0.01 vs. respective scrambled (S) group. x p< 0.05 and xx p< 0.01 vs. respective naive rat in each group (C, S or Aβ). The number of animals in each group is indicated within the columns.

Figure 5

 5 Figure 5

Figure 6

 6 Figure 6 The effects in the PFC of selective GR modulator on the non-amyloidogenic pathways (Panel A) modulated by the icv injection of oAβ 25-35 , were evaluated by Western blot. Variations of sAPPα (100 kDa) (Panels A,B), ADAM10 (α-secretase, 70 kDa) (Panels A,C), Rho-associated coiled-coil kinases (ROCK1 & ROCK2, 160 kDa) (Panels A,D,E) and 3-phosphoinositide-dependent kinase (PDK1, 60 kDa) (Panels A,F) were evaluated in control (C -white column) and in icv injected rats with 10 µg/rat of scrambled peptide (S -orange column) or oAβ 25-35 (Aβ -red column), treated or not with vehicle (sesame oil) or selective GR modulator, CORT113176 (10 mg/kg per ip injection). For experimental protocol see Fig.1A. The variations of sAPPα, ADAM10, ROCK1, ROCK2 and PDK1 in the PFC were normalized with the variations of β-tubulin (β-tub, 50 kDa) and compared with non-injected rats (control group: C). Two-way ANOVA: sAPPα: F 2,49 = 8.65 for group, p< 0.001; F 2,49 = 3.92 for treatment, p< 0.05; and F 4,49 = 4.25 for interaction, p<

Figure 7

 7 Figure 7 Schematic figure recapitulating the central role of GR and the therapeutic potential of selective GR modulators in AD. The icv injection of Aβ oligomers increases APP, C99

  Blue arrow: hypothesis to assess. Red cross: Schematic effects of sGRm. AD: Alzheimer's Disease; ADAM10: A disintegrin and metalloproteinase domain-containing protein 10 (α-secretase); APP: amyloid precursor protein; Aβ: amyloid-β peptide; BACE-1: β-APP cleaving enzyme (β-secretase); Cdk5: cyclin-dependent kinase-5; GC: glucocorticoids; GR: glucocorticoid receptors; GRE: glucocorticoid responsive element; GSK-3β: glycogen synthase kinase 3β; HSP: heat-shock protein; IDE: insulin degrading enzyme; PDK1: 3-phosphoinositidedependent kinase; PS1: Presenilin 1 (γ-secretase); ROCKs: Rho-associated coiled-coil kinases; sGRm: selective GR modulator (CORT113176).

Figure 1

 1 Figure 1

Figure 7

 7 Figure 7

ACKNOWLEDGEMENTS

The authors are thankful to Elisabeth HUETTER and the animal facility staff (CECEMA, University of Montpellier, France) for their daily assistance. They also particularly thank Corcept Therapeutics for generously providing glucocorticoid receptor ligand. Parts of this study were supported by INSERM, University of Montpellier and EPHE annual resources (France), by a grant from "France Alzheimer" and "Fédération pour la Recherche sur le Cerveau" (Grant AAP SM2016#1512 -LG), by a grant from the "Agence Nationale de la Recherche" (ANR) under the program "Investissements d'Avenir" (ANR-11-LABEX-0021-LipSTIC -CD) and by a grant from Corcept Therapeutics (Menlo Park, CA, USA; Exceptional subvention -LG). G. CANET is supported by a PhD Grant from the University of Montpellier, France (CBS2 PhD program). C. HERNANDEZ is supported by a PhD Grant from the University of Montpellier, France (CBS2 PhD program).

Conflict of interest statement: JKB and HH are employees of Corcept Therapeutics (Menlo Park, CA, USA), which develops GR ligands for clinical use. Corcept Therapeutics provided compounds and financed part of the experiments. However, they are not involved in the experimental design, results analysis and conclusions of the present study. All other authors declare that they have no competing interests. and intracellular enzymes (ROCK1, ROCK2 and PDK1).

S-Figure 3

The effects in the PFC of selective GR modulator on the endogenous Aβ