High-content multimodal analysis supports the IL-7/IL-7 receptor axis as a relevant therapeutic target in primary Sjögren's syndrome.

OBJECTIVE: While the involvement of IL-7/IL-7R axis in pSS has been described in relation to T cells, little is known about the contribution of this pathway in relationship with other immune cells, and its implication in autoimmunity. Using high-content multiomics data, we aimed at characterizing IL-7R expressing cells and the involvement of IL-7/IL-7R pathway in pSS pathophysiology. METHODS: An IL-7 signature established using RNA-sequencing of human PBMCs incubated with IL-7 was applied to 304 pSS patients, and on RNA-Seq datasets from tissue biopsies. High-content immunophenotyping using flow and imaging mass cytometry was developed to characterize peripheral and in situ IL-7R expression. RESULTS: We identified a blood 4-gene IL-7 module (IKZF4, KIAA0040, PGAP1 and SOS1) associated with anti-SSA/Ro positiveness in patients as well as disease activity, and a tissue 5-gene IL-7 module (IL7R, PCED1B, TNFSF8, ADAM19, MYBL1) associated with infiltration severity. We confirmed expression of IL-7R on T cells subsets, and further observed upregulation of IL-7R on double-negative (DN) B cells, and especially DN2 B cells. IL-7R expression was increased in pSS compared to sicca patients with variations seen according to the degree of infiltration. When expressed, IL-7R was mainly found on epithelial cells, CD4+ and CD8+ T cells, switched memory B cells, DN B cells and M1 macrophages. CONCLUSION: This exhaustive characterization of the IL-7/IL-7R pathway in pSS pathophysiology established that two IL-7 gene modules discriminate pSS patients with a high IL-7 axis involvement. Their use could guide the implementation of an anti-IL-7R targeted therapy in a precision medicine approach.

Allosteric modulation of AMPA receptors counteracts Tau-related excitotoxic synaptic signaling and memory deficits in stress- and Aß-evoked hippocampal pathology.

Despite considerable progress in the understanding of its neuropathology, Alzheimer's disease (AD) remains a complex disorder with no effective treatment that counteracts the memory deficits and the underlying synaptic malfunction triggered by the accumulation of amyloid beta (Aß) and Tau protein. Mounting evidence supports a precipitating role for chronic environmental stress and glutamatergic excitotoxicity in AD, suggesting that targeting of glutamate receptor signaling may be a promising approach against both stress and AD pathologies. In light of the limited cognitive benefit of the direct antagonism of NMDA receptors in AD, we here focus on an alternative way to modify glutamatergic signaling through positive allosteric modulation of AMPA receptors, by the use of a PAM-AMPA compound. Using non-transgenic animal model of Aß oligomer injection as well as the combined stress and Aß i.c.v. infusion, we demonstrate that positive allosteric modulation of AMPA receptors by PAM-AMPA treatment reverted memory, but not mood, deficits. Furthermore, PAM-AMPA treatment reverted stress/Aß-driven synaptic missorting of Tau and associated Fyn/GluN2B-driven excitotoxic synaptic signaling accompanied by recovery of neurotransmitter levels in the hippocampus. Our findings suggest that positive allosteric modulation of AMPA receptors restores synaptic integrity and cognitive performance in stress- and Aß-evoked hippocampal pathology. As the prevalence of AD is increasing at an alarming rate, novel therapeutic targeting of glutamatergic signaling should be further explored against the early stages of AD synaptic malfunction with the goal of attenuating further synaptic damage before it becomes irreversible.

A 24-week double-blind placebo-controlled study of the efficacy and safety of the AMPA modulator S47445 in patients with mild to moderate Alzheimer's disease and depressive symptoms.

INTRODUCTION: S47445 is a novel positive allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors that may emerge as a favorable candidate for the symptomatic treatment of cognitive and depressive disorders in patients suffering from Alzheimer's disease (AD) of mild to moderate severity and with depressive symptoms. METHODS: For this double-blind, placebo-controlled 24-week phase II trial, 520 outpatients aged between 55 and 85 years, with probable AD at mild to moderate stages (a Mini-Mental State Examination score of 24-15 inclusive) and exhibiting depressive symptoms (Cornell Scale for Depression in Dementia [CSDD] ≥ 8) were recruited in twelve countries and randomized to 3 doses of S47445 (5-15-50 mg) or placebo. The primary end point was the change from baseline in the 11-item Alzheimer's Disease Assessment Scale-Cognitive subscale (ADAS-Cog) total score at week 24. Secondary measures included the Disability Assessment for Dementia, Mini-Mental State Examination, ADAS-Cog 13-item, CSDD, Clinical Global Impression of Change (Alzheimer's Disease Cooperative Study-CGIC), Neuropsychiatric Inventory (NPI), and safety criteria. RESULTS: Baseline characteristics were comparable between the 4 groups. After 24 weeks, no statistically significant treatment difference was demonstrated between S47445 (5, 15 or 50 mg/d) and placebo on cognition (ADAS-Cog), function (Disability Assessment for Dementia), or depressive symptoms (CSDD). An improvement on neuropsychiatric symptoms assessed by NPI was evidenced at the lower dose 5 mg/d (Δ -2.55, P = .023, post hoc analysis) compared to placebo. CSDD and total NPI scores improved in all groups including placebo. There were no specific and/or unexpected safety signals observed with any of the S47445 doses. DISCUSSION: S47445 administered for 24 weeks was safe and well tolerated by patients with mild to moderate AD; the compound did not show significant benefits over placebo on cognition, function, or depressive symptoms.

S 47445 counteracts the behavioral manifestations and hippocampal neuroplasticity changes in bulbectomized mice.

S 47445 is a positive allosteric modulator of glutamate AMPA-type receptors that possesses procognitive, neurotrophic and enhancing synaptic plasticity properties. Its chronic administration promotes antidepressant- and anxiolytic-like effects in different rodent models of depression. We have evaluated the behavioral effects of S 47445 in the bilateral olfactory bulbectomy mice model (OB) and the adaptive changes in those proteins associated to brain neuroplasticity (BDNF and mTOR pathway). Following OB surgery, adult C57BL/6J male mice were chronically administered S 47445 (1, 3 and 10 mg/kg/day; i.p.) and fluoxetine (18 mg/kg/day; i.p.), and then behaviorally tested in the open field test. Afterwards, the expression levels of BDNF, mTOR, phospho-mTOR, 4EBP1 and phospho-4EBP1 were evaluated in hippocampus and prefrontal cortex. Both drugs reduced the OB-induced locomotor activity, a predictive outcome of antidepressant efficacy, with a similar temporal pattern of action. S 47445, but not fluoxetine, showed an anxiolytic effect as reflected by an increased central activity. Chronic administration of S 47445 reversed OB-induced changes in BDNF and phopho-mTOR expression in hippocampus but not in prefrontal cortex. The chronic administration of S 47445 induced antidepressant- and anxiolytic-like effects at low-medium doses (1 and 3 mg/kg/day, i.p.) associated with the reversal of OB-induced changes in hippocampal BDNF and mTOR signaling pathways.

Pharmacological characterisation of S 47445, a novel positive allosteric modulator of AMPA receptors.

S 47445 is a novel positive allosteric modulator of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptors (AMPA-PAM). S 47445 enhanced glutamate's action at AMPA receptors on human and rat receptors and was inactive at NMDA and kainate receptors. Potentiation did not differ among the different AMPA receptors subtypes (GluA1/2/4 flip and flop variants) (EC50 between 2.5-5.4 µM), except a higher EC50 value for GluA4 flop (0.7 µM) and a greater amount of potentiation on GluA1 flop. A low concentration of S 47445 (0.1 µM) decreased receptor response decay time of GluA1flop/GluA2flip AMPA receptors and increased the sensitivity to glutamate. Furthermore, S 47445 (0.1 and 0.3 µM) in presence of repetitive glutamate pulses induced a progressive potentiation of the glutamate-evoked currents from the second pulse of glutamate confirming a rapid-enhancing effect of S 47445 at low concentrations. The potentiating effect of S 47445 (1 µM) was concentration-dependently reversed by the selective AMPA receptor antagonist GYKI52466 demonstrating the selective modulatory effect of S 47445 on AMPA receptors. Using an AMPA-kainate chimera approach, it was confirmed that S 47445 binds to the common binding pocket of AMPA-PAMs. S 47445 did not demonstrate neurotoxic effect against glutamate-mediated excitotoxicity in vitro, in contrast significantly protected rat cortical neurons at 10 µM. S 47445 was shown to improve both episodic and spatial working memory in adult rodents at 0.3 mg/kg, as measured in the natural forgetting condition of object recognition and T-maze tasks. Finally, no deleterious effect on spontaneous locomotion and general behavior was observed up to 1000 mg/kg of S 47445 given acutely in rodents, neither occurrence of convulsion or tremors. Collectively, these results indicate that S 47445 is a potent and selective AMPA-PAM presenting procognitive and potential neuroprotective properties. This drug is currently evaluated in clinical phase 2 studies in Alzheimer's disease and in Major Depressive Disorder.

S 47445 Produces Antidepressant- and Anxiolytic-Like Effects through Neurogenesis Dependent and Independent Mechanisms.

Glutamatergic dysfunctions are observed in the pathophysiology of depression. The glutamatergic synapse as well as the AMPA receptor's (AMPAR) activation may represent new potential targets for therapeutic intervention in the context of major depressive disorders. S 47445 is a novel AMPARs positive allosteric modulator (AMPA-PAM) possessing procognitive, neurotrophic properties and enhancing synaptic plasticity. Here, we investigated the antidepressant/anxiolytic-like effects of S 47445 in a mouse model of anxiety/depression based on chronic corticosterone administration (CORT) and in the Chronic Mild Stress (CMS) model in rats. Four doses of S 47445 (0.3 to 10 mg/kg, oral route, 4 and 5 weeks, respectively) were assessed in both models. In mouse, behavioral effects were tested in various anxiety-and depression-related behaviors : the elevated plus maze (EPM), open field (OF), splash test (ST), forced swim test (FST), tail suspension test (TST), fur coat state and novelty suppressed feeding (NSF) as well as on hippocampal neurogenesis and dendritic arborization in comparison to chronic fluoxetine treatment (18 mg/kg, p.o.). In rats, behavioral effects of S 47445 were monitored using sucrose consumption and compared to those of imipramine or venlafaxine (10 mg/kg, i.p.) during the whole treatment period and after withdrawal of treatments. In a mouse model of genetic ablation of hippocampal neurogenesis (GFAP-Tk model), neurogenesis dependent/independent effects of chronic S 47445 treatment were tested, as well as BDNF hippocampal expression. S 47445 reversed CORT-induced depressive-like state by increasing grooming duration and reversing coat state's deterioration. S 47445 also decreased the immobility duration in TST and FST. The highest doses (3 and 10 mg/kg) seem the most effective for antidepressant-like activity in CORT mice. Furthermore, S 47445 significantly reversed the anxiety phenotype observed in OF (at 1 mg/kg) and EPM (from 1 mg/kg). In the CMS rat model, S 47445 (from 1 mg/kg) demonstrated a rapid onset of effect on anhedonia compared to venlafaxine and imipramine. In the CORT model, S 47445 demonstrated significant neurogenic effects on proliferation, survival and maturation of hippocampal newborn neurons at doses inducing an antidepressant-like effect. It also corrected CORT-induced deficits of growth and arborization of dendrites. Finally, the antidepressant/anxiolytic-like activities of S 47445 required adult hippocampal neurogenesis in the novelty suppressed feeding test contrary to OF, EPM and ST. The observed increase in hippocampal BDNF levels could be one of the mechanisms of S 47445 responsible for the adult hippocampal neurogenesis increase. Altogether, S 47445 displays robust antidepressant-anxiolytic-like properties after chronic administration through neurogenesis dependent/independent mechanisms and neuroplastic activities. The AMPA-PAM S 47445 could have promising therapeutic potential for the treatment of major depressive disorders or generalized anxiety disorders.

The AMPA receptor positive allosteric modulator S 47445 rescues in vivo CA3-CA1 long-term potentiation and structural synaptic changes in old mice.

Positive allosteric modulators of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) are small molecules that decrease deactivation of AMPARs via an allosteric site. These molecules keep the receptor in an active state. Interestingly, this type of modulator has been proposed for treating cognitive decline in ageing, dementias, and Alzheimer's disease (AD). S 47445 (8-cyclopropyl-3-[2-(3-fluorophenyl)ethyl]-7,8-dihydro-3H-[1,3]oxazino[6,5-g][1,2,3]benzotriazine-4,9-dione) is a novel AMPAR positive allosteric modulator (AMPA-PAM). Here, the mechanisms by which S 47445 could improve synaptic strength and connectivity were studied and compared between young and old mice. A single oral administration of S 47445 at 10 mg/kg significantly increased long-term potentiation (LTP) in CA3-CA1 hippocampal synapses in alert young mice in comparison to control mice. Moreover, chronic treatment with S 47445 at 10 mg/kg in old alert animals significantly counteracted the deficit of LTP due to age. Accordingly, chronic treatment with S 47445 at 10 mg/kg seems to preserve synaptic cytoarchitecture in old mice as compared with young control mice. It was shown that the significant decreases in number and size of pre-synaptic buttons stained for VGlut1, and post-synaptic dendritic spines stained for spinophilin, observed in old mice were significantly prevented after chronic treatment with 10 mg/kg of S 47445. Altogether, by its different effects on LTP, VGlut1-positive particles, and spinophilin, S 47445 is able to modulate both the structure and function of hippocampal excitatory synapses known to be involved in learning and memory processes. These results open a new window for the treatment of specific age-dependent cognitive decline and dementias such as AD.

Upregulation of neurotrophins by S 47445, a novel positive allosteric modulator of AMPA receptors in aged rats.

At molecular levels, it has been shown that aging is associated with alterations in neuroplastic mechanisms. In this study, it was examined if the altered expression of neurotrophins observed in aged rats could be corrected by a chronic treatment with S 47445 (1-3-10mg/kg, p.o.), a novel selective positive allosteric modulator of the AMPA receptors. Both the mRNA and the protein levels of the neurotrophins Bdnf, NT-3 and Ngf were specifically measured in the prefrontal cortex and hippocampus (ventral and dorsal) of aged rats. It was found that 2-week-treatment with S 47445 corrected the age-related deficits of these neurotrophins and/or positively modulated their expression in comparison to vehicle aged rats in the range of procognitive and antidepressant active doses in rodents. Collectively, the ability of S 47445 to modulate various neurotrophins demonstrated its neurotrophic properties in two major brain structures involved in cognition and mood regulation suggesting its therapeutic potential for improving several diseases such as Alzheimer's disease and/or Major Depressive Disorders.

Calpain product of WT-CRMP2 reduces the amount of surface NR2B NMDA receptor subunit.

The brain is particularly vulnerable to ischaemia; however, neurons can become tolerant to ischaemic insult. This tolerance has been shown to involve activation of NMDA receptors, but its mechanisms have not yet been fully elucidated. Using a preconditioning protocol, we show that neurons surviving to a transient NMDA exposure become resistant to the glutamatergic agonist. Using a proteomic approach, we found that alterations of the protein pattern of NMDA-resistant neurons are restricted mainly to the five collapsin response mediator proteins (CRMPs). A sustained increase in calpain activity following NMDA treatment is responsible for the production of cleaved CRMPs. Finally, we provide evidence for the involvement of the cleaved form of WT-CRMP2 in the down-regulation of NR2B. Our data suggests that, beside their role in neuronal morphogenesis, CRMPs may contribute to neuronal plasticity.

Differential expression of CRMP1, CRMP2A, CRMP2B, and CRMP5 in axons or dendrites of distinct neurons in the mouse brain.

CRMP1, CRMP2, and CRMP5 have been identified as cytosolic proteins relaying semaphorin 3A signalling, one of the molecular cues conducting axon and dendrite growth and guidance. They are highly expressed during brain ontogenesis, but, because of their lower levels in the adult, their distribution in the mature brain is poorly documented. By using specific antibodies, we investigated the cellular distribution of these CRMPs in different adult brain structures and in neural cell cultures with a special focus on the splice variants CRMP2A and CRMP2B. In brain sections of adult mouse, CRMP1, CRMP2B, and CRMP5 were located predominantly in dendrites of specific neuronal populations, such as cortical pyramidal neurons, hippocampal CA1 pyramidal cells, or Purkinje cerebellar cells. On the contrary, CRMP2A was specifically associated with axons of the corpus callosum, bundles of the striatum, and mossy fibers of the hippocampus. In cultures of cortical neurons, CRMP1, CRMP2A, CRMP2B, and CRMP5 were equally distributed throughout cell bodies, axons, or dendrites of neurons, whereas CRMP2A and CRMP5 were completely absent from Purkinje cerebellar cells in 12-day-old animals. By comparison, oligodendrocytes exclusively express CRMP2B and CRMP5 in cell bodies and processes both in situ in the adult brain and in primary cultures. Overall, our results demonstrate specific subcellular localizations of CRMP1, CRMP2A, CRMP2B, and CRMP5 depending on cell types, neuronal compartment, and developmental stage. This study suggests that, beyond their signalling function in axon outgrowth and guidance, CRMPs also play a role in mature neurons both in axons and in dendrites.