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International audience; Objective: Although epilepsies and neurodegenerative disorders show pathophysiological similarities, their direct functional associations are unclear. Here, we tested the hypothesis that experimental seizures can induce tau hyperphosphorylation and amyloidogenic modifications over time, with intersections with neuroinflammation.Methods: We used a model of mesial temporal lobe epilepsy (MTLE) where unilateral intrahippocampal injection of kainic acid (KA) in C57BL/6 mice elicits epileptogenesis and spontaneous focal seizures. We used a model of generalized status epilepticus (SE) obtained by intraperitoneal KA injection in C57BL/6 mice. We performed analyses and cross-comparisons according to a schedule of 72 h, 1 week, and 8 weeks after KA injection.Results: In experimental MTLE, we show AT100, PHF1, and CP13 tau hyperphosphorylation during epileptogenesis (72 h-1 week) and long-term (8 weeks) during spontaneous seizures in the ipsilateral hippocampi, the epileptogenic zone. These pathological modifications extended to the contralateral hippocampus, a seizure propagating zone with no histological lesion or sclerosis. Two kinases, Cdk5 and GSK3β, implicated in the pathological phosphorylation of tau, were activated. In this MTLE model, the induction of the amyloidogenic pathway (APP, C99, BACE1) was prominent and long-lasting in the epileptogenic zone. These Alzheimer's disease (AD)-relevant markers, established during seizure progression and recurrence, reciprocated an enduring glial (GFAP, Iba1) inflammation and the inadequate activation of the endogenous, anti-inflammatory, glucocorticoid receptor system. By contrast, a generalized SE episode provoked a predominantly transient induction of tau hyperphosphorylation and amyloidogenic markers in the hippocampus, along with resolving inflammation. Finally, we identified overlapping profiles of long-term hippocampal tau hyperphosphorylation by comparing MTLE to J20 mice, the latter a model relevant to AD.Significance: MTLE and a generalized SE prompt persistent and varying tau hyperphosphorylation or amyloidogenic modifications in the hippocampus. In MTLE, an AD-relevant molecular trajectory intertwines with neuroinflammation, spatiotemporally involving epileptogenic and nonlesional seizure propagating zones.

Introduction: Among the risk factors identified in the sporadic forms of Alzheimer’s disease (AD), environmental and lifestyle elements are of growing interest. Clinical observations suggest that stressful events can anticipate AD onset, while stress-related disorders can promote AD. Here, we tested the hypothesis that a chronic treatment with glucocorticoids is sufficient to trigger or exacerbate AD molecular hallmarks. Methods: We first validated a rat model of experimental chronic glucocorticoids (GC) consumption (corticosterone [CORT] in drinking water for 4 weeks). Then, to evaluate the consequences of chronic GC consumption on the onset of amyloid-β (Aβ) toxicity, animals chronically treated with GC were intracerebroventricularly injected with an oligomeric solution of Aβ25–35 (oAβ) (acute model of AD). We evaluated AD-related cognitive deficits and pathogenic mechanisms, with a special emphasis on neuroinflammatory markers. Results: Chronic CORT consumption caused the inhibition of the nonamyloidogenic pathways, the impairment of Aβ clearance processes and the induction of amyloidogenic pathways in the hippocampus. The principal enzymes involved in glucocorticoid receptor activation and Tau phosphorylation were upregulated. Importantly, the AD-like phenotype triggered by chronic CORT was analogous to the one caused by oAβ. These molecular commonalities across models were independent from inflammation, as chronic CORT was immunosuppressive while oAβ was pro-inflammatory. When chronic CORT consumption anticipated the induction of the oAβ pathology, we found a potentiation of neuroinflammatory processes associated with an exacerbation of synaptic and memory deficits but also an aggravation of AD-related hallmarks. Discussion/Conclusion: This study unravels new functional outcomes identifying chronic CORT consumption as a main risk factor for AD and suggests that glucocorticoid-based therapies should be prescribed with caution in populations with AD risk.

G protein-coupled receptors (GPCR), including the metabotrobic glutamate 5 receptor (mGlu5), are important therapeutic targets and the development of allosteric ligands for targeting GPCRs has become a desirable approach toward modulating receptor activity. Traditional pharmacological approaches toward modulating GPCR activity are still limited since precise spatiotemporal control of a ligand is lost as soon as it is administered. Photopharmacology proposes the use of photoswitchable ligands to overcome this limitation, since their activity can be reversibly controlled by light with high precision. As this is still a growing field, our understanding of the molecular mechanisms underlying the light-induced changes of different photoswitchable ligand pharmacology is suboptimal. For this reason, we have studied the mechanisms of action of alloswitch-1 and MCS0331; two freely diffusible, mGlu5 phenylazopyridine photoswitchable negative allosteric modulators. We combined photochemical, cell-based, and in vivo photopharmacological approaches to investigate the effects of trans–cis azobenzene photoisomerization on the functional activity and binding ability of these ligands to the mGlu5 allosteric pocket. From these results, we conclude that photoisomerization can take place inside and outside the ligand binding pocket, and this leads to a reversible loss in affinity, in part, due to changes in dissociation rates from the receptor. Ligand activity for both photoswitchable ligands deviates from high-affinity mGlu5 negative allosteric modulation (in the trans configuration) to reduced affinity for the mGlu5 in their cis configuration. Importantly, this mechanism translates to dynamic and reversible control over pain following local injection and illumination of negative allosteric modulators into a brain region implicated in pain control., We thank Carolina Cera and Teresa Sarrias (SimChem, IQAC-CSIC, Barcelona) and Anna Duran, Roser Borràs and Gloria Somalo (MCS group, IQAC-CSIC, Barcelona) for technical support. We thank Maria José Bleda Hernández (IQAC-CSIC, Barcelona) for statistical support. We thank Jean-Philippe Pin (IGF, Montpellier) for scientific support and contributions to this work. We thank Christine Enjalbal (IBMM, Montpellier), Thierry Durroux (IGF, Montpellier), Jean-Louis Banères (IBMM, Montpellier) and Guillaume Lebon (IGF, Montpellier) for scientific discussion. This research was supported by FEDER/Ministerio de Ciencia, Innovación y Universidades–Agencia Estatal de Investigación (CTQ2017-89222-R and PCI2018-093047), the Catalan government (2017SGR1604), by CSIC (PICS program 08212), by Neuron-ERANET, by grants from the Agence Nationale de la Recherche (ANR-16-CE16-0010 and ANR-17-NEU3-0001 under the frame of Neuron Cofund) and by the Programme International de Collaboration Scientifique of the CNRS (PICS 08212). AEB was supported by the Labex EpiGenMed (program « Investissements d’avenir », ANR-10-LABX-12-01).

Dysregulation of the hypothalamic-pituitary-adrenal (HPA) axis occurs early in Alzheimer's disease (AD), associated with elevated circulating glucocorticoids (GC) and glucocorticoid receptors (GR) signaling impairment. However, the precise role of GR in the pathophysiology of AD remains unclear. Using an acute model of AD induced by the intracerebroventricular injection of amyloid-beta oligomers (oA beta), we analyzed cellular and behavioral hallmarks of AD, GR signaling pathways, processing of amyloid precursor protein, and enzymes involved in Tau phosphorylation. We focused on the prefrontal cortex (PFC), particularly rich in GR, early altered in AD and involved in HPA axis control and cognitive functions. We found that oA beta impaired cognitive and emotional behaviors, increased plasma GC levels, synaptic deficits, apoptosis and neuroinflammatory processes. Moreover, oA beta potentiated the amyloidogenic pathway and enzymes involved both in Tau hyperphosphorylation and GR activation. Treatment with a selective GR modulator (sGRm) normalized plasma GC levels and all behavioral and biochemical parameters analyzed. GR seems to occupy a central position in the pathophysiology of AD. Deregulation of the HPA axis and a feed-forward effect on PFC GR sensitivity could participate in the etiology of AD, in perturbing A beta and Tau homeostasis. These results also reinforce the therapeutic potential of sGRm in AD.

OptoGluNAM4.1, a negative allosteric modulator (NAM) of metabotropic glutamate receptor 4 (mGlu4) contains a reactive group that covalently binds to the receptor and a blue-light-activated, fast-relaxing azobenzene group that allows reversible receptor activity photocontrol in vitro and in vivo. OptoGluNAM4.1 induces light-dependent behavior in zebrafish and reverses the activity of the mGlu4 agonist LSP4-2022 in a mice model of chronic pain, defining a photopharmacological tool to better elucidate the physiological roles of the mGlu4 receptor in the nervous system. © 2016 Elsevier Ltd, We are grateful to C. Serra, L. Muñoz, J. Hernando, F. Malhaire, Y. Pérez, M. Izquierdo-Serra, F. Aguado, S. Laffray, E. Bourinet, F.Codony (GenIUL), and Y. Chomis (Viewpoint) for helpful discussions and technical support. A.F. is supported by a Fondation Lefoulon Delalande postdoctoral fellowship , C.J. by a grant from INSERM ATIP-AVENIR and Marie Curie CIG ( PCIG12-GA-2012-332772 ). A.F. and C.J. are members of the Laboratory of Excellence Ion Channel Science and Therapeutics supported by a grant from the ANR . J.-P.P. is a member of the Laboratory of Excellence Epingenmed. We acknowledge financial support from the European Union's Seventh Framework Program for research, technological development, and demonstration under grant agreements 270483 (Focus), 210355 (Opticalbullet), and 335011 (Theralight) to P.G.; the Federation of European Biochemical Societies ; the Catalan government ( 2012FI_B 01122 to S.P., 2014SGR-1251 to P.G., and 2014SGR-0109 to A. Llebaria); the Spanish Government ( SAF2014-58396-R to J.G., CTQ2014-57020-R to A.L., and CTQ2013-43892R to P.G.); the ERANET Neuron LIGHTPAIN project (to A.L., J.G., and J.-P.P.); the Ramón Areces Foundation , the ERANET SynBio Modulightor project (to P.G.); the HBP Wavescales project (to P.G.); the Fondation Recherche Médicale (FRM team DEQ20130326522 to J.P.P.); the Agence Nationale de la Recherche ( ANR-13-BSV1-006 to C.G.) and the Beatriu de Pinós program of Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR) for the support of X.R.

Overgeneralization of conditioned threat responses is a robust clinical marker of anxiety disorders. In overgeneralization, responses that are appropriate to threat-predicting cues are evoked by perceptually similar safety-predicting cues. Inappropriate learning of conditioned threat responses may thus form an etiological basis for anxiety disorders. The role of dopamine (DA) in memory encoding is well established. Indeed by signaling salience and valence, DA is thought to facilitate discriminative learning between stimuli representing safety or threat. However, the neuroanatomical and biochemical substrates through which DA modulates overgeneralization of threat responses remain poorly understood. Here we report that the modulation of DA D2 receptor (D2R) signaling bidirectionally regulates the consolidation of fear responses. While the blockade of D2R induces generalized threat responses, its stimulation facilitates discriminative learning between stimuli representing safety or threat. Moreover, we show that controlled threat generalization requires the coordinated activation of D2R in the bed nucleus of the stria terminalis and the central amygdala. Finally, we identify the mTORC1 cascade activation as an important molecular event by which D2R mediates its effects. These data reveal that D2R signaling in the extended amygdala constitutes an important checkpoint through which DA participates in the control of threat processing and the emergence of overgeneralized threat responses.Molecular Psychiatry advance online publication, 19 January 2016; doi:10.1038/mp.2015.210.

International audience; Contrary to acute pain, chronic pain does not serve as a warning signal and must be considered as a disease per se. This pathology presents a sensory and psychological dimension at the origin of affective and cognitive disorders. Being largely refractory to current pharmacotherapies, identification of endogenous systems involved in persistent and chronic pain is crucial. The amygdala is a key brain region linking pain sensation with negative emotions. Here, we show that activation of a specific intrinsic neuromodulatory system within the amygdala associated with type 4 metabotropic glutamate receptors (mGlu4) abolishes sensory and affective symptoms of persistent pain such as hypersensitivity to pain, anxiety- and depression-related behaviors, and fear extinction impairment. Interestingly, neuroanatomical and synaptic analysis of the amygdala circuitry suggests that the effects of mGlu4 activation occur outside the central nucleus via modulation of multisensory thalamic inputs to lateral amygdala principal neurons and dorso-medial intercalated cells. Furthermore, we developed optogluram, a small diffusible photoswitchable positive allosteric modulator of mGlu4. This ligand allows the control of endogenous mGlu4 activity with light. Using this photopharmacological approach, we rapidly and reversibly inhibited behavioral symptoms associated with persistent pain through optical control of optogluram in the amygdala of freely behaving animals. Altogether, our data identify amygdala mGlu4 signaling as a mechanism that bypasses central sensitization processes to dynamically modulate persistent pain symptoms. Our findings help to define novel and more precise therapeutic interventions for chronic pain, and exemplify the potential of optopharmacology to study the dynamic activity of endogenous neuromodulatory mechanisms in vivo.

Brain-derived neurotrophic factor (BDNF) appears to be highly involved in hypothalamic-pituitary-adrenal (HPA) axis regulation during adulthood, playing an important role in homeostasis maintenance. The present study aimed to determine the involvement of BDNF in HPA axis activity under basal and stress conditions via partial inhibition of this endogenous neurotrophin. Experiments were conducted in rats and mice with two complementary approaches: (i) BDNF knockdown with stereotaxic delivery of BDNF-specific small interfering RNA (siRNA) into the lateral ventricle of adult male rats and (ii) genetically induced knockdown (KD) of BDNF expression specifically in the central nervous system during the first ontogenesis in mice (KD mice). Delivery of siRNA in the rat brain decreased BDNF levels in the hippocampus (-31%) and hypothalamus (-35%) but not in the amygdala, frontal cortex and pituitary. In addition, siRNA induced no change of the basal HPA axis activity. BDNF siRNA rats exhibited decreased BDNF levels and concomitant altered adrenocortoctrophic hormone (ACTH) and corticosterone responses to restraint stress, suggesting the involvement of BDNF in the HPA axis adaptive response to stress. In KD mice, BDNF levels in the hippocampus and hypothalamus were decreased by 20% in heterozygous and by 60% in homozygous animals compared to wild-type littermates. Although, in heterozygous KD mice, no significant change was observed in the basal levels of plasma ACTH and corticosterone, both hormones were significantly increased in homozygous KD mice, demonstrating that robust cerebral BDNF inhibition (60%) is necessary to affect basal HPA axis activity. All of these results in both rats and mice demonstrate the involvement and importance of a robust endogenous pool of BDNF in basal HPA axis regulation and the pivotal function of de novo BDNF synthesis in the establishment of an adapted response to stress.

Phenylazopyridines are photoisomerizable compounds with high potential to control biological functions with light. We have obtained a series of phenylazopyridines with light dependent activity as negative allosteric modulators (NAM) of metabotropic glutamate receptor subtype 5 (mGlu5). Here we describe the factors needed to achieve an operational molecular photoisomerization and its effective translation into in vitro and in vivo receptor photoswitching, which includes zebrafish larva motility and the regulation of the antinociceptive effects in mice. The combination of light and some specific phenylazopyridine ligands displays atypical pharmacological profiles, including light-dependent receptor overactivation, which can be observed both in vitro and in vivo. Remarkably, the localized administration of light and a photoswitchable compound in the peripheral tissues of rodents or in the brain amygdalae results in an illumination-dependent analgesic effect. The results reveal a robust translation of the phenylazopyridine photoisomerization to a precise photoregulation of biological activity. © 2016 American Chemical Society., We are grateful to C. Serra and L. Muñoz for synthetic and analytical support, F. Malhaire for technical support in cell-based pharmacological assays, and Y. Pérez for NMR support. This research has been supported by RecerCaixa foundation (2010ACUP00378 to P.G., J.G., and A.L.), the Marató de TV3 Foundation (110230 to J.G., 110231 to A.L., 110232 to C.G., and 111531 to P.G.), the Catalan government (2010 BP-A 00194 to X.R., 2012FI_B 01122 to S.P., 2012 CTP 00033 and 2012 BE1 00597 to X.G.-S., 2014SGR-1251 to P.G., and 2014SGR-00109 to A.L.), the Spanish Government (CTQ2014-57020-R and PCIN-2013-017-C03-01 to A.L., PIE14/00034, SAF2014-55700-P, and PCIN-2013-019-C03-03 to F.C., and SAF2014-58396-R to J.G.), AWT (SBO-140028) to F.C., and the ERANET Neuron LIGHTPAIN project (to A.L., F.C., J.G., and J.-P.P.); SynBio MODULIGHTOR, Human Brain Project WAVESCALES and Ramon Areces foundation grants (to P.G.); the Agence Nationale de la Recherche (ANR-16-CE16-0010 to A.L. and C.G.).

International audience

International audience; The coxsackievirus and adenovirus receptor (CAR) serves as a docking factor for some adenovirus (AdV) types and group B coxsackieviruses. Its role in AdV internalisation is unclear as studies suggest that its intracellular domain is dispensable for some AdV serotype infection. We previously showed that in motor neurons, an adenovirus induced CAR internalisation, and their co-transport in axons, suggesting that CAR was linked to endocytic and long-range transport machineries. Here, we characterised the mechanisms of CAR endocytosis in neurons and neuronal cells. We show that ligands that disrupted the homodimeric CAR interactions in its D1 domains triggered an internalisation cascade involving sequences in its intracellular tail. CAR-associated internalisation was also lipid microdomain, actin- and dynamin-dependent, and subsequently followed by CAR degradation by lysosomes.

Elevated cortisol evidence in Alzheimer's disease (AD) patients prompted the hypothesis that stress and glucocorticoids are involved in the development and/or maintenance of AD. We investigated the hypothalamic-pituitary-adrenal (HPA) axis activity, functionality, and reactivity for up to 6 weeks after an intracerebroventricular injection of amyloid-β(25-35) peptide (Aβ(25-35)) in rat, a validated acute model of AD. Aβ(25-35) induces memory impairment, alteration of anxiety responses, HPA axis hyperactivity, and glucocorticoid (GR) and mineralocorticoid (MR) receptor increases in brain regions related to HPA axis functions. GR are progressively translocated in neurons nucleus, while membrane version of MR is evidenced in all structures considered. The MR/GR ratio was modified in all structures considered. Aβ(25-35) induces a subtle disturbance in the feedback of the HPA axis, without modifying its functionality. The reactivity alteration is long-lasting, suggesting that amyloid toxicity affects the HPA axis adaptive response to stress. These findings are evidence of progressive HPA axis deregulation after Aβ(25-35), which is associated with an imbalance of MR/GR ratio and a disruption of the glucocorticoid receptors nucleocytoplasmic shuttling, and suggest that elevated glucocorticoids observed in AD could be first a consequence of amyloid toxicity.

Although we are beginning to understand the late stage of neurodegenerative diseases, the molecular defects associated with the initiation of impaired cognition are poorly characterized. Here, we demonstrate that in the adult brain, the coxsackievirus and adenovirus receptor (CAR) is located on neuron projections, at the presynapse in mature neurons, and on the soma of immature neurons in the hippocampus. In a proinflammatory or diseased environment, CAR is lost from immature neurons in the hippocampus. Strikingly, in hippocampi of patients at early stages of late-onset Alzheimer's disease (AD), CAR levels are significantly reduced. Similarly, in triple-transgenic AD mice, CAR levels in hippocampi are low and further reduced after systemic inflammation. Genetic deletion of CAR from the mouse brain triggers deficits in adult neurogenesis and synapse homeostasis that lead to impaired hippocampal plasticity and cognitive deficits. We propose that post-translational CAR loss of function contributes to cognitive defects in healthy and diseased-primed brains.SIGNIFICANCE STATEMENTThis study addressed the role of the coxsackievirus and adenovirus receptor (CAR), a single-pass cell adhesion molecule, in the adult brain. Our results demonstrate that CAR is expressed by mature neurons throughout the brain. In addition, we propose divergent roles for CAR in immature neurons, during neurogenesis, and at the mature synapse. Notably, CAR loss of function also affects hippocampal plasticity.

Vectors derived from the canine adenovirus serotype 2 (CAV-2) possess a high neurotropism and efficient retrograde transport that lead to widespread neuronal transduction in the central nervous system (CNS) of various animals. These abilities are due to the engagement of virions to the coxsackievirus and adenovirus receptor at the surface of neurons, which is linked to the endocytic and axonal transport machineries. The trafficking of CAV-2 and the coxsackievirus and adenovirus receptor (CAR) can be visualized ex vivo by incubating primary neurons (e.g., motoneurons and hippocampal neurons) with fluorescently labeled virions or recombinant viral proteins. Using this approach, we could recapitulate the mechanisms responsible for long-range transport of adenovirus in neurons.

Vectors derived from the canine adenovirus serotype 2 (CAV-2) possess a high neurotropism and efficient retrograde transport that lead to widespread neuronal transduction in the central nervous system (CNS) of various animals. These abilities are due to the engagement of virions to the coxsackievirus and adenovirus receptor at the surface of neurons, which is linked to the endocytic and axonal transport machineries. The trafficking of CAV-2 and the coxsackievirus and adenovirus receptor (CAR) can be visualized ex vivo by incubating primary neurons (e.g., motoneurons and hippocampal neurons) with fluorescently labeled virions or recombinant viral proteins. Using this approach, we could recapitulate the mechanisms responsible for long-range transport of adenovirus in neurons.

Alzheimer's disease (AD) is a neurodegenerative pathology characterized by the presence of senile plaques and neurofibrillary tangles, accompanied by synaptic and neuronal loss. The major component of senile plaques is an amyloid β protein (Aβ) formed by pathological processing of the Aβ precursor protein. We assessed the time-course and regional effects of a single intracerebroventricular injection of aggregated Aβ fragment 25-35 (Aβ(25-35)) in rats. Using a combined biochemical, behavioral, and morphological approach, we analyzed the peptide effects after 1, 2, and 3 weeks in the hippocampus, cortex, amygdala, and hypothalamus. The scrambled Aβ(25-35) peptide was used as negative control. The aggregated forms of Aβ peptides were first characterized using electron microscopy, infrared spectroscopy, and Congo Red staining. Intracerebroventricular injection of Aβ(25-35) decreased body weight, induced short- and long-term memory impairments, increased endocrine stress, cerebral oxidative and cellular stress, neuroinflammation, and neuroprotective reactions, and modified endogenous amyloid processing, with specific time-course and regional responses. Moreover, Aβ(25-35), the presence of which was shown in the different brain structures and over 3 weeks, provoked a rapid glial activation, acetylcholine homeostasis perturbation, and hippocampal morphological alterations. In conclusion, the acute intracerebroventricular Aβ(25-35) injection induced substantial central modifications in rats, highly reminiscent of the human physiopathology, that could contribute to physiological and cognitive deficits observed in AD.