Analysis of the R451C Neuroligin3 Knock-In mouse, a model of a monogenic form of autism

Autism Spectrum Disorders (ASDs) are neurodevelopmental syndromes, in which several environmental risk factors act on a vulnerable genetic background. Among genes whose mutations have been associated with ASDs, the R451C substitution in the synaptic protein Neuroligin3 (NLGN3) has been highly characterized. It is known from in vitro studies, that the mutation affects folding of the extracellular domain of the protein, causing its retention in the Endoplasmic Reticulum (ER) and the activation of the Unfolded Protein Response (UPR). It has been shown both in vitro and in vivo, that only ~10% of the mutant protein reach the synapse, causing loss of NLGN3 on the cell surface and leading to alterations in synaptic neurotransmission. In this work, we have evaluated whether UPR was activated in vivo, in the brain of the knock-in mouse model carrying the R451C mutation in the endogenous NLGN3. We showed a selective increase of UPR markers levels in the cerebellum of the R451C mice, along with an increase in the frequency of the miniature excitatory currents in the Purkinje cells, that resulted to be UPR-dependent. At the same time, in order to find a strategy to rescue NLGN3 folding and expression on the cell surface, we have generated and characterized a new cell-based model system that allowed studying NLGN3 protein trafficking. By using this system, we have screened an FDA-approved library of compounds for improving impaired protein folding. Among the compounds that have been tested, several members of the glucocorticoid family showed efficacy in increasing mutant protein trafficking and restoring membrane localization. Collectively, our data indicated that the ER-retention of R451C NLGN3 in vivo, caused UPR activation and alterations of synaptic function in the cerebellum of a mouse model of a monogenic form of autism. Furthermore, we identified compounds improving NLGN3 folding and rescuing impaired trafficking

Mir-34a-5p Mediates Cross-Talk between M2 Muscarinic Receptors and Notch-1/EGFR Pathways in U87MG Glioblastoma Cells: Implication in Cell Proliferation

Glioblastoma (GBM) is the most aggressive human brain tumor. The high growth potential and decreased susceptibility to apoptosis of the glioma cells is mainly dependent on genetic amplifications or mutations of oncogenic or pro-apoptotic genes, respectively. We have previously shown that the activation of the M2 acetylcholine muscarinic receptors inhibited cell proliferation and induced apoptosis in two GBM cell lines and cancer stem cells. The aim of this study was to delve into the molecular mechanisms underlying the M2-mediated cell proliferation arrest. Exploiting U87MG and U251MG cell lines as model systems, we evaluated the ability of M2 receptors to interfere with Notch-1 and EGFR pathways, whose activation promotes GBM proliferation. We demonstrated that the activation of M2 receptors, by agonist treatment, counteracted Notch and EGFR signaling, through different regulatory cascades depending, at least in part, on p53 status. Only in U87MG cells, which mimic p53-wild type GBMs, did M2 activation trigger a molecular circuitry involving p53, Notch-1, and the tumor suppressor mir-34a-5p. This regulatory module negatively controls Notch-1, which affects cell proliferation mainly through the Notch-1/EGFR axis. Our data highlighted, for the first time, a molecular circuitry that is deregulated in the p53 wild type GBM, based on the cross-talk between M2 receptor and the Notch-1/EGFR pathways, mediated by mir-34a-5p

Neurolign 3 misfolding mutations and activation of the unfolded protein response

Several forms of monogenic heritable autism spectrum disorders are associated with mutations in the neuroligin genes. The autism-linked substitution R451C in neuroligin3 induces local misfolding of its extracellular domain, causing partial retention in the ER (endoplasmic reticulum) of expressing cells. We have generated a PC12 Tet-On cell model system with inducible expression of wild-type or R451C neuroligin3 to investigate whether there is activation of the UPR (unfolded protein response) as a result of misfolded protein retention. As a positive control for protein misfolding, we also expressed the mutant G221R neuroligin3, which is known to be completely retained within the ER. Our data show that overexpression of either R451C or G221R mutant proteins leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6 (activating transcription factor 6), IRE1 (inositol-requiring enzyme 1) and PERK [PKR (dsRNA-dependent protein kinase)-like endoplasmic reticulum kinase]. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that up-regulation of BiP (immunoglobulin heavy-chain-binding protein) and CHOP [C/EBP (CCAAT/enhancer-binding protein)-homologous protein] was induced by both mutant proteins but not by wild-type neuroligin3, both in proliferative cells and cells differentiated to a neuron-like phenotype. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in a novel cell model system, suggesting that this cellular response may have a role in monogenic forms of autism characterized by misfolding mutations.This work was supported by: Compagnia San Paolo, Sapienza University of Rome and Pasteur Institute - Cenci Bolognetti Foundation grants to ADJ. SJM is a MRC Senior Clinical Research Fellow [MRC Ref G1002610]. This work was also supported by National Institutes of Health grants (MH092906), from the Robert Wood Johnson Foundation to the Child Health Institute of New Jersey [grant #67038] and to the Governor's Council for Medical Research and Treatment of Autism [CAUT14APL028] to D.C.This is the final version of the article. It first appeared from Portland Press via https://doi.org/http://dx.doi.org/10.1042/BJ2015027

Comparative mapping of selected structural determinants on the extracellular domains of cholinesterase-like cell-adhesion molecules.

International audienceCell adhesion generally involve formation of homophilic or heterophilic protein complexes between two cells to form transcellular junctions. Neural cell-adhesion members of the α/β-hydrolase fold superfamily of proteins use their extracellular or soluble cholinesterase-like domain to bind cognate partners across cell membranes, as illustrated by the neuroligins. These cell-adhesion molecules currently comprise the synaptic organizers neuroligins found in all phyla, along with three proteins found only in invertebrates: the guidance molecule neurotactin, the glia-specific gliotactin, and the basement membrane protein glutactin. Although these proteins share a cholinesterase-like fold, they lack one or more residues composing the catalytic triad responsible for the enzymatic activity of the cholinesterases. Conversely, they are found in various subcellular localisations and display specific disulfide bonding and N-glycosylation patterns, along with individual surface determinants possibly associated with recognition and binding of protein partners. Formation of non-covalent dimers typical of the cholinesterases is documented for mammalian neuroligins, yet whether invertebrate neuroligins and their neurotactin, gliotactin and glutactin relatives also form dimers in physiological conditions is unknown. Here we provide a brief overview of the localization, function, evolution, and conserved versus individual structural determinants of these cholinesterase-like cell-adhesion proteins

LINKING ENDOPLASMIC RETICULUM STRESS TO NEURODEVELOPMENTAL DISORDERS

Autism spectrum disorders are a group of neurodevelopmental disorders with a strong genetic background. One of the most characterized autism-linked mutations is the R451C substitution in the synaptic protein Neuroligin3 (NLGN3). The mutation induces a local misfolding in the extracellular domain causing the retention of NLGN3 in the Endoplasmic Reticulum (ER)1. The presence of misfolded protein in the ER can lead to the activation of the Unfolded Protein Response (UPR), implicated in several neurological diseases and in the regulation of neurotransmission and plasticity2. Our aim is to ascertain whether the ER retention of the R451C NLGN3 mutant protein activates the UPR. We have generated a new PC12 Tet-On model system with inducible expression of NLGN3, either wild type or R451C proteins, for studying the UPR signaling in time-course experiments. PC12 clones were characterized for NLGN3 expression, by western blots and immunofluorescence. Wild type NLGN3 protein is correctly trafficked to the cell surface, with the R451C NLGN3 being retained in the ER, as shown by sensitivity to endoglycosidase H. Our results indicate that PC12 clones expressing the R451C mutant NLGN3, activate all UPR signaling pathways downstream of the ATF6, IRE1 and PERK stress sensors. Synthesis of R451C NLGN3 induces the up-regulation of UPR target genes, such as BiP and CHOP, before and after differentiating the cells to a neuronal phenotype. In order to understand the potential role of UPR in neurodevelopmental disorders, we are currently investigating its activation in the Knock In mouse model of autism, carrying the R451C mutation in the NLGN3 endogenous gene. Our data represent the first evidence on the effects of the R451C NLGN3 in activating the UPR and represent a solid link between UPR and neurodevelopmental disorders characterized by the retention of misfolded proteins in the ER

The neurobiological bases of autism spectrum disorders: the R451C-neuroligin 3 mutation hampers the expression of long-term synaptic depression in the dorsal striatum

Autism spectrum disorders (ASDs) comprise a heterogeneous group of disorders with a complex genetic etiology. Current theories on the pathogenesis of ASDs suggest that they might arise from an aberrant synaptic transmission affecting specific brain circuits and synapses. The striatum, which is part of the basal ganglia circuit, is one of the brain regions involved in ASDs. Mouse models of ASDs have provided evidence for an imbalance between excitatory and inhibitory neurotransmission. Here, we investigated the expression of long-term synaptic plasticity at corticostriatal glutamatergic synapses in the dorsal striatum of the R451C-NL3 phenotypic mouse model of autism. This mouse model carries the human R451C mutation in the neuroligin 3 (NL3) gene that has been associated with highly penetrant autism in a Swedish family. The R451C-NL3 mouse has been shown to exhibit autistic-like behaviors and alterations of synaptic transmission in different brain areas. However, excitatory glutamatergic transmission and its long-term plasticity have not been investigated in the dorsal striatum so far. Our results indicate that the expression of long-term synaptic depression (LTD) at corticostriatal glutamatergic synapses in the dorsal striatum is impaired by the R451C-NL3 mutation. A partial rescue of LTD was obtained by exogenous activation of cannabinoid CB1 receptors or enhancement of the endocannabinoid tone, suggesting that an altered cannabinoid drive might underlie the deficit of synaptic plasticity in the dorsal striatum of R451C-NL3 mice

Neural stem cell properties and adult hippocampal neurogenesis in a knock-in mice model, expressing an autism-associated mutation.

Alteration of adult neurogenesis has been associated with neuropsychiatric disorders, including autism spectrum disorders (ASDs). Particularly, in the hippocampus of few ASD mice models, the properties of adult neural stem/progenitor cells (aNSPC) pool and the formation of new neurons have been found altered, suggesting a link between deregulated neurogenesis and some of the behavioural deficits found in these mice. In order to investigate neurogenesis in association to ASDs, we have been using the R451C Neuroligin3 (NLG3) knock-in mice, a model of a monogenic form of ASDs carrying the R451C substitution found in autistic patients. NLG3 is a postsynaptic protein involved in maturation, specification and plasticity of neural networks and the R451C knock-in mice display excitatory/inhibitory balance alterations in different brain regions, behavioural deficits, and structural brain abnormalities. We focused our study on the subgranular zone of the hippocampal dentate gyrus (DG), a neurogenic niche of the adult brain. Specifically, we compared proliferation and differentiation of new neurons between knock-in and wild-type mice, both in vivo and in vitro. In vitro data demonstrate that NSPC cultures derived from the DG of two-month-old knock-in mice contained a higher number of cells compared to the wild-type. However, BrdU cell number in the DGs was unchanged between KI and WT mice. In vivo data also show a decrease in the number of newly formed differentiated mature neurons (BrdU-NeuN double positive) in the hippocampus of the knock-in compared to wild-type mice. The mechanisms underlying the neurogenesis reduction in the knock-in mice are currently under investigation

Effects of Glucocorticoid treatment on the impaired trafficking of R451C Neuroligin3

Autism spectrum disorders are developmental syndromes (ASDs) characterized, at the neuronal level, by alterations in neurotransmission. Among the risk genes associated with ASD, mutations have been found in synaptic molecules such as the Neuroligins that are post-synaptic adhesion proteins. In particular, the R451C substitution in Neuroligin3 (NLGN3) was found in a Swedish family affected by ASD. It was shown by in vitro and in vivo studies, that this mutation affects the folding of the extracellular domain of NLGN3, causing a reduced exposure of the protein on the cell surface with its retention in the Endoplasmic Reticulum (ER) and the consequent activation of the unfolded protein response (UPR). We selected, from a library of 2662 drugs approved by the FDA, few compounds belonging to the family of Glucocorticoids (GCs) such as Aclometasone Dipropionate (AD), Denoside (D), Prednisolone Sodium Phosphate (PSP) and Dexamethasone (DEX) for improving the impaired trafficking of NLGN3 R451C protein. Our data show that GCs increase selectively protein levels of mutant NLGN3 by activating the glucocorticoid receptor (GR) in a dose and time-dependent manner. In particular, DEX promotes protein stability and its exposure on the cell surface. Our data also shows that, by reducing the ER-retention of the mutant protein, GCs mitigate ER stress, by lowering the activation of the three branches of UPR. Moreover, PDI levels were decreased by the GCs treatment, but levels of BiP and Grp94 were unchanged

R451C autism linked substitution in Neuroligin3 activates the unfolded protein response in a neuronal inducible system

Several forms of monogenic autism spectrum disorders (ASDs) are associated to mutations in the Neuroligin (NLGNs) genes. The autism-linked substitution of arginine 451 by a cysteine (R451C) in NLGN3 induces local misfolding of the extracellular domain, causing partial retention in the endoplasmic reticulum (ER). ER stress due to the accumulation of misfolded proteins can result in the unfolded protein response (UPR). We have generated a PC12 Tet-On cell lines with inducible expression WT or R451C NLGN3 to investigate if there is activation of the unfolded protein response (UPR) as a result of misfolded protein retention. Our data show that overexpression of R451C mutant protein leads to the activation of all three signalling branches of the UPR downstream of the stress sensors ATF6, IRE1 and PERK. Each branch displayed different activation profiles that partially correlated with the degree of misfolding caused by each mutation. We also show that upregulation of BiP and CHOP was induced by both mutant proteins but not by wild type neuroligin3, both in proliferative cells and cells differentiated to a neuronal–like phenotype. Downstream effects of UPR have been found in PC12 NLGN3 Tet-On cells differentiated to a neuronal phenotype. This supports the relevance of the UPR elicited by the ER retention of misfolded R451C NLGN3, which might in turn play a role in neuronal behaviour. In fact, although the UPR is classically linked to protein folding stress under pathological conditions, it is becoming clear that UPR signalling also regulates various processes, including synaptic functions. At the molecular levels, subtype-selective modulation of cell surface receptors by CHOP has been reported and the phosphorylation of eIF2α has been associated to synaptic plasticity, learning and memory. Moreover, in vivo administration of GSK2606414 has been shown to affect memory consolidation, supporting the role of UPR and its mediators in mediating synaptic functions. Collectively, our data show that mutant R451C neuroligin3 activates the UPR in PC12 cells, suggesting that this response may lead to neuronal circuit alterations and consequently have a role in the autistic phenotype

The neuroligins and the synaptic pathway in Autism Spectrum Disorder.

International audienceThe genetics underlying autism spectrum disorder (ASD) is complex and heterogeneous, and de novo variants are found in genes converging in functional biological processes. Neuronal communication, including trans-synaptic signaling involving two families of cell-adhesion proteins, the presynaptic neurexins and the postsynaptic neuroligins, is one of the most recurrently affected pathways in ASD. Given the role of these proteins in determining synaptic function, abnormal synaptic plasticity and failure to establish proper synaptic contacts might represent mechanisms underlying risk of ASD. More than 30 mutations have been found in the neuroligin genes. Most of the resulting residue substitutions map in the extracellular, cholinesterase-like domain of the protein, and impair protein folding and trafficking. Conversely, the stalk and intracellular domains are less affected. Accordingly, several genetic animal models of ASD have been generated, showing behavioral and synaptic alterations. The aim of this review is to discuss the current knowledge on ASD-linked mutations in the neuroligin proteins and their effect on synaptic function, in various brain areas and circuits