Glucocorticoids rescue cell surface trafficking of R451C Neuroligin3 and enhance synapse formation.

Neuroligins are synaptic cell adhesion proteins with a role in synaptic function, implicated in neurodevelopmental disorders. The autism spectrum disorder-associated substitution Arg451Cys (R451C) in NLGN3 promotes a partial misfolding of the extracellular domain of the protein leading to retention in the endoplasmic reticulum (ER) and the induction of the unfolded protein response (UPR). The reduced trafficking of R451C NLGN3 to the cell surface leads to altered synaptic function and social behavior. A screening in HEK-293 cells overexpressing NLGN3 of 2662 compounds (FDA-approved small molecule drug library), led to the identification of several glucocorticoids such as alclometasone dipropionate, desonide, prednisolone sodium phosphate, and dexamethasone (DEX), with the ability to favor the exit of full-length R451C NLGN3 from the ER. DEX improved the stability of R451C NLGN3 and trafficking to the cell surface, reduced the activation of the UPR, and increased the formation of artificial synapses between HEK-293 and hippocampal primary neurons. The effect of DEX was validated on a novel model system represented by neural stem progenitor cells and differentiated neurons derived from the R451C NLGN3 knock-in mouse, expressing the endogenous protein. This work shows a potential rescue strategy for an autism-linked mutation affecting cell surface trafficking of a synaptic protein.

Adult hippocampal neurogenesis and social behavioural deficits in the R451C Neuroligin3 mouse model of autism are reverted by the antidepressant fluoxetine.

Neuron generation persists throughout life in the hippocampus but is altered in animal models of neurological and neuropsychiatric diseases, suggesting that disease-associated decline in cognitive and emotional hippocampal-dependent behaviours might be functionally linked with dysregulation of postnatal neurogenesis. Depletion of the adult neural stem/progenitor cell (NSPCs) pool and neurogenic decline have been recently described in mice expressing synaptic susceptibility genes associated with autism spectrum disorder (ASDs). To gain further insight into mechanisms regulating neurogenesis in mice carrying mutations in synaptic genes related to monogenic ASDs, we used the R451C Neuroligin3 knock-in (Nlgn3 KI) mouse, which is characterized by structural brain abnormalities, deficits in synaptic functions and reduced sociability. We show that the number of adult-born neurons, but not the size of the NSPC pool, was reduced in the ventral dentate gyrus in knock-in mice. Notably, this neurogenic decline was rescued by daily injecting mice with 10 mg/Kg of the antidepressant fluoxetine for 20 consecutive days. Sustained treatment also improved KI mice's sociability and increased the number of c-Fos active adult-born neurons, compared with vehicle-injected KI mice. Our study uncovers neurogenesis-mediated alterations in the brain of R451C KI mouse, showing that the R451C Nlgn3 mutation leads to lasting, albeit pharmacologically reversible, changes in the brain, affecting neuron formation in the adult hippocampus. Our results suggest that fluoxetine can ameliorate social behaviour in KI mice, at least in part, by rescuing adult hippocampal neurogenesis, which may be relevant for the pharmacological treatment of ASDs.

Exposure to Antibiotics and Neurodevelopmental Disorders: Could Probiotics Modulate the Gut-Brain Axis?

In order to develop properly, the brain requires the intricate interconnection of genetic factors and pre-and postnatal environmental events. The gut-brain axis has recently raised considerable interest for its involvement in regulating the development and functioning of the brain. Consequently, alterations in the gut microbiota composition, due to antibiotic administration, could favor the onset of neurodevelopmental disorders. Literature data suggest that the modulation of gut microbiota is often altered in individuals affected by neurodevelopmental disorders. It has been shown in animal studies that metabolites released by an imbalanced gut-brain axis, leads to alterations in brain function and deficits in social behavior. Here, we report the potential effects of antibiotic administration, before and after birth, in relation to the risk of developing neurodevelopmental disorders. We also review the potential role of probiotics in treating gastrointestinal disorders associated with gut dysbiosis after antibiotic administration, and their possible effect in ameliorating neurodevelopmental disorder symptoms.

Retinoic acid-induced 1 gene haploinsufficiency alters lipid metabolism and causes autophagy defects in Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a neurodevelopmental disorder characterized by cognitive and behavioral symptoms, obesity, and sleep disturbance, and no therapy has been developed to alleviate its symptoms or delay disease onset. SMS occurs due to haploinsufficiency of the retinoic acid-induced-1 (RAI1) gene caused by either chromosomal deletion (SMS-del) or RAI1 missense/nonsense mutation. The molecular mechanisms underlying SMS are unknown. Here, we generated and characterized primary cells derived from four SMS patients (two with SMS-del and two carrying RAI1 point mutations) and four control subjects to investigate the pathogenetic processes underlying SMS. By combining transcriptomic and lipidomic analyses, we found altered expression of lipid and lysosomal genes, deregulation of lipid metabolism, accumulation of lipid droplets, and blocked autophagic flux. We also found that SMS cells exhibited increased cell death associated with the mitochondrial pathology and the production of reactive oxygen species. Treatment with N-acetylcysteine reduced cell death and lipid accumulation, which suggests a causative link between metabolic dyshomeostasis and cell viability. Our results highlight the pathological processes in human SMS cells involving lipid metabolism, autophagy defects and mitochondrial dysfunction and suggest new potential therapeutic targets for patient treatment.

Postsynaptic autism spectrum disorder genes and synaptic dysfunction.

This review provides an overview of the synaptic dysfunction of neuronal circuits and the ensuing behavioral alterations caused by mutations in autism spectrum disorder (ASD)-linked genes directly or indirectly affecting the postsynaptic neuronal compartment. There are plenty of ASD risk genes, that may be broadly grouped into those involved in gene expression regulation (epigenetic regulation and transcription) and genes regulating synaptic activity (neural communication and neurotransmission). Notably, the effects mediated by ASD-associated genes can vary extensively depending on the developmental time and/or subcellular site of expression. Therefore, in order to gain a better understanding of the mechanisms of disruptions in postsynaptic function, an effort to better model ASD in experimental animals is required to improve standardization and increase reproducibility within and among studies. Such an effort holds promise to provide deeper insight into the development of these disorders and to improve the translational value of preclinical studies.

The neuroligins and the synaptic pathway in Autism Spectrum Disorder.

The 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.

A Lipophilic 4-Phenylbutyric Acid Derivative That Prevents Aggregation and Retention of Misfolded Proteins.

Chemical chaperones prevent protein aggregation. However, the use of chemical chaperones as drugs against diseases due to protein aggregation is limited by the very high active concentrations (mm range) required to mediate their effect. One of the most common chemical chaperones is 4-phenylbutyric acid (4-PBA). Despite its unfavorable pharmacokinetic properties, 4-PBA was approved as a drug to treat ornithine cycle diseases. Here, we report that 2-isopropyl-4-phenylbutanoic acid (5) has been found to be 2-10-fold more effective than 4-PBA in several in vitro models of protein aggregation. Importantly, compound 5 reduced the secretion rate of autism-linked Arg451Cys Neuroligin3 (R451C NLGN3).

A Proteomic Screen of Neuronal Cell-Surface Molecules Reveals IgLONs as Structurally Conserved Interaction Modules at the Synapse.

In the developing brain, cell-surface proteins play crucial roles, but their protein-protein interaction network remains largely unknown. A proteomic screen identified 200 interactions, 89 of which were not previously published. Among these interactions, we find that the IgLONs, a family of five cell-surface neuronal proteins implicated in various human disorders, interact as homo- and heterodimers. We reveal their interaction patterns and report the dimeric crystal structures of Neurotrimin (NTRI), IgLON5, and the neuronal growth regulator 1 (NEGR1)/IgLON5 complex. We show that IgLONs maintain an extended conformation and that their dimerization occurs through the first Ig domain of each monomer and is Ca2+ independent. Cell aggregation shows that NTRI and NEGR1 homo- and heterodimerize in trans. Taken together, we report 89 unpublished cell-surface ligand-receptor pairs and describe structural models of trans interactions of IgLONs, showing that their structures are compatible with a model of interaction across the synaptic cleft.

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.

Production and characterization of CSSI003 (2961) human induced pluripotent stem cells (iPSCs) carrying a novel puntiform mutation in RAI1 gene, Causative of Smith-Magenis syndrome.

Smith-Magenis syndrome (SMS) is a complex genetic disorder characterized by developmental delay, behavioural problems and circadian rhythm dysregulation. About 90% of SMS cases are due to a 17p11.2 deletion containing retinoic acid induced1 (RAI1) gene, 10% are due to heterozygous mutations affecting RAI1 coding region. Little is known about RAI1 role.

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.

Unbalance between Excitation and Inhibition in Phenylketonuria, a Genetic Metabolic Disease Associated with Autism.

Phenylketonuria (PKU) is the most common genetic metabolic disease with a well-documented association with autism spectrum disorders. It is characterized by the deficiency of the phenylalanine hydroxylase activity, causing plasmatic hyperphenylalaninemia and variable neurological and cognitive impairments. Among the potential pathophysiological mechanisms implicated in autism spectrum disorders is the excitation/inhibition (E/I) imbalance which might result from alterations in excitatory/inhibitory synapse development, synaptic transmission and plasticity, downstream signalling pathways, and intrinsic neuronal excitability. Here, we investigated functional and molecular alterations in the prefrontal cortex (pFC) of BTBR-Pahenu2 (ENU2) mice, the animal model of PKU. Our data show higher frequency of inhibitory transmissions and significant reduced frequency of excitatory transmissions in the PKU-affected mice in comparison to wild type. Moreover, in the pFC of ENU2 mice, we reported higher levels of the post-synaptic cell-adhesion proteins neuroligin1 and 2. Altogether, our data point toward an imbalance in the E/I neurotransmission favouring inhibition in the pFC of ENU2 mice, along with alterations of the molecular components involved in the organization of cortical synapse. In addition to being the first evidence of E/I imbalance within cortical areas of a mouse model of PKU, our study provides further evidence of E/I imbalance in animal models of pathology associated with autism spectrum disorders.

Neuroserpin polymers cause oxidative stress in a neuronal model of the dementia FENIB.

The serpinopathies are human pathologies caused by mutations that promote polymerisation and intracellular deposition of proteins of the serpin superfamily, leading to a poorly understood cell toxicity. The dementia FENIB is caused by polymerisation of the neuronal serpin neuroserpin (NS) within the endoplasmic reticulum (ER) of neurons. With the aim of understanding the toxicity due to intracellular accumulation of neuroserpin polymers, we have generated transgenic neural progenitor cell (NPC) cultures from mouse foetal cerebral cortex, stably expressing the control protein GFP (green fluorescent protein), or human wild type, G392E or delta NS. We have characterised these cell lines in the proliferative state and after differentiation to neurons. Our results show that G392E NS formed polymers that were mostly retained within the ER, while wild type NS was correctly secreted as a monomeric protein into the culture medium. Delta NS was absent at steady state due to its rapid degradation, but it was easily detected upon proteasomal block. Looking at their intracellular distribution, wild type NS was found in partial co-localisation with ER and Golgi markers, while G392E NS was localised within the ER only. Furthermore, polymers of NS were detected by ELISA and immunofluorescence in neurons expressing the mutant but not the wild type protein. We used control GFP and G392E NPCs differentiated to neurons to investigate which cellular pathways were modulated by intracellular polymers by performing RNA sequencing. We identified 747 genes with a significant upregulation (623) or downregulation (124) in G392E NS-expressing cells, and we focused our attention on several genes involved in the defence against oxidative stress that were up-regulated in cells expressing G392E NS (Aldh1b1, Apoe, Gpx1, Gstm1, Prdx6, Scara3, Sod2). Inhibition of intracellular anti-oxidants by specific pharmacological reagents uncovered the damaging effects of NS polymers. Our results support a role for oxidative stress in the cellular toxicity underlying the neurodegenerative dementia FENIB.

Alpha-7 Nicotinic Receptors in Nervous System Disorders: From Function to Therapeutic Perspectives.

BACKGROUND: The α7 nicotinic receptor consists of identical subunits and is one of the most abundant acetylcholine receptors in the mammalian central nervous system. However its expression is also found in the peripheral nervous system as well as in the immune system and various peripheral tissues. Nicotinic Receptors: They are involved in the regulation of several activities ranging from excitatory neurotransmission, the modulation of the release of several neurotransmitters, regulation of neurite outgrowth, and even neuronal survival/death. Its expression is found in brain areas that underlie learning and memory, suggesting their involvement in regulating cognitive functions. The α7-nicotinic receptor has a strategic role during development in regulating molecular pathways activated during neurogenesis. Because of its pleiotropic effects, receptor dysfunction or dysregulated expression is found in pathophysiological conditions of the nervous system including neurodegenerative diseases and neurodevelopmental disorders. CONCLUSION: Here we review the physiological and pathological roles of alpha-7 nicotinic receptor in different nervous system disorders and the current therapeutic strategies developed to target selectively this receptor for potentiating or reducing its functions.

Structural Characterization of the Extracellular Domain of CASPR2 and Insights into Its Association with the Novel Ligand Contactin1.

Contactin-associated protein-like 2 (CNTNAP2) encodes for CASPR2, a multidomain single transmembrane protein belonging to the neurexin superfamily that has been implicated in a broad range of human phenotypes including autism and language impairment. Using a combination of biophysical techniques, including small angle x-ray scattering, single particle electron microscopy, analytical ultracentrifugation, and bio-layer interferometry, we present novel structural and functional data that relate the architecture of the extracellular domain of CASPR2 to a previously unknown ligand, Contactin1 (CNTN1). Structurally, CASPR2 is highly glycosylated and has an overall compact architecture. Functionally, we show that CASPR2 associates with micromolar affinity with CNTN1 but, under the same conditions, it does not interact with any of the other members of the contactin family. Moreover, by using dissociated hippocampal neurons we show that microbeads loaded with CASPR2, but not with a deletion mutant, co-localize with transfected CNTN1, suggesting that CNTN1 is an endogenous ligand for CASPR2. These data provide novel insights into the structure and function of CASPR2, suggesting a complex role of CASPR2 in the nervous system.

Autism-associated R451C mutation in neuroligin3 leads to activation of the unfolded protein response in a PC12 Tet-On inducible system.

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.

Cystatin B and SOD1: protein­protein interaction and possible relation to neurodegeneration.

Cystatin B (CSTB), an inhibitor of the cysteine proteases, belongs to the cathepsin family and it is known to interact with a number of proteins involved in cytoskeletal organization. CSTB has an intrinsic tendency to form aggregates depending on the redox environment. The gene encoding for CSTB is frequently mutated in association with the rare neurodegenerative condition progressive myoclonus epilepsy. Increased levels of CSTB have been observed in the spinal cord of transgenic mice modeling SOD1-linked familial amyotrophic lateral sclerosis, a fatal neurodegenerative disease affecting motoneurons. In the present study, we have investigated the relationship occurring between the expression of SOD1 and CSTB either wild-type or double-cysteine substitution mutant (Cys 3 and Cys 64). Whether or not there is a physical interaction between the two proteins was also investigated in overexpression experiments using a human neuroblastoma cell line and mouse-immortalized motoneurons. Here we report evidences for a reciprocal influence of CSTB and SOD1 at the gene expression level and for a direct interaction of the two proteins.

Nicotine exposure during adolescence: cognitive performance and brain gene expression in adult heterozygous reeler mice.

RATIONALE: We have recently reported nicotine-induced stimulation of reelin and glutamic acid decarboxylase 67 (GAD67) mRNA expression levels in the brain of heterozygous reeler mice (HRM), a putative animal model for the study of symptoms relevant to major behavioral disorders. OBJECTIVES: We aimed to evaluate long-term behavioral effects and brain molecular changes as a result of adaptations to nicotine exposure in the developing HRM males. METHODS: Adolescent mice (pnd 37-42) were exposed to oral nicotine (10 mg/l) in a 6-day free-choice drinking schedule. As expected, no differences in total nicotine intake between WT (wild-type) mice and HRM were found. RESULTS: Long-term behavioral effects and brain molecular changes, as a consequence of nicotine exposure during adolescence, were only evidenced in HRM. Indeed, HRM perseverative exploratory behavior and poor cognitive performance were modulated to WT levels by subchronic exposure to nicotine during development. Furthermore, the expected reduction in the expression of mRNA of reelin and GAD67 in behaviorally relevant brain areas of HRM appeared persistently restored by nicotine. For brain-derived neurotrophic factor (BDNF) mRNA expression, no genotype-dependent changes appeared. However, expression levels were increased by previous nicotine in brains from both genotypes. The mRNA encoding for nicotine receptor subunits (α7, ß2 and α4) did not differ between genotypes and as a result of previous nicotine exposure. CONCLUSION: These findings support the hypothesis of pre-existing vulnerability (based on haploinsufficiency of reelin) to brain and behavioral disorders and regulative short- and long-term effects associated with nicotine modulation.

Cholinesterase confabs and cousins: approaching forty years.

In the past four decades of cholinesterase (ChE) research, we have seen substantive evolution of the field from one centered around substrate and inhibitor kinetic profiles and compound characterizations to the analysis of ChE structure, first through the gene families and then by X-ray crystallographic determinations of the free enzymes and their complexes and conjugates. Indeed, these endeavors have been facilitated by recombinant DNA technologies, structure determinations and parallel studies in related proteins in the α/ß-hydrolase fold family. This approach has not only contributed to a fundamental understanding of structure and function of a large family of hydrolase-like proteins possessing functions other than catalysis, but also has been used to develop new practical strategies for scavenging and antidotal activity in cases of organophosphate insecticide or nerve agent exposure.