Aberrant neuronal activity-induced signaling and gene expression in a mouse model of RASopathy

Noonan syndrome (NS) is characterized by reduced growth, craniofacial abnormalities, congenital heart defects, and variable cognitive deficits. NS belongs to the RASopathies, genetic conditions linked to mutations in components and regulators of the Ras signaling pathway. Approximately 50% of NS cases are caused by mutations in PTPN11. However, the molecular mechanisms underlying cognitive impairments in NS patients are still poorly understood. Here, we report the generation and characterization of a new conditional mouse strain that expresses the overactive Ptpn11D61Y allele only in the forebrain. Unlike mice with a global expression of this mutation, this strain is viable and without severe systemic phenotype, but shows lower exploratory activity and reduced memory specificity, which is in line with a causal role of disturbed neuronal Ptpn11 signaling in the development of NS-linked cognitive deficits. To explore the underlying mechanisms we investigated the neuronal activity-regulated Ras signaling in brains and neuronal cultures derived from this model. We observed an altered surface expression and trafficking of synaptic glutamate receptors, which are crucial for hippocampal neuronal plasticity. Furthermore, we show that the neuronal activity-induced ERK signaling, as well as the consecutive regulation of gene expression are strongly perturbed. Microarray-based hippocampal gene expression profiling revealed profound differences in the basal state and upon stimulation of neuronal activity. The neuronal activity-dependent gene regulation was strongly attenuated in Ptpn11D61Y neurons. In silico analysis of functional networks revealed changes in the cellular signaling beyond the dysregulation of Ras/MAPK signaling that is nearly exclusively discussed in the context of NS at present. Importantly, changes in PI3K/AKT/mTOR and JAK/STAT signaling were experimentally confirmed. In summary, this study uncovers aberrant neuronal activity-induced signaling and regulation of gene expression in Ptpn11D61Y mice and suggests that these deficits contribute to the pathophysiology of cognitive impairments in NS

Towards the convergent therapeutic potential of GPCRs in autism spectrum disorders

Changes in genetic and/or environmental factors to developing neural circuits and subsequent synaptic functions are known to be a causative underlying the varied socio-emotional behavioural patterns associated with autism spectrum disorders (ASD). Seven transmembrane G protein-coupled receptors (GPCRs) comprising the largest family of cell-surface receptors, mediate the transfer of extracellular signals to downstream cellular responses. Disruption of GPCR and their signalling have been implicated as a convergent pathologic mechanism of ASD. Here, we aim to review the literature about the 23 GPCRs that are genetically associated to ASD pathology according to Simons Foundation Autism Research Initiative (SFARI) database such as oxytocin (OXTR) and vasopressin (V1A, V1B) receptors, metabotropic glutamate (mGlu5, mGlu7) and gamma-aminobutyric acid (GABAB) receptors, dopamine (D1, D2), serotoninergic (5-HT1B and additionally included the 5-HT2A, 5-HT7 receptors for their strong relevance to ASD), adrenergic ($\beta$2) and cholinergic (M3) receptors, adenosine (A2A, A3) receptors, angiotensin (AT2) receptors, cannabinoid (CB1) receptors, chemokine (CX3CR1) receptors, orphan (GPR37, GPR85) and olfactory (OR1C1, OR2M4, OR2T10, OR52M1) receptors. We discussed the genetic variants, relation to core ASD behavioural deficits and update on pharmacological compounds targeting these 23 GPCRs. Of these OTR, V1A, mGlu5, D2, 5-HT2A, CB1, and GPR37 serve as the best therapeutic targets and have potential towards core domains of ASD pathology. With a functional crosstalk between different GPCRs and converging pharmacological responses, there is an urge to develop novel therapeutic strategies based on multiple GPCRs to reduce the socioeconomic burden associated with ASD and we strongly emphasize the need to prioritize the increased clinical trials targeting the multiple GPCRs

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype

Effect of the social environment on olfaction and social skills in WT and mouse model of autism

Autism spectrum disorders are complex, polygenic and heterogenous neurodevelopmental conditions, imposing a substantial economic burden. Genetics are influenced by the environment, specifically the social experience during the critical neurodevelopmental period. Despite efficacy of early behavior interventions targeted specific behaviors in some autistic children, there is no sustainable treatment for the two core symptoms: deficits in social interaction and communication, and stereotyped or restrained behaviors or interests. In this study, we investigated the impact of the social environment on both wild-type (WT) and Shank3 knockout (KO) mice, a mouse model that reproduces core autism-like symptoms. Our findings revealed that WT mice raised in an enriched social environment maintained social interest towards new conspecifics across multiple trials. Additionally, we observed that 2 hours or chronic social isolation induced social deficits or enhanced social interaction and olfactory neuron responses in WT animals, respectively. Notably, chronic social isolation restored both social novelty and olfactory deficits, and normalized self-grooming behavior in Shank3 KO mice. These results novel insights for the implementation of behavioral intervention and inclusive classrooms programs for children with ASD

The Presynaptic Scaffold Protein Bassoon in Forebrain Excitatory Neurons Mediates Hippocampal Circuit Maturation: Potential Involvement of TrkB Signalling

A presynaptic active zone organizer protein Bassoon orchestrates numerous important functions at the presynaptic active zone. We previously showed that the absence of Bassoon exclusively in forebrain glutamatergic presynapses (BsnEmx1cKO) in mice leads to developmental disturbances in dentate gyrus (DG) affecting synaptic excitability, morphology, neurogenesis and related behaviour during adulthood. Here, we demonstrate that hyperexcitability of the medial perforant path-to-DG (MPP-DG) pathway in BsnEmx1cKO mice emerges during adolescence and is sustained during adulthood. We further provide evidence for a potential involvement of tropomyosin-related kinase B (TrkB), the high-affinity receptor for brain-derived neurotrophic factor (BDNF), mediated signalling. We detect elevated TrkB protein levels in the dorsal DG of adult mice (~3-5 months-old) but not in adolescent (~4-5 weeks-old) mice. Electrophysiological analysis reveals increased field-excitatory-postsynaptic-potentials (fEPSPs) in the DG of the adult, but not in adolescent BsnEmx1cKO mice. In line with an increased TrkB expression during adulthood in BsnEmx1cKO, blockade of TrkB normalizes the increased synaptic excitability in the DG during adulthood, while no such effect was observed in adolescence. Accordingly, neurogenesis, which has previously been found to be increased in adult BsnEmx1cKO mice, was unaffected at adolescent age. Our results suggest that Bassoon plays a crucial role in the TrkB-dependent postnatal maturation of the hippocampus

Towards the convergent therapeutic potential of G protein‐coupled receptors in autism spectrum disorders

International audienceAutism spectrum disorders (ASDs) are diagnosed in 1/100 children worldwide, based on two core symptoms: deficits in social interaction and communication, and stereotyped behaviours. G protein-coupled receptors (GPCRs) are the largest family of cellsurface receptors that transduce extracellular signals to convergent intracellular signalling and downstream cellular responses that are commonly dysregulated in ASD. Despite hundreds of GPCRs being expressed in the brain, only 23 are genetically associated with ASD according to the Simons Foundation Autism Research Initiative (SFARI) gene database: oxytocin OTR; vasopressin V 1A and V 1B ; metabotropic glutamate mGlu 5 and mGlu 7 ; GABA B2 ; dopamine D 1 , D 2 and D 3 ; serotoninergic 5-HT 1B ; β 2-adrenoceptor; cholinergic M 3 ; adenosine A 2A and A 3 ; angiotensin AT 2 ; cannabinoid CB 1 ; chemokine CX 3 CR1; orphan GPR37 and GPR85; and olfactory OR1C1, OR2M4, OR2T10 and OR52M1. Here, we review the therapeutic potential of these 23 GPCRs, as well as 5-HT 2A and 5-HT 7 , for ASD. For each GPCR, we discuss its genetic association, genetic and pharmacological manipulation in animal models, pharmacopoeia for core symptoms of ASD and rank them based on these factors. Among these GPCRs, we highlight D 2 , 5-HT 2A , CB 1 , OTR and V 1A as the more promising targets for ASD. We discuss that the dysregulation of GPCRs and their signalling is a convergent pathological mechanism of ASD. Their therapeutic potential has only begun as multiple GPCRs could mitigate ASD

Towards the convergent therapeutic potential of GPCRs in autism spectrum disorders

Changes in genetic and/or environmental factors to developing neural circuits and subsequent synaptic functions are known to be a causative underlying the varied socio-emotional behavioural patterns associated with autism spectrum disorders (ASD). Seven transmembrane G protein-coupled receptors (GPCRs) comprising the largest family of cell-surface receptors, mediate the transfer of extracellular signals to downstream cellular responses. Disruption of GPCR and their signalling have been implicated as a convergent pathologic mechanism of ASD. Here, we aim to review the literature about the 23 GPCRs that are genetically associated to ASD pathology according to Simons Foundation Autism Research Initiative (SFARI) database such as oxytocin (OXTR) and vasopressin (V1A, V1B) receptors, metabotropic glutamate (mGlu5, mGlu7) and gamma-aminobutyric acid (GABAB) receptors, dopamine (D1, D2), serotoninergic (5-HT1B and additionally included the 5-HT2A, 5-HT7 receptors for their strong relevance to ASD), adrenergic (β2) and cholinergic (M3) receptors, adenosine (A2A, A3) receptors, angiotensin (AT2) receptors, cannabinoid (CB1) receptors, chemokine (CX3CR1) receptors, orphan (GPR37, GPR85) and olfactory (OR1C1, OR2M4, OR2T10, OR52M1) receptors. We discussed the genetic variants, relation to core ASD behavioural deficits and update on pharmacological compounds targeting these 23 GPCRs. Of these OTR, V1A, mGlu5, D2, 5-HT2A, CB1, and GPR37 serve as the best therapeutic targets and have potential towards core domains of ASD pathology. With a functional crosstalk between different GPCRs and converging pharmacological responses, there is an urge to develop novel therapeutic strategies based on multiple GPCRs to reduce the socioeconomic burden associated with ASD and we strongly emphasize the need to prioritize the increased clinical trials targeting the multiple GPCRs

Ablation of the presynaptic organizer Bassoon in excitatory neurons retards dentate gyrus maturation and enhances learning performance

Bassoon is a large scaffolding protein of the presynaptic active zone involved in the development of presynaptic terminals and in the regulation of neurotransmitter release at both excitatory and inhibitory brain synapses. Mice with constitutive ablation of the Bassoon (Bsn) gene display impaired presynaptic function, show sensory deficits and develop severe seizures. To specifically study the role of Bassoon at excitatory forebrain synapses and its relevance for control of behavior, we generated conditional knockout (Bsn cKO) mice by gene ablation through an Emx1 promoter-driven Cre recombinase. In these animals, we confirm selective loss of Bassoon from glutamatergic neurons of the forebrain. Behavioral assessment revealed that, in comparison to wild-type littermates, Bsn cKO mice display selectively enhanced contextual fear memory and increased novelty preference in a spatial discrimination/pattern separation task. These changes are accompanied by an augmentation of baseline synaptic transmission at medial perforant path to dentate gyrus (DG) synapses, as indicated by increased ratios of field excitatory postsynaptic potential slope to fiber volley amplitude. At the structural level, an increased complexity of apical dendrites of DG granule cells can be detected in Bsn cKO mice. In addition, alterations in the expression of cellular maturation markers and a lack of age-dependent decrease in excitability between juvenile and adult Bsn cKO mice are observed. Our data suggest that expression of Bassoon in excitatory forebrain neurons is required for the normal maturation of the DG and important for spatial and contextual memory

Linking epileptic phenotypes and neural extracellular matrix remodeling signatures in mouse models of epilepsy

Epilepsies are multifaceted neurological disorders characterized by abnormal brain activity, e.g. caused by imbalanced synaptic excitation and inhibition. The neural extracellular matrix (ECM) is dynamically modulated by physiological and pathophysiological activity and critically involved in controlling the brain's excitability. We used different epilepsy models, i.e. mice lacking the presynaptic scaffolding protein Bassoon at excitatory, inhibitory or all synapse types as genetic models for rapidly generalizing early-onset epilepsy, and intra-hippocampal kainate injection, a model for acquired temporal lobe epilepsy, to study the relationship between epileptic seizures and ECM composition. Electroencephalogram recordings revealed Bassoon deletion at excitatory or inhibitory synapses having diverse effects on epilepsy-related phenotypes. While constitutive Bsn mutants and to a lesser extent GABAergic neuron-specific knockouts (BsnDlx5/6cKO) displayed severe epilepsy with more and stronger seizures than kainate-injected animals, mutants lacking Bassoon solely in excitatory forebrain neurons (BsnEmx1cKO) showed only mild impairments. By semiquantitative immunoblotting and immunohistochemistry we show model-specific patterns of neural ECM remodeling, and we also demonstrate significant upregulation of the ECM receptor CD44 in null and BsnDlx5/6cKO mutants. ECM-associated WFA-binding chondroitin sulfates were strongly augmented in seizure models. Strikingly, Brevican, Neurocan, Aggrecan and link proteins Hapln1 and Hapln4 levels reliably predicted seizure properties across models, suggesting a link between ECM state and epileptic phenotype