Genetic targeting of NRXN2 in mice unveils role in excitatory cortical synapse function and social behaviors

Human genetics has identified rare copy number variations and deleterious mutations for all neurexin genes (NRXN1-3) in patients with neurodevelopmental diseases, and electrophysiological recordings in animal brains have shown that Nrxns are important for synaptic transmission. While several mouse models for Nrxn1α inactivation have previously been studied for behavioral changes, very little information is available for other variants. Here, we validate that mice lacking Nrxn2α exhibit behavioral abnormalities, characterized by social interaction deficits and increased anxiety-like behavior, which partially overlap, partially differ from Nrxn1α mutant behaviors. Using patch-clamp recordings in Nrxn2α knockout brains, we observe reduced spontaneous transmitter release at excitatory synapses in the neocortex. We also analyse at this cellular level a novel NRXN2 mouse model that carries a combined deletion of Nrxn2α and Nrxn2β. Electrophysiological analysis of this Nrxn2-mutant mouse shows surprisingly similar defects of excitatory release to Nrxn2α, indicating that the β-variant of Nrxn2 has no strong function in basic transmission at these synapses. Inhibitory transmission as well as synapse densities and ultrastructure remain unchanged in the neocortex of both models. Furthermore, at Nrxn2α and Nrxn2-mutant excitatory synapses we find an altered facilitation and N-methyl-D-aspartate receptor (NMDAR) function because NMDAR-dependent decay time and NMDAR-mediated responses are reduced. As Nrxn can indirectly be linked to NMDAR via neuroligin and PSD-95, the trans-synaptic nature of this complex may help to explain occurrence of presynaptic and postsynaptic effects. Since excitatory/inhibitory imbalances and impairment of NMDAR function are alledged to have a role in autism and schizophrenia, our results support the idea of a related pathomechanism in these disorders

Persistent gating deficit and increased sensitivity to NMDA receptor antagonism after puberty in a new mouse model of the human 22q11.2 micro-deletion syndrome – a study in male mice

Background: The hemizygous 22q11.2 micro-deletion is a common copy number variant in humans. The deletion confers high risk of neurodevelopmental disorders including autism and schizophrenia. Up to 41% of deletion carriers experience psychotic symptoms. Methods: We present a new mouse model (Df(h22q11)/+) of the deletion syndrome (22q11.2DS) and report on the most comprehensive study undertaken in 22q11.2DS models. The study was conducted in male mice. Results: We found elevated post-pubertal NMDA receptor antagonist induced hyper-locomotion, age-independent prepulse inhibition (PPI) deficits and increased acoustic startle response (ASR). The PPI deficit and increased ASR was resistant to antipsychotic treatment. The PPI deficit was not a consequence of impaired hearing measured by auditory brain stem responses. The Df(h22q11)/+ mice also displayed increased amplitude of loudness-dependent auditory evoked potentials. Prefrontal cortex and dorsal striatal (DStr) elevations of the dopamine metabolite DOPAC and increased DStr expression of the AMPA receptor subunit GluR1 was found. The Df(h22q11)/+ mice did not deviate from wild-type mice in a wide range of other behavioural and biochemical assays. Limitations: The 22q11.2 micro-deletion has incomplete penetrance in humans and the severity of disease depends on the complete genetic makeup in concert with environmental factors. In order to obtain more marked phenotypes reflecting the severe conditions related to 22q11.2DS it is suggested to expose the Df(h22q11)/+ mice to environmental stressors which may unmask latent psychopathology. Conclusion: The Df(h22q11)/+ model will be a valuable tool for increasing our understanding of the aetiology of schizophrenia and other psychiatric disorders associated with the 22q11DS.The research leading to these results was conducted as part of NEWMEDS and received support from the Innovative Medicine Initiative Joint Undertaking under grant agreement n° 115008 of which resources are composed of EFPIA in-kind contribution and financial contribution from the European Union’s Seventh Framework Programme (FP7/2007-2013). This work was further supported by grants from the Danish Advanced Technology Foundation (File no. 001-2009-2) and by the Instituto de Salud Carlos III, Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM)

NRXN1 deletions identified by array comparative genome hybridisation in a clinical case series – further understanding of the relevance of NRXN1 to neurodevelopmental disorders

BACKGROUND: Microdeletions in the NRXN1 gene have been associated with a range of neurodevelopmental disorders, including autism spectrum disorders, schizophrenia, intellectual disability, speech and language delay, epilepsy and hypotonia. RESULTS: In the present study we performed array CGH analysis on 10,397 individuals referred for diagnostic cytogenetic analysis, using a custom oligonucleotide array, which included 215 NRXN1 probes (median spacing 4.9 kb). We found 34 NRXN1 deletions (0.33% of referrals) ranging from 9 to 942 kb in size, of which 18 were exonic (0.17%). Three deletions affected exons also in the beta isoform of NRXN1. No duplications were found. Patients had a range of phenotypes including developmental delay, learning difficulties, attention deficit hyperactivity disorder (ADHD), autism, speech delay, social communication difficulties, epilepsy, behaviour problems and microcephaly. Five patients who had deletions in NRXN1 had a second CNV implicated in neurodevelopmental disorder: a CNTNAP2 and CSMD3 deletion in patients with exonic NRXN1 deletions, and a Williams-Beuren syndrome deletion and two 22q11.2 duplications in patients with intronic NRXN1 deletions. CONCLUSIONS: Exonic deletions in the NRXN1 gene, predominantly affecting the alpha isoform, were found in patients with a range of neurodevelopmental disorders referred for diagnostic cytogenetic analysis. The targeting of dense oligonucleotide probes to the NRXN1 locus on array comparative hybridisation platforms provides detailed characterisation of deletions in this gene, and is likely to add to understanding of the importance of NRXN1 in neural development

Deletion of Nrxn1α causes reduced locomotor activity in the light/dark box.

<p>Data shown are mean (± sem) number of transmissions between the light and dark compartments for male (A) and female (B) Nrxn1α mice. Data is derived from 23 WT (12M, 11F) 29 HET (15M, 14F) 18 KO (9M, 9F) mice, and levels of significance indicated by ** and *** as p<0.1 and p<0.001, respectively, compared to WT mice.</p

Anxiety is elevated in male Nrxn1α mice.

<p>Data shown are anxiety measures taken from the open field, light/dark box and elevated plus maze. Panels are mean (± sem) time spent in the central area of the open field for male (A) and female (B) Nrxn1α mice, time spent in the light compartment of the light/dark box for male (C) and female (D) Nrxn1α mice and time spent in the open arms of the elevated plus maze for male (E) and female (F) Nrxn1α mice. Data derived from 23 WT (12M, 11F) 29 HET (15M, 14F) 18 KO (9M, 9F) mice. Levels of significance indicated by *** as p<0.001, compared to WT mice, and ### as p<0.001, compared to HET mice.</p

Altered Social Behaviours in Neurexin 1α Knockout Mice Resemble Core Symptoms in Neurodevelopmental Disorders

<div><p>Background</p><p>Copy number variants have emerged as an important genomic cause of common, complex neurodevelopmental disorders. These usually change copy number of multiple genes, but deletions at 2p16.3, which have been associated with autism, schizophrenia and mental retardation, affect only the neurexin 1 gene, usually the alpha isoform. Previous analyses of neurexin 1α (Nrxn1α) knockout (KO) mouse as a model of these disorders have revealed impairments in synaptic transmission but failed to reveal defects in social behaviour, one of the core symptoms of autism.</p><p>Methods</p><p>We performed a detailed investigation of the behavioural effects of Nrxn1α deletion in mice bred onto a pure genetic background (C57BL/6J<b>)</b> to gain a better understanding of its role in neurodevelopmental disorders. Wildtype, heterozygote and homozygote Nrxn1α KO male and female mice were tested in a battery of behavioural tests (n = 9–16 per genotype, per sex).</p><p>Results</p><p>In homozygous Nrxn1α KO mice, we observed altered social approach, reduced social investigation, and reduced locomotor activity in novel environments. In addition, male Nrxn1α KO mice demonstrated an increase in aggressive behaviours.</p><p>Conclusions</p><p>These are the first experimental data that associate a deletion of Nrxn1α with alterations of social behaviour in mice. Since this represents one of the core symptom domains affected in autism spectrum disorders and schizophrenia in humans, our findings suggest that deletions within NRXN1 found in patients may be responsible for the impairments seen in social behaviours, and that the Nrxn1α KO mice are a useful model of human neurodevelopmental disorder.</p></div

Deletion of Nrxn1α affects the social approach behaviours in mice.

<p>Data shown are social approach behaviours of the Nrxn1α mice during the three-chamber social approach task. A – Trial 1 - Mean (± sem) time spent (s) in the chamber containing the mouse, the centre chamber or the chamber containing the object. B – Trial 2 - Mean (± sem) time spent (s) in the chamber containing the familiar mouse, the centre chamber or the chamber containing the novel mouse. Data is derived from 23 WT (12M, 11F) 29 HET (15M, 14F) 18 KO (9M, 9F) mice, and levels of significance indicated by ** and *** as p<0.01 and p<0.001, respectively, compared to WT mice, and ## and ### as p<0.01 and p<0.001, respectively, compared to HET mice.</p

Nrxn1α mice show no impairment in spatial working.

<p>Data shown are mean (± sem) behaviours of the Nrxn1α mice during the Morris water maze. Latency to reach the platform for male (A) and female (B) mice, path length for male (C) and female (D) mice, and swim speed for male (E) and female (F) mice. Data is derived from 23 WT (12M, 11F) 29 HET (15M, 14F) 18 KO (9M, 9F) mice, and levels of significance indicated by ** and *** as p<0.01 and p<0.0001, respectably, compared to WT mice.</p

Male Nrxn1α mice show increased aggressive behaviours and social investigation towards a juvenile conspecific.

<p>Data shown are the mean (± sem) social investigation behaviours of the Nrxn1α mice. A - Time spent (s) carrying out aggressive behaviours towards the juvenile conspecific by the male Nrxn1α mice in the main study. B - Time spent (s) carrying out aggressive behaviours towards the juvenile conspecific by the male Nrxn1α mice in the replication study. Time spent (s) carrying out social sniffing of the juvenile conspecific mice by the male (C) and female (E) Nrxn1α mice in the main study. Time spent (s) carrying out social sniffing of the juvenile conspecific mice by the male (D) and female (F) Nrxn1α mice in the replication study. Data is derived from 23 WT (12M, 11F) 29 HET (15M, 14F) 18 KO (9M, 9F) mice for the main study, and 16 WT (7M, 9F) 12 HET (6M, 6F) 13 KO (8M, 5F) mice in the replication study, and levels of significance indicated by * and ** as p<0.05 and p<0.01, respectively, compared to WT mice, and # and ## as p<0.05 and p<0.01, respectively, compared to HET mice.</p