Deletion of α-neurexin II results in autism-related behaviors in mice

Abstract

Autism is a common and frequently disabling neurodevelopmental disorder with a strong genetic basis. Human genetic studies have discovered mutations disrupting exons of the NRXN2 gene, which encodes the synaptic adhesion protein α-neurexin II (Nrxn2α), in two unrelated individuals with autism, but a causal link between NRXN2 and the disorder remains unclear. To begin to test the hypothesis that Nrxn2α deficiency contributes to the symptoms of autism, we employed Nrxn2α knockout (KO) mice that genetically model Nrxn2α deficiency in vivo. We report that Nrxn2α KO mice displayed deficits in sociability and social memory when exposed to novel conspecifics. In tests of exploratory activity, Nrxn2α KO mice displayed an anxiety-like phenotype in comparison with wild-type littermates, with thigmotaxis in an open field, less time spent in the open arms of an elevated plus maze, more time spent in the enclosure of an emergence test and less time spent exploring novel objects. However, Nrxn2α KO mice did not exhibit any obvious changes in prepulse inhibition or in passive avoidance learning. Real-time PCR analysis of the frontal cortex and hippocampus revealed significant decreases in the mRNA levels of genes encoding proteins involved in both excitatory and inhibitory transmission. Quantification of protein expression revealed that Munc18-1, encoded by Stxbp1, was significantly decreased in the hippocampus of Nrxn2α KO mice, which is suggestive of deficiencies in presynaptic vesicular release. Our findings demonstrate a causal role for the loss of Nrxn2α in the genesis of autism-related behaviors in mice.

Figure 1

Nrxn2α KO mice exhibit deficits in sociability and social memory. During the first phase (a), whereby the test mouse had to discriminate between a novel mouse and an empty but identical cage, Nrxn2α KO mice (n=16) display no significant preference for exploring the novel mouse compared with the empty cage, whereas WT (n=31) showed a very clear discrimination (repeated measure analysis of variance (RM ANOVA), significant genotype × discrimination interaction; F_(1,45)=14.89, P<0.0001). Tests of simple main effects found a significant effect of genotype when exploring the novel mouse (F_(1,45)=18.51, P<0.0001) but not the empty cage (F_(1,45)<1, P>0.05). In stage 2 (b), the preference of the test mouse to discriminate between the previously explored mouse (Stranger 1) and a second novel mouse (Stranger 2) was measured. Nrxn2α KO mice spent a similar time as WT exploring Stranger 1, but showed significantly less exploration of Stranger 2 (RM ANOVA, significant genotype × discrimination interaction; F_(1,45)=8.08, P=0.007). Tests of simple main effects confirmed a significant effect of genotype on time exploring the novel mouse (F_(1,45)=8.92, P=0.005) but not on time exploring the previously explored mouse (F_(1,45)<1, P>0.05). Nrxn2α KO mice were also unable to discriminate between exploring soiled vs clean bedding (c). Nrxn2α KO mice (n=13) showed no preference for either cage, whereas WT (n=11) spent a proportionately longer time exploring the cage containing the soiled bedding (RM ANOVA, significant genotype × discrimination interaction; F_(1,22)=8.37, P=0.008). Tests of simple main effects found a significant effect of genotype on time exploring the soiled bedding (F_(1,22)=5.01, P=0.036) but not the clean bedding (F_(1,22)=3.81, P>0.05). KO, knockout; Nrxn2α, α-neurexin II; NS, not significant; WT, wild type. *P<0.05; **P<0.01; ***P<0.0001.

Figure 2

Activity of Nrxn2α KO mice in the open field. (a) Nrxn2α KO mice (n=16) display a marginal but non-significant increase in locomotion compared with WT mice (n=33) over 30 min of free exploration (two-way repeated measures analysis of variance, main effect of time block (F_(5,235)=18.12, P<0.0001), no effect of genotype (F_(1,47)=2.44, P>0.05) or interactions (F_(5,235)<1, P>0.05)). During the trial, the arena floor was divided into three zones (outer, intermediate, center) and the mice were tracked automatically. Nrxn2α KO mice spent significantly more time within the outer zone (b; t₍₄₇₎=2.54, P=0.015; thigmotaxis) and significantly less time in the intermediate zone (c; t₍₄₇₎=2.59, P=0.013). There was also a trend for Nrxn2α KO mice to spend less time in the center zone (d; t₍₄₇₎=1.76, P=0.085). KO, knockout; Nrxn2α, α-neurexin II; WT, wild type. *P<0.05.

Figure 3

Nrxn2α KO mice show an anxiety-like phenotype. In the elevated plus maze, KO mice (n=16) spent significantly less time in the open arms (a; t₍₄₇₎=2.62, P=0.012) and significantly more time in the closed arms (b; t₍₄₇₎=6.84, P<0.0001) compared with WT (n=33). KO mice also make significantly fewer head dips (c; t₍₄₇₎=4.68, P<0.0001). In the emergence test, the latency to emerge from an enclosed shelter into an open arena was significantly longer in Nrxn2α KO mice (n=16) compared with WT (n=33) (d; t₍₄₇₎=4.16, P<0.0001) and, overall, they spent significantly less time out of the enclosed shelter over the 15 min trial (e; two-way repeated measures analysis of variance, main effect of time block (F_(2,94)=14.34, P<0.0001) and genotype (F_(1,47)=12.30, P=0.001), no significant interactions (F_(2,94)=2.01, P>0.05)). In a familiar environment, Nrxn2α KO mice also spent significantly less time engaging with novel objects (f; t₍₄₇₎=2.86, P=0.006). KO, knockout; Nrxn2α, α-neurexin II; WT, wild type. *P<0.05; **P<0.01; ***P<0.0001.

Figure 4

Altered mRNA transcript and protein expression levels in the frontal cortex and hippocampus of Nrxn2α KO brain. The 13 genes examined were divided into groups of inhibitory-related (a), excitatory-related (b) and synaptic scaffold-related (c). Within the frontal cortex, Nrxn2α KO mice showed significant reductions in the mRNA level of Dlg4, whereas in the hippocampus, Pvalb, Grin2a, Stxbp1 and Dlg4 were also reduced. Gad1 and Slc17a7 (VGlut1) both had reductions approaching significance (note in a and b, ^#P=0.059 and ^#P=0.086, respectively). (d) Summary of the significantly altered genes (unpaired t-test). (e and f) Within the hippocampus, western blotting confirmed a decrease in the protein expression of Munc18-1 (Stxbp1) (t₍₂₂₎=2.31, P=0.031), although there was no significant difference in the cortex (P>0.05). KO, knockout; Nrxn2α, α-neurexin II; RQ, relative quantification; WT, wild type. *P<0.05.