In the present study, we generated a unique mouse model carrying a single point mutation R215H of NLGN2 gene that was originally identified from human schizophrenia patients. The NL2 R215H KI mice have impaired GABAergic synapse development, reduced inhibitory synaptic transmission, and decreased hippocampal activation in response to stress. Moreover, the R215H KI mice display anxiety-like behavior, impaired pre-pulse inhibition, cognitive deficits and abnormal stress response, partially recapitulating some of the core symptoms of schizophrenia patients. These results suggest that this newly generated R215H KI mouse line may provide a unique animal model for studying molecular mechanisms underlying schizophrenia and related neuropsychiatric disorders.
GABAergic and behavioral deficits in NL2 R215H KI mice
NL2 plays important roles in regulating perisomatic GABAergic synapse development, phasic GABAergic transmission, and neural excitability [
1,
3,
9,
19,
24,
25,
30,
40,
47,
59,
61]. Consistent with our previous in vitro studies, the current in vivo work demonstrates that R215H mutation disrupts GABAergic synapse development. Functionally, NL2 R215H mutation caused a reduction of both frequency and amplitude of inhibitory neurotransmission. These results suggest that the R215H KI mice display more GABAergic deficits than the reported NL2 KO mice [
1,
9,
19,
30,
47], which might explain why our KI mice display more behavioral deficits than the NL2 KO mice, such as PPI impairment, cognitive deficits, and abnormal stress response. Coincidentally, previous studies reported that NL3 R451C KI mouse also displayed stronger phenotypes than the NL3 KO mice [
14,
16,
57,
64]. These evidences suggest that genetic mouse models based on mutations identified from patients may be more suitable than the germline KO mouse models for studying pathological mechanisms of human diseases, because of less compensation from other genes in KI mice than in KO mice.
Behaviorally, NL2 R215H KI mice display an anxiety phenotype, which may be the result of decreased GABAergic inhibition [
3,
11,
63]. Interestingly, R215H KI mice also show impaired startle responses and deficits in pre-pulse inhibition (PPI). Previous study in rats has reported that disturbance of PV neuron development in the hippocampal DG region may cause reduction of PPI [
21]. A recent study also demonstrates that specific inhibition of PV neurons in the ventral hippocampus results in a reduction of both startle response and PPI [
45]. Consistent with these findings, we demonstrate here that our R215H KI mice display a significant reduction of PV innervation in the hippocampus, which may underlie the deficits of PPI. In contrast, the NL2 KO mice lack PPI deficit, which might be related to an insufficient loss of PV innervation at hippocampal regions [
61]. Besides PV neurons, CCK (cholecystokinin) neurons are another type of inhibitory neurons mainly innervate CA1/2/3 pyramidal cells and DG proximal dendrites. CCK neurons can release GABA to act on GABA
A receptor α2 subunits that are known to mediate anxiolytic effect, or release CCK to act on CCK2 receptors and induce anxiogenic effect [
18]. It would be important to further investigate the expression and functional alteration of CCK neurons in our R215H KI mice in future studies.
Surprisingly, our former collaborator Dr. Chia-Hsiang Chen’s group recently reported that their NL2 R215H KI mice displayed an increased pre-pulse inhibition phenotype [
8]. However, because the startle response of their KI mice was not reported, it makes the data difficult to compare with ours. Additionally, they reported that their KI mice didn’t express NL2 and resembled global NL2 KO mice, but they did not present the actual comparison with NL2 KO mice. In contrast, our R215H KI mice are clearly different from the NL2 KO mice, because our KI mice showed small amount of NL2 expression, particularly during early developmental stages. The low expression level of NL2 in our R215H KI mice distinguishes our KI mice from the NL2 KO mice, which showed completely absent expression of NL2 in our Western blot analysis. Furthermore, our R215H KI mice also showed clear GABAergic deficits as expected, but it is unknown whether their KI mice have any GABAergic deficits or not [
8].
Another interesting observation is that the R215H KI mice are hyperactive after acute stress and are associated with impaired hippocampal activation. It has been reported that robust neuron activation requires low background activity before stimulus [
33,
49]. However, due to the reduction of GABAergic inhibition in our R215H KI mice, the background activity of hippocampal neurons may be chronically elevated, which will dampen further activation of the hippocampus by external stimulation [
40]. The impaired activation of hippocampal neurons in R215H KI mice may contribute to the abnormal stress response we observed, as hippocampus acts like a “brake” during acute stress to prevent HPA axis from over activation [
22]. Besides hippocampus, sensitized HPA-axis involves brain regions such as hypothalamus and amygdala. Loss of NL2 in these areas could directly affect their GABAergic transmission and releasing of corticosteroids into the circulation, which is worth of further investigation as well.
NL2 R215H mutation and schizophrenia
It is well documented that schizophrenia patient’s show impaired pre-pulse inhibition as an abnormal sensorimotor gating deficit [
5,
20]. Many patients also have emotional symptoms such as anxiety and depression [
39]. Additionally, patients are hypersensitive toward stress and certain patients have been found with altered HPA axis function [
4]. Intriguingly, R215H KI mice recapitulated these SCZ-like behaviors, suggesting a potential role of NL2 R215H in the development of schizophrenia symptoms. Furthermore, reduction of PV expression and PV-positive synapses is a prominent phenotype observed in SCZ patients [
36‐
38,
62]. The R215H mutation KI mice also show a significant reduction of PV innervation, consistent with the pathogenic deficit of SCZ patients. These GABAergic deficits, together with cognition and PPI deficits manifested in the KI mice, support the hypothesis that GABA dysfunction makes an important contribution to the cognitive and attention deficits of SCZ. Taken together, NLGN2 R215H single point mutation has a significant impact on GABAergic synapse development and the pathogenesis of neuropsychiatric disorders. Our newly generated NL2 R215H KI mice may provide a useful mouse model for the study of molecular mechanisms and drug development of neuropsychiatric disorders including schizophrenia.