Discussion
Our behavioral analysis in mice uncovered some novel roles of Zic2 related to higher brain function. A summary of the results from this behavioral analysis and that of previous studies is provided in Table 1.
The locomotor activity differences between wild-type and Zic2kd/+ mice were context-dependent. In home cages, the mutant mice showed reduced locomotor activity in the early dark phase compared to wild-type mice, but in the open field test the mutant mice showed higher locomotor activity than wild-type mice. The tendency for Zic2kd/+ mice to display higher activity in a novel environment, compared to wild-type mice was also observed in the the Y-maze test and the light-dark box test (Figure S1C). Therefore, Zic2kd/+ mice appear to be generally hyperactive upon exposure to a novel environment. This hyperactivity is possibly consistent with symptoms of schizophrenia in humans; hyperactivity in response to a novel environment has been suggested as a useful animal correlate of schizophrenia symptoms34 and has been noted in some genetically engineered mouse models of schizophrenia353637.
We demonstrated cognitive dysfunction in Zic2kd/+ mice by the water maze test, the fear-conditioning test, and the Y-maze test. In addition, abnormal PPI, which is deemed to reflect impaired sensorimoter-gating function seen in schizophrenia, is reported in Zic2kd/+ 12. These results corroborate that the cognitive function deficits in Zic2kd/+ mice are not simple, but multimodal ones including sensorimotor gating function.
Social behavioral abnormalities in Zic2kd/+ mice were characterized by a reduction in aggressive behavior compared to the wild-type controls in the absence of clear deficits in the affiliative behaviors. The aggressivity assessed in the resident-intruder and social dominance tube tests may be related to their territory protecting behavior. The absence of depression-like behavior in these mice excludes the possibility that their reduced aggressivity was the result of a general loss in motivation.
Collectively, the behavioral phenotypes of Zic2kd/+ mice seem to be implicated in the three classes of schizophrenia symptoms (positive/negative symptoms and cognitive dysfunction). When we compare the Zic2kd/+ mice phenotype with those of other typical schizophrenia model mice (Table 3), novelty-induced hyperactivity and prepulse inhibition reduction were commonly found in the dominant negative DISC1 transgenic38and NRG1 transmembrane KO35and conditional KO of ErbB4 in PV-positive interneuron39. In addition, the enlargement of lateral ventricle and decrement of working memory were shared with Zic2kd/+ and some of them (Table 3).
The morphological abnormalities in the brain of Zic2kd/ include a reduction in the septal mass, thinning of the cerebral cortex and corpus callosum, narrowing of the fimbria hippocampi, and a regional reduction of amygdalar nuclei. These abnormalities have a pathophysiological resemblance to neuropsychiatric disorders in humans. In particular, enlarged lateral ventricles and decrease in whole brain volume are a symptom of the first episode of schizophrenia161718, and have been observed in some genetically-engineered mouse models of schizophrenia1537384041. These finding add further support for the genetic involvement of ZIC2 in the pathogenesis of schizophrenia.
Regarding the basis of neural circuits underlying the higher brain function abnormalities observed in Zic2kd/+mice, we consider the following two observations to be significant. Firstly, we observed a reduction in the number of cholinergic neurons in the basal forebrain, which raises the possibility that abnormal cholinergic regulation of higher brain function underlies the behavioral abnormalities seen in Zic2kd/+ mice. Basal forebrain cholinergic neurons are thought to be capable of regulating the cortical processing of sensory stimuli within various domains42. In addition, recent studies indicate that the cholinergic system modulates cognitive deficits in schizophrenia and that cholinergic transmission is a potential target of therapeutics for the improvement of cognitive functions43. Thus, further evaluation of the cholinergic transmission dynamics in Zic2 mutants would be beneficial for a better understanding of the role of Zic2 in cognitive function. We also examined the distribution of PV-positive cells in medial and dorsolateral prefrontal cortices and in the hippocampus (Figure S3) because the distribution of PV-positive cortical neurons, which represent a subset of GABAergic inhibitory neurons, is altered in some animal models of schizophrenia4044 and is thought to be a key abnormality underlying the pathogenesis of schizophrenia45. However, we did not observe any significant alterations in the distribution of PV-positive cells in Zic2kd/+ cortices (Figure S3).
Our second key observation relates to those implying that abnormalities of the amygdala underlie the social behavior abnormalities in Zic2kd/+ mice. The reduced aggressivity of Zic2kd/+ mice was indicative of abnormal social behavior and we hypothesized that abnormalities of the amygdala were involved for a number of reasons. Firstly, it is well known that the amygdala is essential for controlling aggressive behaviors46. Also, lesions in the rat medial amygdala cause a reduction in aggressive behavior47. Adding further support, a recent study showed that the AHA and medial amygdala project into the hypothalamic aggression area (mediobasal hypothalamus), which plays a central role in the control of aggressive behavior48. These facts led us to hypothesize that the reduced aggressivity in Zic2kd/+ mice is related to the altered morphology of AHA. However, there have been limited studies focusing on the role of AHA in aggressive behavior. Therefore, further investigation of the amygdalar abnormalities in Zic2kd/+ mice would contribute to our understanding of the neural circuits controlling aggressive behavior.
The molecular mechanism of developmental disturbances that lead to the cholinergic neuronal loss and amygdalar dysgenesis remains elusive. As one interpretation, these abnormalities may reflect a milder representation of the HPE-like abnormality8 and cortical dysgenesis partly as a result of the abnormal Zic2-expressing meningeal progenitor cells11 in Zic2kd/kd mice. In terms of forebrain cholinergic neuron development, we found that the p75-expressing cholinergic progenitor neurons in the prospective medial septum and diagonal band are missing in Zic1/Zic3 compound mutant mice49. Since the structure and function of the vertebrate Zic family of proteins is highly conserved3, Zic2 might function to expand the medial forebrain cholinergic neural progenitor cells by inhibiting their exit from the proliferating cell cycle in a manner analogous to that in the Zic1/Zic3 compound mutant mice49.
Resequencing analysis of Zic2 in Japanese patients with schizophrenia revealed three novel nonsynonymous mutations in ZIC2. Functional analysis of these mutations in the Zic2kd/+ mouse model of schizophrenia indicated that the R409P mutation results in severe loss-of-function. We showed that the transcriptional activation capacity of the Zic2-R409P protein was about 20% that of the wild-type protein; which corresponds to the decreased protein production from the Zic2kd allele shown previously8. This finding in turn validates Zic2kd/+ mice as an animal model of the R409P mutation in schizophrenia. The patient with the R409P mutation was diagnosed with residual-type schizophrenia.
Many studies have investigated the ZIC2 mutations in patients with HPE. A recent meta-analysis study of previously published results showed that the vast majority of ZIC2 mutations (98%) cause significant loss-of-function7. This suggests that HPE is caused by severely impaired function of ZIC2. Interestingly, only the very few cases (three families), in which the function of ZIC2 was shown to be null, included two independent parents with normal brain imaging despite the identification of ZIC2 missense mutations (Q36P or D152F)7. Together with these results, our findings raise the possibility that mildly impaired ZIC2 function does not result in HPE, but in psychiatric illnesses.
In summary, behavioral and morphological phenotypes in Zic2kd/+ mice were reminiscent of those of schizophrenia. Additionally, the detection of rare, but significantly defective, missense mutations in patients with schizophrenia suggests that further analysis of ZIC2 in neuropsychiatric patients is meaningful. Since this study focused on missense mutations, there still remains the possibility that mutations in introns and/or flanking regions that provoke partial loss of function are associated with schizophrenia.