Motoko Maekawa

Concise Review: Pax6 Transcription Factor Contributes to both Embryonic and Adult Neurogenesis as a Multifunctional Regulator

Pax6 is a highly conserved transcription factor among vertebrates and is important in various developmental processes in the central nervous system (CNS), including patterning of the neural tube, migration of neurons, and formation of neural circuits. In this review, we focus on the role of Pax6 in embryonic and postnatal neurogenesis, namely, production of new neurons from neural stem/progenitor cells, because Pax6 is intensely expressed in these cells from the initial stage of CNS development and in neurogenic niches (the subgranular zone of the hippocampal dentate gyrus and the subventricular zone of the lateral ventricle) throughout life. Pax6 is a multifunctional player regulating proliferation and differentiation through the control of expression of different downstream molecules in a highly context‐dependent manner.

Fabp7 maps to a quantitative trait locus for a schizophrenia endophenotype.

Deficits in prepulse inhibition (PPI) are a biological marker for schizophrenia. To unravel the mechanisms that control PPI, we performed quantitative trait loci (QTL) analysis on 1,010 F2 mice derived by crossing C57BL/6 (B6) animals that show high PPI with C3H/He (C3) animals that show low PPI. We detected six major loci for PPI, six for the acoustic startle response, and four for latency to response peak, some of which were sex-dependent. A promising candidate on the Chromosome 10-QTL was Fabp7 (fatty acid binding protein 7, brain), a gene with functional links to the N-methyl-D-aspartic acid (NMDA) receptor and expression in astrocytes. Fabp7-deficient mice showed decreased PPI and a shortened startle response latency, typical of the QTLs proposed effects. A quantitative complementation test supported Fabp7 as a potential PPI-QTL gene, particularly in male mice. Disruption of Fabp7 attenuated neurogenesis in vivo. Human FABP7 showed altered expression in schizophrenic brains and genetic association with schizophrenia, which were both evident in males when samples were divided by sex. These results suggest that FABP7 plays a novel and crucial role, linking the NMDA, neurodevelopmental, and glial theories of schizophrenia pathology and the PPI endophenotype, with larger or overt effects in males. We also discuss the results from the perspective of fetal programming.

Pax6 is required for production and maintenance of progenitor cells in postnatal hippocampal neurogenesis

Neurogenesis is crucial for brain formation and continues to take place in certain regions of the postnatal brain including the subgranular zone (SGZ) of the hippocampal dentate gyrus (DG). Pax6 transcription factor is a key player for patterning the brain and promoting embryonic neurogenesis, and is also expressed in the SGZ. In the DG of wild‐type rats, more than 90% of total BrdU‐incorporated cells expressed Pax6 at 30 min time point after BrdU injection. Moreover, approximately 60% of Pax6+ cells in the SGZ exhibited as GFAP+ cells with a radial glial phenotype and about 30% of Pax6+ cells exhibited as PSA‐NCAM+ cells in clusters. From BrdU labeling for 3 days, we found that cell proliferation was 30% decreased at postnatal stages in Pax6‐deficient rSey2/+ rat. BrdU pulse/chase experiments combined with marker staining revealed that PSA‐NCAM+ late progenitor cells increased at the expense of GFAP+ early progenitors in rSey2/+ rat. Furthermore, expression of Wnt ligands in the SGZ was markedly reduced in rSey2/+ rat. Taken all together, an appropriate dosage of Pax6 is essential for production and maintenance of the GFAP+ early progenitor cells in the postnatal hippocampal neurogenesis.

Arachidonic acid drives postnatal neurogenesis and elicits a beneficial effect on prepulse inhibition, a biological trait of psychiatric illnesses.

Prepulse inhibition (PPI) is a compelling endophenotype (biological markers) for mental disorders including schizophrenia. In a previous study, we identified Fabp7, a fatty acid binding protein 7 as one of the genes controlling PPI in mice and showed that this gene was associated with schizophrenia. We also demonstrated that disrupting Fabp7 dampened hippocampal neurogenesis. In this study, we examined a link between neurogenesis and PPI using different animal models and exploring the possibility of postnatal manipulation of neurogenesis affecting PPI, since gene-deficient mice show biological disturbances from prenatal stages. In parallel, we tested the potential for dietary polyunsaturated fatty acids (PUFAs), arachidonic acid (ARA) and/or docosahexaenoic acid (DHA), to promote neurogenesis and improve PPI. PUFAs are ligands for Fabp members and are abundantly expressed in neural stem/progenitor cells in the hippocampus. Our results are: (1) an independent model animal, Pax6 (+/−) rats, exhibited PPI deficits along with impaired postnatal neurogenesis; (2) methylazoxymethanol acetate (an anti-proliferative drug) elicited decreased neurogenesis even in postnatal period, and PPI defects in young adult rats (10 weeks) when the drug was given at the juvenile stage (4–5 weeks); (3) administering ARA for 4 weeks after birth promoted neurogenesis in wild type rats; (4) raising Pax6 (+/−) pups on an ARA-containing diet enhanced neurogenesis and partially improved PPI in adult animals. These results suggest the potential benefit of ARA in ameliorating PPI deficits relevant to psychiatric disorders and suggest that the effect may be correlated with augmented postnatal neurogenesis.

A deficit of brain dystrophin impairs specific amygdala GABAergic transmission and enhances defensive behaviour in mice

Duchenne muscular dystrophy (DMD) is accompanied by cognitive deficits and psychiatric symptoms. In the brain, dystrophin, the protein responsible for DMD, is localized to a subset of GABAergic synapses, but its role in brain function has not fully been addressed. Here, we report that defensive behaviour, a response to danger or a threat, is enhanced in dystrophin-deficient mdx mice. Mdx mice consistently showed potent defensive freezing responses to a brief restraint that never induced such responses in wild-type mice. Unconditioned and conditioned defensive responses to electrical footshock were also enhanced in mdx mice. No outstanding abnormality was evident in the performances of mdx mice in the elevated plus maze test, suggesting that the anxiety state is not altered in mdx mice. We found that, in mdx mice, dystrophin is expressed in the amygdala, and that, in the basolateral nucleus (BLA), the numbers of GABA(A) receptor alpha2 subunit clusters are reduced. In BLA pyramidal neurons, the frequency of norepinephrine-induced GABAergic inhibitory synaptic currents was reduced markedly in mdx mice. Morpholino oligonucleotide-induced expression of truncated dystrophin in the brains of mdx mice, but not in the muscle, ameliorated the abnormal freezing response to restraint. These results suggest that a deficit of brain dystrophin induces an alteration of amygdala local inhibitory neuronal circuits and enhancement of fear-motivated defensive behaviours in mice.

Development of the mouse amygdala as revealed by enhanced green fluorescent protein gene transfer by means of in utero electroporation

The amygdala is located in the caudal part of the ventral telencephalon. It is composed of many subdivisions and is involved in the control of emotion. It is important to know the mechanisms of amygdalar development in order to analyze the pathogenesis of emotional disorders, but they are still not adequately understood. In the present study the migration, differentiation, and distribution of amygdalar neurons in the mouse embryo were investigated by means of in utero electroporation. Ventricular zone cells in restricted regions, that is, the caudal ganglionic eminence (CGE), the ventral pallium, the lateral pallium, and the diencephalon, were labeled with an expression vector of the enhanced green fluorescent protein (EGFP) gene. Labeling at embryonic day (E)10 revealed that the central nucleus originates from the neuroepithelium in the ganglionic eminence and the labeling at E11 and E12 revealed that the basolateral complex originates from the neuroepithelium of the ventral and lateral pallia. The introduction of the EGFP gene into the neuroepithelium of the third ventricle at E11 showed that the medial nucleus originates, at least in part, from the neuroepithelium of the diencephalon and migrates over the diencephalo–telencephalic boundary. The radial glial arrangement corresponded well with the initial migration of amygdalar neurons, and the radial processes later formed the boundary demarcating the basolateral complex. These findings indicate that the neurons originating from the temporally and spatially restricted neuroepithelium in both the telencephalon and diencephalon migrate and differentiate to form the mosaic of amygdalar subdivisions. J. Comp. Neurol. 513:113–128, 2009.

The effects of Fabp7 and Fabp5 on postnatal hippocampal neurogenesis in the mouse.

New neurons are continually produced after birth from neural stem/progenitor cells (NSCs/NPCs) in the hippocampal dentate gyrus (DG). Recent studies have reported that fatty acid binding protein 7 (Fabp7/brain lipid binding protein (BLBP)) is required for the maintenance of embryonic NSCs/NPCs and have identified an association between the Fabp7 gene and behavioral paradigms that correlate with hippocampal functions. However, the specific roles of Fabps in postnatal neurogenesis remain unknown. Herein, we demonstrate the effects of Fabp7, and another Fabp, Fabp5, on postnatal neurogenesis. Fabp7 and Fabp5 were detected in the subgranular zone (SGZ) of the DG, and Fabp7+ cells were less differentiated than Fabp5+ cells. We analyzed the differentiation state of NSCs/NPCs in the SGZ of 4‐week‐old (4w) Fabp7 knockout (7KO), Fabp5 KO (5KO), and Fabp7/Fabp5 double KO (7/5KO) mice and found that the number of NSCs/NPCs was dramatically reduced compared with wild‐type mice. Although the uptake of BrdU 1 day after injection was decreased in all KO mice, the survival of BrdU+ cells 1 month after injection was increased in the 7/5KO mice compared to other three genotypes. We also observed an enhancement of neuronal differentiation in all Fabp KO mice. In addition, the proliferation and survival of NSCs/NPCs differed along the anterior‐posterior axis (A‐P axis). A greater number of newborn cells in the posterior region became extinct, but this tendency was not apparent in the Fabps KO mice. These data suggest that Fabp7 and Fabp5 have differential roles for proliferation and survival of the NSCs/NPCs during postnatal DG neurogenesis. STEM Cells2012;30:1532–1543

Genome-wide association study of schizophrenia in Japanese population.

Schizophrenia is a devastating neuropsychiatric disorder with genetically complex traits. Genetic variants should explain a considerable portion of the risk for schizophrenia, and genome-wide association study (GWAS) is a potentially powerful tool for identifying the risk variants that underlie the disease. Here, we report the results of a three-stage analysis of three independent cohorts consisting of a total of 2,535 samples from Japanese and Chinese populations for searching schizophrenia susceptibility genes using a GWAS approach. Firstly, we examined 115,770 single nucleotide polymorphisms (SNPs) in 120 patient-parents trio samples from Japanese schizophrenia pedigrees. In stage II, we evaluated 1,632 SNPs (1,159 SNPs of p<0.01 and 473 SNPs of p<0.05 that located in previously reported linkage regions). The second sample consisted of 1,012 case-control samples of Japanese origin. The most significant p value was obtained for the SNP in the ELAVL2 [(embryonic lethal, abnormal vision, Drosophila)-like 2] gene located on 9p21.3 (p = 0.00087). In stage III, we scrutinized the ELAVL2 gene by genotyping gene-centric tagSNPs in the third sample set of 293 family samples (1,163 individuals) of Chinese descent and the SNP in the gene showed a nominal association with schizophrenia in Chinese population (p = 0.026). The current data in Asian population would be helpful for deciphering ethnic diversity of schizophrenia etiology.

NMDA receptor antagonist memantine promotes cell proliferation and production of mature granule neurons in the adult hippocampus

Memantine, which is used clinically for the treatment of Alzheimers disease (AD), is classified as an N-methyl-d-aspartate (NMDA) receptor antagonist. Since previous studies have shown that NMDA receptor antagonists promote neurogenesis in the adult brain, we examined the effect of memantine on neurogenesis in the adult mouse hippocampus. We intraperitoneally injected 3-month-old mice with memantine (at 10 or 50 mg/kg body weight) followed by 5-bromo-2-deoxyuridine (BrdU) injections (3x) after 3 days. We then examined the number of BrdU+ cells in the dentate gyrus (DG) of the hippocampus at different time points. The number of BrdU+ cells in the 50 mg/kg memantine-injected group increased by 2.1-fold (1 day after BrdU-injection), 3.4-fold (after 7 days), and 6.8-fold (after 28 days), whereas the 10 mg/kg dose of memantine had little effect on labeling compared to the control group. Immunohistochemical staining at 28 days after BrdU-injection revealed that the newly generated cells in the 50 mg/kg memantine-group had differentiated into mature granule neurons. Moreover, when 12-month-old mice were injected with memantine, cell proliferation was promoted in the DG (3.7-fold). These findings demonstrate that memantine promotes the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus.

Evaluation of Pax6 Mutant Rat as a Model for Autism

Autism is a highly variable brain developmental disorder and has a strong genetic basis. Pax6 is a pivotal player in brain development and maintenance. It is expressed in embryonic and adult neural stem cells, in astrocytes in the entire central nervous system, and in neurons in the olfactory bulb, amygdala, thalamus, and cerebellum, functioning in highly context-dependent manners. We have recently reported that Pax6 heterozygous mutant (rSey2/+) rats with a spontaneous mutation in the Pax6 gene, show impaired prepulse inhibition (PPI). In the present study, we further examined behaviors of rSey2/+ rats and revealed that they exhibited abnormality in social interaction (more aggression and withdrawal) in addition to impairment in rearing activity and in fear-conditioned memory. Ultrasonic vocalization (USV) in rSey2+ rat pups was normal in male but abnormal in female. Moreover, treatment with clozapine successfully recovered the defects in sensorimotor gating function, but not in fear-conditioned memory. Taken together with our prior human genetic data and results in other literatures, rSey2/+ rats likely have some phenotypic components of autism.