Oral Session

Oral Session 9 一般口演9

O9-1 Macrocephaly of Chd8^+/- mice shows an increased number of cells and a decreased number of neurons in the cortex and the amygdala Chd8ヘテロ欠損マウス巨脳では大脳皮質と扁桃体の細胞数が増加し神経細胞が減少する Kasai Atsushi（笠井淳司）¹，中雄一郎¹，片山雄太²，彌永祐輔¹，山浦港生¹，田沼将人¹，勢力薫^1,3，中澤敬信^1,4，中山敬一²，橋本均^1,5,6,7 ¹Dept. Mol. Neuropharmacol., Grad. Sch. Pharm.Sci., Osaka Univ. ²Dept. Mol. Cell Biol., Med. Inst. Bioreg., Kyushu Univ. ³Inst. Acad. Init., Osaka Univ. ⁴Dept. Pharmacol., Grad. Sch. Dent., Osaka Univ. ⁵Mol. Res. Cent. Child Ment. Dev., United Grad. Sch. Child. Dev., Osaka Univ. ⁶Inst. Datability Sci., Osaka Univ. ⁷Open and Transdisciplinary Res. Init., Osaka Univ. Individuals with CHD8 mutations, strongly associated with neurological developmental disorders, present macrocephaly. Heterozygous knockout of Chd8 (Chd8^+/-) in mice and zebrafish also leads to macrocephaly. However, detailed pathological changes in macrocephaly with Chd8 mutations remain unclear. Here, we investigated morphological changes in the brains of Chd8^+/- mice using our newly developed imaging system, FAST (block-face serial microscopy tomography), which acquires whole-brain images to distinguish all individual cells. Fixed brains of 4-week-old Chd8^+/- mice and wild-type littermates were stained with propidium iodide to address the precise spatial arrangement of cells in the whole brain tissues, and were then imaged by the FAST system. We automatically measured the volume of brain regions, such as the cerebral cortex, and counted the cell numbers in each region using an image processing software. The volumes of the cerebral cortex and the amygdala in Chd8^+/- mice were significantly increased compared with wild-type littermates, whereas the volumes of the hippocampus, the striatum and the septum in both genotypes did not differ. The cell numbers in the cortex and the amygdala of Chd8^+/- mice were significantly increased. The cell density in layers II/III, V and VI of the cortex and the amygdala of Chd8^+/- mice were significantly increased. We then conducted immunostaining of the brain sections with NeuN, a neuronal marker, after FAST imaging. Surprisingly, the cell numbers and densities of neurons in layers II/III, V and VI of the cortex were significantly decreased in Chd8^+/- mice. These data indicate that macrocephaly in Chd8^+/- mice is caused by an increased number of cells, but the number of neurons are decreased in specific brain regions.		O9-2 Functional analysis of gene mutation of SRF coactivator MKL2 which was found in patients with autism spectrum disorders 自閉症スペクトラム障害で発見されたSRFコアクチベーターMKL2遺伝子変異の機能解析 Ihara Daisuke（伊原大輔）¹，山崎雄哉¹，佐藤夏美¹，福地守^1,2，津田正明¹，田渕明子¹ ¹Lab. Mol. Neurobiol., Grad. Sch. of Med. and Pharm. Sci., Univ. of Toyama, Toyama, Japan ²Lab. Mol. Neurosci., Fac. Pharmacy, Takasaki Univ. of Health and Welfare, Gunma, Japan Megakaryoblastic leukemia (MKL) family members, which are highly expressed in the brain, have actin-binding motifs and function as transcriptional cofactors of serum response factor (SRF). It has been proposed that MKL1, released from G-actin via Rho signaling, translocates into nucleus and increases SRF-mediated gene expression. Therefore, MKL is suggested to act as a linker between "morphological change" and "gene expression". Recently, it has been reported that de novo mutation of MKL2 gene is a risk factor of autism spectrum disorder (ASD). In this report, it is showed that the de novo mutation of cytosine in the MKL2 gene encoding B1 domain into thymine resulted in the substitution of 299^th arginine into tryptophan. However, it remains unclear how the mutation of MKL2 gene mentioned above affects their functions. Therefore, we constructed vectors for expressing human MKL2 (hMKL2-WT) and its mutant (hMKL2-MUT) and analyzed their functions in neurons. As the result of reporter assays using NIH3T3 cells, expression of hMKL2-WT increased the SRF-mediated transcriptional activity, but the activity was significantly reduced by expression of hMKL2-MUT. As the result of Sholl analysis for evaluation of dendritic complexity, the number of dendrites of rat cortical neurons was increased by expression of hMKL2-WT, but decreased by expression of hMKL2-MUT. These results indicate that hMKL2 which is introduced the mutation found in patients with ASD negatively regulates the dendritic morphology of neurons, probably due to the decrease in MKL/SRF-mediated gene expression.

O9-3 Pathophysiological significance of a missense mutation of PER3, a circadian clock gene, in autism spectrum disorders 時計遺伝子Per3の変異による自閉症発症メカニズムの解析 Noda Mariko（野田万理子），水野誠，田畑秀典，永田浩一 Dept. of Mol. Neurobiol., Inst. for Developmental Res., Aichi Human Service Center We recently screened autism spectrum disorder (ASD) patients for mutations in the coding regions of circadian-relevant genes and detected mutations in several clock genes including PER3. Based on in silico prediction analyses, we focused on the pathophysiological significance of c.1361G >A/p.(Arg366Gln) mutation of PER3 in ASD in the development of mouse cerebral cortex. This mutant was unstable when expressed in primary cultured cortical neurons, strongly suggesting haploinsufficiency leading to loss-of-function effects. Acute knockdown of Per3 with in utero electroporation caused abnormal positioning of cortical neurons during mouse corticogenesis, which was rescued by wild type Per3. Time-lapse imaging revealed abnormal migration phenotypes of Per3-deficient cortical neurons during corticogenesis. When Per3 was knocked down, axon extension and dendritic arbor formation of cortical neurons were suppressed while proliferation of neuronal progenitors and stem cells at the ventricular zone was not affected. In addition, acute Per3-knockdown mice showed defects in active avoidance learning. Taken together, Per3 was found to play a pivotal role in corticogenesis via regulation of excitatory neuron migration and synaptic network formation. Loss-of-function of Per3 was supposed to relate to ASD etiology and clinical features.