Deciphering sugar chain-based signals regulating integrative neuronal functions

金沢大学医薬保健研究域医学系精巧な神経回路網は高次神経機能の構造的基盤である。約２０年前に神経軸索ガイダンス分子が発見され、神経軸索が（中間）標的細胞からの軸索ガイダンス分子に導かれるという軸索誘導の基本メカニズムは明らかにされた。しかし、脳発生期に複雑で精巧な脳回路がどのようにして形成されるのかについては分かっていない。この１０年間に、軸索ガイダンス分子であるnetrin, Slit, Ephrinの働きが糖鎖により制御されることが報告され、糖鎖によるより高度な軸索ガイダンス機構の存在が示された。脳発生期にはヘパラン硫酸（HS）やコンドロイチン硫酸（CS）の糖鎖構造が時空間的にダイナミックに変化していく事は明らかになっているが、それらの軸索ガイダンスにおける役割については不明な点が多い。本研究では、我々が独自に発見した軸索ガイダンス分子draxinとプロテオグリカンとの関連性を調べた。ノックアウトマウスの解析から、draxinが脳回路形成に重要であることは分かっていたが、その活性制御についてはほとんど分かっていなかった。本研究において、draxinが視床神経の軸索伸長に対して濃度依存的な活性を保持し、その活性制御にヘパラン硫酸プロテオグリカンが重要であることが分かった。研究課題/領域番号:26110715, 研究期間(年度):2014-04-01 – 2016-03-3

An essential role of SVZ progenitors in cortical folding in gyrencephalic mammals

Because folding of the cerebral cortex in the mammalian brain is believed to be crucial for higher brain functions, the mechanisms underlying its formation during development and evolution are of great interest. Although it has been proposed that increased neural progenitors in the subventricular zone (SVZ) are responsible for making cortical folds, their roles in cortical folding are still largely unclear, mainly because genetic methods for gyrencephalic mammals had been poorly available. Here, by taking an advantage of our newly developed in utero electroporation technique for the gyrencephalic brain of ferrets, we investigated the role of SVZ progenitors in cortical folding. We found regional differences in the abundance of SVZ progenitors in the developing ferret brain even before cortical folds began to be formed. When Tbr2 transcription factor was inhibited, intermediate progenitor cells were markedly reduced in the ferret cerebral cortex. Interestingly, outer radial glial cells were also reduced by inhibiting Tbr2. We uncovered that reduced numbers of SVZ progenitors resulted in impaired cortical folding. When Tbr2 was inhibited, upper cortical layers were preferentially reduced in gyri compared to those in sulci. Our findings indicate the biological importance of SVZ progenitors in cortical folding in the gyrencephalic brain

CRISPR/Cas9-mediated gene knockout in the mouse brain using in utero electroporation

The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor and is responsible for callosal axon projections in the developing mouse brain. We first confirmed that these constructs efficiently induced double-strand breaks (DSBs) in target sites of exogenous plasmids both in vitro and in vivo. We then found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Furthermore, we found that Cas9-mediated targeting of the Satb2 gene induced abnormalities in axonal projection patterns, which is consistent with the phenotypes previously observed in Satb2 mutant mice. Introduction of pX330-NeuN using our procedure also resulted in the efficient disruption of the NeuN gene. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo. © 2016, Nature Publishing Group. All rights reserved

Pathophysiological analyses of cortical malformation using gyrencephalic mammals

One of the most prominent features of the cerebral cortex of higher mammals is the presence of gyri. Because malformations of the cortical gyri are associated with severe disability in brain function, the mechanisms underlying malformations of the cortical gyri have been of great interest. Combining gyrencephalic carnivore ferrets and genetic manipulations using in utero electroporation, here we successfully recapitulated the cortical phenotypes of thanatophoric dysplasia (TD) by expressing fibroblast growth factor 8 in the ferret cerebral cortex. Strikingly, in contrast to TD mice, our TD ferret model showed not only megalencephaly but also polymicrogyria. We further uncovered that outer radial glial cells (oRGs) and intermediate progenitor cells (IPs) were markedly increased. Because it has been proposed that increased oRGs and/or IPs resulted in the appearance of cortical gyri during evolution, it seemed possible that increased oRGs and IPs underlie the pathogenesis of polymicrogyria. Our findings should help shed light on the molecular mechanisms underlying the formation and malformation of cortical gyri in higher mammals. © 2015 Macmillan Publishers Limited

Three-dimensional visualization of intrauterine conceptus through the uterine wall by tissue clearing method

金沢大学医薬保健研究域医学系Visualization of specific cells in the three-dimensional organ architecture is one of the key steps to develop our knowledge about pathophysiological mechanisms in various organs. In this study, we successfully obtained stereoscopic whole images of the intrauterine murine embryo and placenta through the uterus using a modified tissue clearing CUBIC method. By this procedure, we can recognize the three-dimensional relationships among various tissues within the pregnant uterus and analyze free-angle images of cross-sections with single-cell resolution using a computer system. Based on these data, we can select optimal cross-section angles and then produce the corresponding tissue slices that are adequate for further immunohistochemical examination. Furthermore, using transgenic mice, distinct images of an EGFP-positive embryo and the placenta can be obtained, confirming the precise three-dimensional location of invading trophoblasts in the feto-maternal interface in the uterus. These results indicate that this procedure will significantly contribute to analyzing pathophysiological mechanisms in reproductive organs. © 2017 The Author(s)

ERAD components Derlin-1 and Derlin-2 are essential for postnatal brain development and motor function

Derlin family members (Derlins) are primarily known as components of the endoplasmic reticulum-associated degradation pathway that eliminates misfolded proteins. Here we report a function of Derlins in the brain development. Deletion of Derlin-1 or Derlin-2 in the central nervous system of mice impaired postnatal brain development, particularly of the cerebellum and striatum, and induced motor control deficits. Derlin-1 or Derlin-2 deficiency reduced neurite outgrowth in vitro and in vivo and surprisingly also inhibited sterol regulatory element binding protein 2 (SREBP-2)-mediated brain cholesterol biosynthesis. In addition, reduced neurite outgrowth due to Derlin-1 deficiency was rescued by SREBP-2 pathway activation. Overall, our findings demonstrate that Derlins sustain brain cholesterol biosynthesis, which is essential for appropriate postnatal brain development and function

Folding of the Cerebral Cortex Requires Cdk5 in Upper-Layer Neurons in Gyrencephalic Mammals

金沢大学医薬保健研究域医学系Folds in the cerebral cortex in mammals are believed to be key structures for accommodating increased cortical neurons in the cranial cavity. However, the mechanisms underlying cortical folding remain largely unknown, mainly because genetic manipulations for the gyrencephalic brain have been unavailable. By combining in utero electroporation and the CRISPR/Cas9 system, we succeeded in efficient gene knockout of Cdk5, which is mutated in some patients with classical lissencephaly, in the gyrencephalic brains of ferrets. We show that Cdk5 knockout in the ferret cerebral cortex markedly impaired cortical folding. Furthermore, the results obtained from the introduction of dominant-negative Cdk5 into specific cortical layers suggest that Cdk5 function in upper-layer neurons is more important for cortical folding than that in lower-layer neurons. Cdk5 inhibition induced severe migration defects in cortical neurons. Taken together, our findings suggest that the appropriate positioning of upper-layer neurons is critical for cortical folding. © 2017 The Author(s

大脳皮質特異的なノックアウト動物作成法の開発

金沢大学医薬保健研究域医学系本研究では、子宮内エレクトロポレーションとCRISPR/Cas9システムとを組み合わせることにより、大脳皮質特異的な遺伝子ノックアウト法の確立を目指した。マウス大脳皮質にSatb2遺伝子に対するCRISPRコンストラクトを導入し、Satb2の発現を免疫染色により調べた。その結果、Satb2に対するCRISPRコンストラクトが導入された細胞のほとんどで、Satb2の発現が消失していた。これらの結果から、子宮内エレクトロポレーション法を用いたCRISPR/Cas9の導入により大脳皮質神経細胞において効果的に標的遺伝子をノックアウトできることが分かった。The CRISPR/Cas9 system has recently been adapted for generating knockout mice to investigate physiological functions and pathological mechanisms. Here, we report a highly efficient procedure for brain-specific disruption of genes of interest in vivo. We constructed pX330 plasmids expressing humanized Cas9 and single-guide RNAs (sgRNAs) against the Satb2 gene, which encodes an AT-rich DNA-binding transcription factor. We found that the introduction of pX330-Satb2 into the developing mouse brain using in utero electroporation led to a dramatic reduction of Satb2 expression in the transfected cerebral cortex, suggesting DSBs had occurred in the Satb2 gene with high efficiency. Thus, our procedure combining the CRISPR/Cas9 system and in utero electroporation is an effective and rapid approach to achieve brain-specific gene knockout in vivo.研究課題/領域番号:15K14332, 研究期間(年度):2015-04-01 - 2017-03-3

新規神経ガイダンス分子Draxin受容体の固定とその機能解析

本研究では、cDNAライブラリーの発現スクリーニングにより新規ガイダンス分子Draxin受容体の同定を試みた。FACSを用いた発現スクリーニングを行ったが、受容体のクローニングには至らなかった。一方、既知の受容体タンパクからのスクリーニングを行った(candidate approach)結果、Draxinと強く結合する膜タンパクを見出した。現在、この分子が真にDraxin受容体であるかどうかを詳細に解析している。研究課題/領域番号:19770196, 研究期間(年度):2007-2008出典：「新規神経ガイダンス分子Draxin受容体の固定とその機能解析」研究成果報告書　課題番号19770196（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/report/KAKENHI-PROJECT-19770196/19770196seika/)を加工して作