染色质重塑蛋白Duplin在大脑皮层神经元放射状迁移和发育中的调控作用和机制

Neurodevelopmental Disabilities (NDD) is a group of heterogeneous neurodevelopmental disorders and has a high prevalence in neonatal stages， with poor prognosis during the life span. There is still no effective strategies of diagnosis and treatment for NDD so far. Defect in cortical development is a major phenomenon involved in the pathogenesis of NDD. However, the underlying cellular and molecular mechanisms remain obscure. In our preliminary clinical study, it was found a patient with NDD harbors de novo mutation in gene encoding a novel chromatin remodeling protein, Duplin. But little is known about the biological function of Duplin in brain till now. Our pilot experiments showed that Duplin was selectively and highly expressed in the early stages of brain development, and may acts as a new important regulator of neuronal radial migration during mouse cortical development. To clarify the specific roles of Duplin in developing brain, here we will use multiple tools from molecular biology, histology and immunochemistry, and especially resort to human brain tissues, to investigate the expression pattern of Duplin in the nervous system. Furthermore, we will perform in utero electroporation, neuronal culture, virus transfection, genetic manipulation, molecular cloning, and biochemical analysis to investigate the specific role and the underlying molecular cellular mechanisms of Duplin in the migration and development of cerebral cortical neurons. This work will be helpful for us to understand the novel biological function of Duplin, and provide new insights into our knowledge on cerebral cortical development, as well as great translational potential in drug development for neonatal encephalopathy such as NDD.

神经发育障碍性疾病（NDD）是新生儿高发疾病，严重影响生存质量，亟需有效诊疗方法。大脑皮层发育异常是其重要病理现象，目前相关分子调控机制仍不明确。研究表明染色质重塑蛋白可能在大脑发育中发挥关键作用，我们在前期临床中新发现染色质重塑蛋白Duplin的编码基因突变与NDD相关，但该基因在大脑中的功能尚无详细研究。预实验数据表明Duplin在大脑发育早期呈特定表达并可能是调控皮层发育特别是放射状迁移的重要因子。为深入阐明Duplin对脑发育的作用和机制，本项目拟用分子生物学、组织学和免疫化学方法，结合人脑样品，研究Duplin在发育期脑内的表达分布规律；综合胚胎电转、细胞培养、病毒转染、基因敲除/过表达等手段，解析Duplin调控神经元放射状迁移和发育的分子细胞机制。这不仅开拓我们对Duplin分子功能的了解，还丰富我们对皮层发育机制的认识，也为NDD等新生儿脑病的诊疗和药物筛选提供新思路。

神经发育障碍性疾病（NDD）是新生儿高发疾病，严重影响生存质量，亟需有效诊疗方法。大脑皮层发育异常是其重要病理现象，目前相关分子调控机制仍不明确。研究表明染色质重塑蛋白可能在大脑发育中发挥关键作用，我们在前期临床中新发现染色质重塑蛋白Duplin的编码基因突变与NDD相关，但该基因在大脑中的功能尚无详细研究。Duplin蛋白又称CHD8蛋白。.我们使用免疫组织化学和原位杂交实验检查了CHD8在人和小鼠大脑中的表达，shRNA进行了胚胎电转，构建了RNAi载体，分析CHD8基因功能缺失的神经学功能影响。我们发现，CHD8在人脑和小鼠的神经元中均高表达，在胶质细胞中低表达。CHD8主要位于MAP2和小白蛋白阳性神经元的细胞核中。在发育期的小鼠大脑中，Chd8的表达从胚胎期16天到胚胎期18天达到峰值，从生后第14天到成年期显着下降。敲减Chd8导致神经元轴突和树突生长抑制，且破坏神经元轴突投射到对侧皮层的功能，在胚胎期第18.5天时导致神经元迁移延迟，但是在生后第3天和生后第7天迁移延迟恢复。.对人CHD8突变进行了功能学研究，模拟患者在体基因突变的情况，用点突变的方法构建c.5661_5662 del CT的CHD8突变质粒，转染到293细胞后用Western Blot检测发现CHD8突变质粒产生截短的CHD8蛋白；免疫细胞化学实验表明，CHD8突变质粒转染神经元后，其主树突的复杂程度降低。.我们通过胚胎电转和体外电转的实验，表明增强Wnt/β-catenin信号通路活性可以修复Chd8蛋白敲减引起的皮层神经元迁移延迟和神经元轴突、树突生长抑制的缺陷。.我们的研究数据表明CHD8在树突和轴突发育以及神经元的迁移过程中具有重要作用。进一步剖析由CHD8缺乏引起NDD的潜在分子作用机制，为NDD患者的临床表型提供细胞和分子水平上的初步解释。