Deciphering molecular pathomechanisms of alpha-tubulin mutation-based neuronal disorders.

神经细胞通过被称为轴突的电缆状突起将神经系统连接到全身。它们需要保养一辈子。因此，在神经退行性疾病中，意外事故造成的轴突损伤或轴突丢失对神经元功能具有破坏性影响。肌动蛋白和微管（MT）细胞骨架构成轴突的结构骨干；尤其是mt，提供了维持生命的细胞运输高速公路。已知的人类α -小管蛋白突变可导致无脑畸形；轴突生长、神经元迁移和引导受到影响。然而，这些突变的分子病理机制尚不清楚。该项目旨在利用果蝇神经元组织和体内分析，阐明这些病理机制以及α 1-微管蛋白突变下游蛋白质网络的变化。alpha1-Tubulin是高度保守的（人类和苍蝇之间97%的同源性）；因此，鉴定的蛋白质相互作用有望从果蝇到人类的高度翻译。特异性无脑相关的α - 1微管蛋白氨基酸位点突变将在果蝇中产生，并用于研究其在分子、细胞和有机体水平上的影响。为此，果蝇神经元α 1-微管蛋白基因的遗传零背景将产生。利用crispr - cas9同源性定向修复，phi31 /attP着陆平台将取代内源性基因位点。这将随后允许通过重组酶介导的盒式交换容易地重新引入具有不同工程氨基酸替代突变的α - 1小管蛋白。在细胞水平上，免疫组织化学和生化染色、实时qRT-PCR、活细胞成像和遗传相互作用研究将用于确定表达突变α 1微管蛋白版本的原代神经元培养中的微管功能。为此，将分析微管稳定性、动力学、翻译后修饰、轴突运输和微管结合蛋白与mts的相互作用。在生物水平上，轴突引导、束状结构、神经元生长和神经元功能将在动物发育和衰老过程中进行研究。这将通过使用UAS-Gal4和克隆MARCM系统，结合先进的免疫组织化学成像技术和行为分析，表达突变的α - 1微管蛋白版本来实现。提出的研究策略将揭示在基于α - 1微管蛋白突变的神经疾病中蛋白质与微管相互作用的变化。这种研究策略可以在未来很容易地适应于研究在微管蛋白上发现的额外翻译后修饰的重要性，分析非神经元组织中无脑相关突变的分子方面，并确定微管蛋白突变相关疾病和神经退行性疾病的新分子靶点。

Nerve cells electrically wire the nervous system throughout the body via cable-like protrusions called axons. They have to be maintained for a lifetime. Therefore, damage to axons through accidents or their loss in neurodegenerative diseases has a devastating impact on neuronal function.The actin and microtubule (MT) cytoskeleton forms the structural backbone of axons; MTs in particular, provide life-sustaining cellular transport highways. Known mutations in human alpha-tubulins lead to lissencephaly; where axonal outgrowth, neuronal migration and guidance are affected. However, the molecular pathomechanisms of these mutations are poorly understood. The proposed project aims to clarify these pathomechanisms as well as changes in protein networks downstream of alpha1-Tubulin-mutations, using Drosophila neuronal tissue and in vivo analysis. alpha1-Tubulin is strongly conserved (97% identity between human and fly versions); therefore, identified protein interactions are expected to be highly translational from flies to humans. Specific lissencephaly-related alpha1-tubulin amino acid site mutations will be generated in flies and used to study their impact at a molecular, cellular and organismic level. To that end, a genetic null background of the Drosophila neuronal alpha1-tubulin gene will be generated. Using CRISPR-Cas9-homology directed repair, a PhiC31/attP landing platform will replace the endogenous gene locus. This will subsequently allow the easy reintroduction of alpha1-tubulins with varying engineered amino acid substitution mutations via recombinase mediated cassette exchange. At the cellular level, immunohistochemical and biochemical stainings, real-time qRT-PCR, live cell imaging and genetic interactions studies will be used to determine microtubule function in primary neuron cultures expressing the mutated alpha1-tubulin versions. To that end, microtubule stability, dynamics, posttranslational modifications, axonal transport and microtubule binding protein interactions with MTs. will be analysed. At an organismic level, axon guidance, fasciculation, neuronal growth and neuronal function will be studied during animal development and aging. This will be achieved by using the UAS-Gal4 and clonal MARCM systems for the expression of the mutated alpha1-tubulin versions in combination with advanced immunohistochemical imaging techniques and behavioural assays.The proposed research strategy will unravel changes in protein interactions with microtubules in alpha1-tubulin mutation based neuronal disorders. This research strategy can be easily adapted in the future to study the importance of additional posttranslational modifications found on tubulins, to analyse molecular aspects of lissencephaly-linked mutations in non-neuronal tissues, and to identify new molecular targets in tubulin mutation-linked disorders and neurodegenerative diseases.