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

破译基于α-微管蛋白突变的神经元疾病的分子病理机制。

• 批准号: 269898187
• 负责人: Dr. André Voelzmann
• 金额: --
• 依托单位: Wellcome Trust Centre for Cell-Matrix Research
• 依托单位国家: 德国
• 项目类别: Research Fellowships
• 财政年份: 2014
• 资助国家: 德国
• 起止时间: 2013-12-31 至 2016-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/269898187?language=en
• 关键词: Deciphering; molecular; pathomechanisms; alpha; tubulin

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.

神经细胞通过被称为轴突的电缆状突起将神经系统电连接到全身。它们必须维持一辈子。因此，轴突的损伤或神经退行性疾病的损失对神经元功能具有毁灭性的影响。肌动蛋白和微管（MT）细胞骨架形成轴突的结构骨架;特别是MT，提供维持生命的细胞运输公路。人类α-微管蛋白的已知突变导致无脑回畸形;其中轴突生长、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知之甚少。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。α 1-微管蛋白是高度保守的（人和果蝇版本之间97%的同一性）;因此，预期所鉴定的蛋白质相互作用是从果蝇到人类的高度翻译。特定的lissencephalys相关的α 1-微管蛋白氨基酸位点突变将在苍蝇中产生，并用于研究其在分子，细胞和器官水平的影响。为此，将产生果蝇神经元α 1-微管蛋白基因的遗传零背景。使用CRISPR-Cas9同源定向修复，PhiC 31/attP着陆平台将取代内源基因座。这随后将允许通过重组酶介导的盒交换容易地重新引入具有不同工程化氨基酸取代突变的α 1-微管蛋白。在细胞水平，免疫组织化学和生物化学染色、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的α 1-微管蛋白版本的原代神经元培养物中的微管功能。为此，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MT的相互作用。将被分析。在器官水平，轴突导向，成束，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-Gal 4和克隆MARCM系统表达突变的α 1-微管蛋白版本，结合先进的免疫组织化学成像技术和行为分析来实现。拟议的研究策略将揭示在α 1-微管蛋白突变为基础的神经元疾病中蛋白质与微管相互作用的变化。这种研究策略可以很容易地适应在未来的研究中发现的微管蛋白的额外的翻译后修饰的重要性，分析在非神经元组织中的lissencephaly-linked突变的分子方面，并确定新的分子靶点微管蛋白突变相关的疾病和神经退行性疾病。

项目成果

Tau and spectraplakins promote synapse formation and maintenance through Jun kinase and neuronal trafficking

• DOI: 10.7554/elife.14694
• 发表时间: 2016-08
• 期刊: eLife
• 影响因子: 7.7
• 作者: André Voelzmann;Pilar Okenve-Ramos;Yue Qu;Monika Chojnowska-Monga;Manuela del Caño-Espinel;A. Prokop;N. Sánchez-Soriano