Tubulin Mutations in Neuronal Migration Disorders

描述（由申请人提供）：微管由微管蛋白异源二聚体组装而成。在哺乳动物中，一个小的多基因家族编码微管蛋白和微管蛋白，每个基因产物（称为同型）具有不同的发育调节和组织特异性表达模式。最近，在编码这些同型的基因（如TUBA1A和TUBB2B）中发现了突变，这些突变导致某些人类神经元迁移障碍，所有这些都与严重的认知障碍有关。这些发现强化了这样一种观点，即基于微管的事件在大脑发育过程中的神经元迁移中起着核心作用，关键微管过程的破坏导致皮质发育异常。对TUBA1A致病突变特性的分析表明两大类机制：1。该途径涉及新合成的微管蛋白多肽与多种伴侣蛋白（包括胞质伴侣蛋白CCT和五种微管蛋白特异性伴侣蛋白TBCA-TBCE）的顺序相互作用。CCT通过提供一个隔离的环境来促进高效折叠，在这种环境中，折叠可以在没有可能导致聚集的通路外相互作用的情况下发生，而微管蛋白特异性伴侣蛋白在CCT下游作为微管蛋白异源二聚体组装机发挥作用。2. 微管动力学缺陷和/或与微管相关蛋白（MAPs）的相互作用疾病引起的突变可能损害微管动力学或干扰微管和MAPs之间的相互作用，而这些相互作用对于指导适当的神经元迁移至关重要。这里提出的实验构成了一个多方面的方法来理解这些疾病的机制。1)我们将利用cct产生的微管蛋白折叠中间体和TBCB之间突变诱导的缺陷相互作用来定义这种相互作用。这些实验将确定TBCB在适当的皮质生成中起关键作用的机制。2)我们将产生等典型同质野生型和突变型微管蛋白异源二聚体群体。这些将用于识别map（如微管聚合酶TOGp），它们与野生型和致病突变异源二聚体聚合的微管结合方式不同。3)对于那些不干扰异源二聚体组装机制的致病微管蛋白突变，我们将在体内单细胞实验（包括培养的神经元）中研究它们对微管动力学的影响。4)通过构建和分析将野生型tuba1a （tuba1a的小鼠同源基因）的一个拷贝替换为具有致病突变的相同等位基因的转基因小鼠，探索疾病的机制。这些小鼠的大脑将在体外和体内进行发育和微管行为方面的研究。

DESCRIPTION (provided by applicant): Microtubules are assembled from ¿/¿ tubulin heterodimers. In mammals, a small multigene family encodes ¿- and ¿-tubulins, with each gene product (termed an isotype) having a distinct developmentally regulated and tissue-specific pattern of expression. Recently, mutations have been identified in genes (e.g. TUBA1A and TUBB2B) encoding these isotypes that cause certain human neuronal migration disorders, all of which are associated with severe cognitive disabilities. These discoveries reinforce the notion that microtubule based events play a central role in neuronal migration during brain development, and that disruption of critical microtubule processes results in cortical dysgeneses. Analysis of the properties of disease causing mutations in TUBA1A suggests two broad classes of mechanism: 1. Defects in the complex tubulin heterodimer assembly pathway This pathway involves sequential interactions of newly synthesized ¿- and ¿-tubulin polypeptides with multiple chaperone proteins (including the cytosolic chaperonin CCT and five tubulin specific chaperones, TBCA-TBCE). CCT facilitates productive folding by providing a sequestered environment in which folding can occur in the absence of off-pathway interactions that might otherwise lead to aggregation, while the tubulin specific chaperones function downstream of CCT as an ¿/¿ tubulin heterodimer assembly machine. 2. Defective microtubule dynamics and/or interactions with Microtubule Associated Proteins (MAPs) Disease causing mutations might compromise microtubule dynamics or interfere with interaction(s) between microtubules and MAPs that are critical for directing proper neuronal migration. The experiments proposed here constitute a multifaceted approach towards understanding the mechanism of these diseases. 1) We will exploit the mutation-induced defective interactions between CCT-generated ¿-tubulin folding intermediates and TBCB to define this interaction. These experiments will establish the mechanism of action of TBCB as a critical player in proper corticogenesis. 2) We will generate populations of isotypically homogeneous wild type and mutant tubulin heterodimers. These will be used to identify MAPs (such as the microtubule polymerase TOGp) that bind differently to microtubules polymerized from wild type and disease-causing mutant heterodimers. 3) For those disease-causing tubulin mutations that do not interfere with the heterodimer assembly machinery, we will examine their effect on microtubule dynamics in single cell experiments (including cultured neurons) in vivo. 4) The mechanism of disease will be explored via the construction and analysis of transgenic mice in which one copy of wild type tuba1a (the mouse homolog of TUBA1A) is replaced with the same allele harboring a disease-causing mutation. The brains of these mice will be examined in terms of their development and the behavior of their microtubules in vitro and in vivo.