Role of alpha-Tubulin Mutations in Lissencephaly

无脑是一种灾难性的发育性疾病，其特征是大脑表面光滑，四个皮质层异常，小脑发育不良。这种情况与顽固性癫痫、低眼压和婴儿时期严重的精神发育迟缓有关。至少有三个基因的突变--Lis1、DCX和RELN--会导致与这种疾病相关的神经元迁移的非常相似的缺陷。总而言之，这些病例约占无脑畸形病例的70%，但其余病例的潜在原因尚不清楚。我们最近发现，在Tuba1α-微管蛋白基因的GTP结合口袋附近的一个突变会导致小鼠异常的神经元迁移事件，这让人想起无脑。事实上，对一组没有携带Lis1、DCX或RELN突变的患者进行的筛查显示，TUBA1A(人类TUBA1的同源物)的突变会导致神经元迁移缺陷，包括无脑和厚脑回。这些数据强化了这样一种观点，即基于微管的事件在发育过程中的神经元迁移中发挥着核心作用，皮质迁移障碍是关键微管过程中断的结果。我们发现，10种不同的致病α-微管蛋白突变分为两类：可能导致单倍性功能不全的突变(I类)和不会导致单倍性功能不全的突变(II类)。1)在I类突变的情况下，我们建议在异二聚体组装途径中建立导致微管蛋白异二聚体产量减少的缺陷(S)。为此，我们将分析使用单个纯化成分(伴侣)进行的体外折叠反应的产物，这些成分是组装异源二聚体所需的。2)在第二类突变的情况下，我们将检验这样一种假设，即微管行为的关键元件可能通过一种机制发生破坏，在这种机制中，α-微管蛋白与微管功能的关键效应器之间的适当相互作用受到损害。A)我们将产生一批均匀的微管蛋白异源二聚体，因为它将包含α-和β-亚基，仅由一种野生型或突变型的同功型组成。B)我们将比较同型纯野生型和突变型TUBA1A的动力学性质。

Lissencephaly is a catastrophic developmental disease characterized by a smooth surface of the brain, four abnormal cortical layers, and a variably hypoplastic cerebellum. The condition is associated with intractable epilepsy, hypotonia, and profound mental retardation from infancy. Mutations in at least three genes – LIS1, DCX and RELN – lead to very similar defects in neuronal migration that are associated with the disease. Together, these account for about 70% of cases of lissencephaly, but the underlying cause of the remaining cases is unknown. We have recently discovered that a mutation in close proximity to the GTP binding pocket of the tuba1 α-tubulin gene causes abnormal neuronal migration events in mice that are reminiscent of lissencephaly. Indeed, screening of a cohort of patients who do not carry mutations in either LIS1, DCX or RELN has revealed that mutations in TUBA1A, the human homolog of tuba1, cause neuronal migration defects including lissencephaly and pachygyria. These data reinforce the notion that microtubule based events play a central role in neuronal migration during development, and that cortical migration disorders results from disruption of critical microtubule processes. We have found that 10 different disease-causing α-tubulin mutations fall into two categories: those that could lead to haploinsufficiency (Class I), and those that do not (Class II). 1) In the case of Class I mutations, we propose to establish the defect(s) in the heterodimer assembly pathway that lead to a diminished yield of tubulin heterodimers. To do this, we will analyze the products of in vitro folding reactions done using individual purified components (chaperones) required for de novo heterodimer assembly. 2) In the case of Class II mutations, we will test the hypothesis that disruption of key elements of microtubule behavior can occur via a mechanism in which proper interactions between α-tubulin and critical effectors of microtubule function are compromised. a) We will generate a supply of tubulin heterodimers that is homogeneous in that it will contain α- and β-subunits consisting of only a single isotype that is either wild type or mutant. b) We will compare the dynamic properties of isotypically pure wild type and mutant forms of TUBA1A.