MECHANISM OF ACTION OF THE LISSENCEPHALY GENES LIS-1

描述（改编自申请人摘要）：无脑病变是一种 大脑发育障碍：一类人类大脑发育疾病，被认为是由于 神经元胞体迁移。LIS-1基因突变导致 Miller-Dieker无脑畸形和孤立性无脑畸形序列。LIS-1编码 一种在其整个长度上与真菌蛋白质同源的多肽 与细胞质动力蛋白功能有关，但也与PAF共纯化 乙酰水解酶，一种负责灭活脂质介质PAF的酶 （血小板活化因子）。本研究的总体目标是确定 LIS-1在细胞质动力蛋白功能中的作用及其机制 LIS-1控制神经元迁移。在初步研究中， 哺乳动物细胞，LIS-1过表达，LIS-1反义核酸 寡核苷酸和抗LIS-1抗体的显微注射，观察到 产生明显的表型效应。其中值得注意的是一个戏剧性的 有丝分裂指数增加，有丝分裂纺锤体染色体附着缺陷， 有丝分裂微管的结构和组织的改变，以及 动力蛋白和动力蛋白相关复合物分布的变化 在细胞皮层和微管末端的动力蛋白。 具体目标1旨在定义LIS-1的生理功能 （i）通过真实的时间监测这些效应;（ii）通过测定 LIS-1的亚细胞分布;和（iii）通过比较LIS-1和 细胞质动力蛋白表型，包括对核迁移的影响， 初级神经元、脑切片和非神经元细胞。具体目标2旨在 明确PAF在细胞质动力蛋白功能中的作用（i）通过检查 PAF对细胞器分布和微管动力学的影响;（ii）通过 确定LIS-1表达的变化如何改变这些效应;以及 (iii)通过确定PAF如何影响 动力蛋白具体目标3寻求（i）进一步界定 在初步研究中鉴定出具有细胞质动力蛋白的LIS-1，以及（ii） 确定这种相互作用与涉及PAF的相互作用的关系 乙酰水解酶和其它蛋白质。这些研究应该提供重要的新信息， 阐明严重脑发育疾病的机制，并提供 对微管和马达蛋白在 神经元迁移是大脑发育的基本特征。

DESCRIPTION (Adapted from applicant's abstract): The lissencephalopathies are a class of human brain development diseases thought to result from defects in neuronal cell body migration. Mutations in the LIS-1 gene are responsible for Miller-Dieker Lissencephaly and Isolated Lissencephaly Sequence. LIS-1 encodes a polypeptide which is homologous throughout its length to fungal proteins implicated in cytoplasmic dynein function but which also copurifies with PAF acetylhydrolase, an enzyme responsible for inactivating the lipid mediator PAF (platelet activating factor). The overall goal of this study is to determine the role of LIS-1 in cytoplasmic dynein function and the mechanism through which LIS-1 controls neuronal migration. In preliminary studies in cultured mammalian cells, overexpression of LIS-1, exposure to LIS-1 antisense oligonucleotides, and microinjection of anti-LIS-1 antibody were observed to produce pronounced phenotypic effects. Noteworthy among these were a dramatic increase in mitotic index, defects in chromosome attachment to mitotic spindle, alterations in the structure and organization of mitotic microtubules, and changes in the distribution of dynein and the dynein-associated complex dynactin at the cell cortex and at microtubule ends. Specific Aim 1 seeks to define the physiological function of the LIS-1 polypeptide (i) by monitoring these effects in real time; (ii) by determining the subcellular distribution of LIS-1; and (iii) by comparing LIS-1 and cytoplasmic dynein phenotypes, including effects on nuclear migration, in primary neurons, brain slices and nonneuronal cells. Specific Aim 2 seeks to define the role of PAF in cytoplasmic dynein function (i) by examining the effects of PAF on organelle distribution and microtubule dynamics; (ii) by determining how these effects are altered by changes in LIS-1 expression; and (iii) by determining how PAF affects the post-translational modification of dynein. Specific Aim 3 seeks (i) to define further the physical interaction of LIS-1 with cytoplasmic dynein identified in preliminary studies, and (ii) to determine the relationship of this interaction with those involving PAF acetylhydrolase and other proteins. These studies should shed important new light on the mechanism of a serious brain developmental disease, and provide critical new insight into the role of microtubules and motor proteins in neuronal migration, a fundamental feature of brain development.