MECHANISM OF ACTION OF THE LISSENCEPHALY GENES LIS-1

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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.