Our understanding of the molecular mechanisms governing the development of the brain has been facilitated by genetic approaches in human and mouse that have identified several genes and protein products required for neocortical development and neuronal migration. The first identified neuronal migration gene was LIS1. Heterozygous loss-of-function of LIS1 resulted in the severe neuronal migration defect lissencephaly, or smooth brain. Recent studies by others and us have identified some important interactions that define at least some of the cellular function of LIS1. For example, LIS1 and the NudE homologue NDEL1 (coded by the Ndel1 gene, formerly NudE1) together bind the cytoplasmic dynein heavy chain (CDHC) to regulate dynein motor function. NDEL is phosphorylated by Cdk5/p35, suggesting that the activity of the LIS1/NDEL1/CDHC complex is regulated by phosphorylation. Cdk5-phosphorylated NDEL1 (P- NDEL1) binds to 14-3-3E, a member of a large family of binding proteins that regulate stability and subcellular localization of phosphoproteins, to protect P-NDEL1 from phosphatase attack. In this resubmission, we will focus our studies on the role of NDEL1 in the LIS1/NDEL1/CDHC complex because of its apparent central role in coordinating the function of LIS1 and cytoplasmic dynein These studies will provide further insight into neuronal migration and neocortical development by providing a more comprehensive understanding of the molecular intricacies that govern neocortical development, and will also provide insight into human lissencephaly. Therefore, we propose to investigate the function of NDEL1 in integrating these pathways in vivo by the following Specific Aims:
Aim 1. Test the hypothesis that NDEL1 has several important functions during brain development and in the adult by examining the dosage dependent effects of NDEL1 during neurogenesis, neuronal migration, cell survival and adult neuronal function in vivo. Based on our published and preliminary data after loss of LIS1, we predict that NDEL1 is critical for processes at all stages of brain development, and even in the postmitotic adult brain, although not in non-neuronal somatic tissues.
Aim 2. Test the hypothesis that the phosphorylation of NDEL1 by Cdk5/p35 and binding to 14-3-3e are critical for neuronal development and migration in vivo by producing specific Cdk5 phosphorylation site mutants in mice by BAG transgenesis.
Aim 3. Test the hypothesis that NDEL1 binding to LIS1 and CDHC regulates the function of dynein, and this binding is responsible for the in vivo phenotypes of mice with mutations in the LIS1/NUDEL/CDHC complex by producing Ndell alleles defective in binding for LIS1 and CDHC. ? ? ? ? ?
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