Apoptosis plays a key role in eliminating neurons that lack trophic support, thereby controlling the total number of neurons. Many adult human illnesses including Alzheimer's disease (AD), and Parkinson's disease and amyotrophic lateral sclerosis (ALS) involve pathologic change of neurons, which results in their loss through apoptosis. The long-term objective of this research in our laboratory is to understand how transcription-dependent mechanisms regulate neuronal survival and apoptosis during development and neurodegeneration. Taking advantage of the key role of MEF2 in the survival of both developing and mature neurons, we propose in the present application to explore the critical mechanisms by which cyclin-dependent kinase 5 (Cdk5) inhibits MEF2 function. Our specific aims are: 1. to explore the molecular mechanisms by which CDK5-mediated phosphorylation regulate MEF2 stability; 2. to study the effects and mechanisms of Cdk5-mediated phosphorylation on the regulation of MEF2 by its co-regulators; and 3. to study the effects and mechanisms of Cdk5-mediated phosphorylation on the subcellular and subnuclear distribution of MEF2. Neurotoxicity-induced apoptosis of cerebellar granule neurons will be used as a model for our investigation. A combined biochemical, molecular biological, and cell biological approach will be used to determine MEF2 de-stabilization by glutamate, and the role of Cdk5-mediated phosphorylation in this process. The role of caspase in MEF2 instability will be assessed by caspase cleavage assay. The unique regulatory mechanisms that control MEF2 stability will be studied through domain mapping. Cdk5-dependent regulation of the interaction between MEF2 and its co-regulators, both activators and inhibitors, will be investigated by in vitro pull down assays, co-immunoprecipitation, and chromatin immunoprecipitation. Signaling pathways that may antagonize Cdk5 function will be studied. Finally, sub nuclear and subcellular distribution of MEF2 will be investigated by immunocytochemistry analysis and subcellular fractionation. The effect of Cdk5 on MEF2 DNA binding will be studied by DNA binding assay and promoter recruiting assay. These experiments will define specific mechanisms by which neurotoxin and Cdk5-induced phosphorylation regulate MEF2 function and neuronal death, which are essential to our understanding of the process of neuronal apoptosis under pathological conditions.