Serious mitochondrial injury has been proposed as underlying mechanism of many acute and chronic neurodegenerative diseases. However, how exactly mitochondria participate in the pathogenesis of neurodegeneration is unclear. Mitochondria are dynamic organelles, undergoing frequent fission (separation) and fusion. New findings in tumor cell lines, indicate that mitochondria rapidly fragment in response to apoptotic stimuli, a process that may facilitate the release of apoptogenic factors from mitochondria, an increase in reactive oxygen species (ROS), and changes in energy supply. Mitochondrial fission is regulated by evolutionary, highly conserved machinery, involving Dynamin-related GTPases. Among them is Drp-1, which mediates mitochondrial fission. The purpose of this proposal is to explore the new concept that mitochondrial fission is a key component of excitotoxic neuronal cell death. Among the specific questions that will be addressed are: (1) Do mitochondria undergo fission in neuronal cell death evoked by NMDA or NO exposure? (2) Does Drp-1 regulate mitochondrial fission in neuronal cell death? (3) How do pro- and anti-apoptotic Bcl-2 family members regulate mitochondrial fission? To address these questions primary cerebrocortical neurons will be analyzed with multi-disciplinary approaches, including cell biology, time-lapse fluorescence deconvolution microscopy, electron tomography, molecular genetics (dominant-negative mutants and siRNA), and biochemistry. This study will improve our general understanding of the cellular and molecular mechanism of neuronal cell death. In particular, it will address how mitochondria contribute to this process. In addition, it will provide new insights into how the mitochondrial fission machinery recruits the cell death program. Most importantly, drugs targeting the mitochondrial fission/fusion machinery may open new therapeutic opportunities to combat chronic and acute neurodegeneration.