Nickolay Brustovetsky - US grants

DESCRIPTION (provided by applicant): There is a fundamental gap in our understanding of the mechanisms of the release of mitochondrial apoptogenic factors induced by elevated Ca2+ and by pro-apoptotic proteins. Our long-term goal is to establish the role of mitochondria in neuronal apoptosis. The objective of this study is to delineate the mechanisms of release of apoptogenic proteins from brain mitochondria initiated by elevated Ca2+ or by pro-apoptotic proteins. The central hypothesis of the proposed research is that an increased generation of reactive oxygen species, augmentation of lipid peroxidation, activation of phospholipase A2, and K+ influx in brain mitochondria are the major processes leading to the release of apoptogenic proteins induced by elevated Ca2+ or pro-apoptotic proteins tBID and BAX. In Specific Aim 1 we will establish K+-dependent mechanisms of the Ca2+-induced swelling of brain mitochondria and release of apoptogenic proteins. Inhibitors of mitochondrial K+ channels and the adenine nucleotide translocase will be applied to isolated brain mitochondria or to cultured neurons to establish their role in the Ca2+-induced swelling, and release of apoptogenic proteins. In Specific Aim 2 we will determine the extent to which an activation of mitochondrial K+ channels and the permeability transition contributes to the release of apoptogenic factors induced by pro-apoptotic proteins tBID and BAX. Inhibitors of the permeability transition and blockers of K+ channels will be used to identify their role in the release of the apoptogenic proteins. In Specific Aim 3 we will establish the role of reactive oxygen species, lipid peroxidation and phospholipase A2 in the release of apoptogenic proteins induced by tBID and BAX. Various antioxidants and inhibitors of phospholipase A2 will be used to inhibit the release of apoptogenic proteins. In Specific Aim 4 we will determine the role of caspases in the release of apoptogenic proteins from brain mitochondria exposed to tBID and BAX. Isolated brain mitochondria exposed to tBID and BAX and treated with recombinant caspases will be used to test this hypothesis. The proposed research lays the foundation for a better understanding of the molecular mechanisms of the permeabilization of the outer mitochondrial membrane induced by elevated Ca 2+ or pro-apoptotic proteins tBID and BAX and contributes to filling in a gap in our knowledge of these phenomena.

Mitochondrial dynamics, manifest as ability of mitochondria to change morphology and motility, play a vital role in neuronal response to fluctuating energy demands. Impairment of mitochondrial dynamics contributes to different disorders such as Alzheimer?s, Parkinson?s, and Huntington?s diseases (HD). In HD, interaction of mutant huntingtin (mHtt) with dynamin related protein 1 (Drp1) results in an increased Drp1 activity, leading to augmented mitochondrial fission, accompanied by reduced mitochondrial traffic. Despite significant effort, the molecular mechanisms, leading to mHtt-induced changes in mitochondrial morphology and motility are not completely understood. In preliminary experiments, we found that CRMP2, a protein implicated in axon guidance and regulation of neurite outgrowth, regulates mitochondrial dynamics. A mechanistic link between CRMP2 and regulation of mitochondrial dynamics has never been investigated. CRMP2 binds to neuronal mitochondria and in its dephosphorylated form to mHtt. CRMP2 physically interacts with Drp1, Mitofusin 2, and Miro 2, proteins involved in regulation of mitochondrial fission, fusion, and motility, respectively. Downregulation of CRMP2 with siRNA leads to increased fission and reduced mitochondrial traffic, implicating CRMP2 in regulation of mitochondrial dynamics. CRMP2 hyperphosphorylation after inhibition of protein phosphatases 1 and 2A correlates with augmented fission and reduced mitochondrial traffic. Conversely, decreasing CRMP2 phosphorylation can prevent these alterations. Finally, we found CRMP2 downregulation and hyperphosphorylation in striatal tissues from YAC128 HD mouse model and in postmortem striatal tissues of HD patients. Overall, the literature and our preliminary data strongly suggest that CRMP2 is involved in regulation of mitochondrial morphology and motility and CRMP2 hyperphosphorylation contributes to HD pathogenesis leading to excessive fission, reduced mitochondrial traffic, and neuronal loss. Dephosphorylated CRMP2 binds to mHtt and to proteins involved in mitochondrial dynamics and reduces their activities, whereas CRMP2 downregulation and hyperphosphorylation disrupts these protein-protein interactions, liberates binding partners of CRMP2, and increases their activities. In Aim 1, we will determine CRMP2 localization in mitochondria, establish protein interaction partners, and assess the extent to which CRMP2 regulates mitochondrial dynamics in neurons. In Aim 2, the mechanisms of CRMP2-medited regulation of mitochondrial dynamics will be determined. In Aim 3, we will establish CRMP2-mediated mechanisms contributing to defects of mitochondrial dynamics and cell death in human neurons expressing mHtt. Finally, in Aim 4, we will assess to what extent CRMP2 dephosphorylation alters protein-protein interactions, protects neurons, and corrects behavioral deficits in animal models of HD. The proposed study will considerably improve our understanding of HD pathophysiology, lay a solid foundation for identifying new mechanisms of HD pathogenesis, and open novel avenues in HD research.