Acute injury due to cerebrovascular disease is responsible for significant morbidity and mortality. During ischemic brain injury mitochondria undergo fission resulting in the release of proapoptotic factors which contribute to cell death. The mitochondrial fission machinery, specifically dynamin-related protein 1 (Drp1), has been implicated as a mediator of cell death. Drp1 is regulated by a complex series of posttranslational modifications;however, a full understanding of Drp1 regulation remains to be developed. My long term goal is to characterize the regulatory interaction between the calcium-dependent protein phosphates calcineurin (CaN) and Drp1. It is my hypothesis that calcium influx during ischemia activates CaN to dephosphorylate Drp1, which in turn fragments mitochondria to promote apoptotic and necrotic neuronal death.
The specific aims of this proposal are designed to develop a comprehensive model of CaN regulation of Drp1 and investigate the pathophysiological consequences of this regulation in neurons following acute ischemic injury.
Aim 1 will identify the molecular determinants for CaN binding to and dephosphorylation of Drp1. Recombinant CaN will be used for phosphates assays and isothermal titration calorimetry to characterize how modification of the Can docking site on Drp1 influences the Can reaction cycle. Further, the molecular determinants of CaN::Drp1 complex formation will be investigated in intact cells by genetic manipulation of a CaN docking site on Drp1 using co-immunoprecipitation studies and western blot analysis with a phospho-state specific Drp1. The objective of these studies will be to first characterize how Can activity is modulated by the Can docking site on target substrates and second to develop a detailed mechanistic understanding of calcium-induced mitochondrial fragmentation in ischemic injury.
Aim 2 will characterize the path physiologic impact of Drp1 regulation by CaN in hippocampal neurons. To model the regulation of Drp1 by CaN following stroke, cultured hippocampal neurons will be exposed to toxic levels of glutamate or simulated ischemia in the background of controlled formation and activity of the CaN::Drp1 complex. Following neuronal injury, advanced fluorescence microscopy techniques will be used to probe the role of CaN regulation of Drp1 in dictating mitochondrial morphology, intracellular calcium management and cell survival. The objective of these studies is to assess the potential of the CaN::Drp1 complex as a novel molecular target for therapeutic intervention following neuronal injury.
Mitochondrial fission plays a key role mediating cell death following ischemic injury to neurons. As cerebrovascular disease is currently the fourth leading cause of mortality in the United States, understanding the pathophysiology of injury due to cerebrovascular disease is paramount for the development of novel neuroprotective therapeutics. The regulatory processing of the mitochondrial fission machinery, specifically regulation of the mitochondrial fission factor dynamin related protein-1 (Drp1) by the calcium sensitive protein phosphatase calcineurin, is one potential mechanism which could be exploited for therapeutic gain.
|Wilson, Theodore J; Slupe, Andrew M; Strack, Stefan (2013) Cell signaling and mitochondrial dynamics: Implications for neuronal function and neurodegenerative disease. Neurobiol Dis 51:13-26|
|Merrill, Ronald A; Slupe, Andrew M; Strack, Stefan (2013) N-terminal phosphorylation of protein phosphatase 2A/B*2 regulates translocation to mitochondria, dynamin-related protein 1 dephosphorylation, and neuronal survival. FEBS J 280:662-73|
|Slupe, Andrew M; Merrill, Ronald A; Flippo, Kyle H et al. (2013) A calcineurin docking motif (LXVP) in dynamin-related protein 1 contributes to mitochondrial fragmentation and ischemic neuronal injury. J Biol Chem 288:12353-65|