Investigating mitochondrial dynamics in cerebral ischemia/reperfusion injury using novel morphological analyses and computational modeling

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PROJECT SUMMARY Cerebral ischemia/reperfusion (I/R) injury, mainly caused by cardiac arrest and stroke, induces debilitative neurological damage. A key component of cerebral I/R injury is dysfunction in mitochondrial metabolism and maintenance. The mitochondrial network is maintained by two balancing forces: fission and fusion. Under basal conditions, these dynamic processes work to stabilize the mitochondrial network and maintain efficient energy production. However, the processes of mitochondrial dynamics and quality control are severely disrupted by cerebral I/R injury. The objective of the present research proposal is to identify the phases of disrupted mitochondrial dynamics and the related molecular mechanisms in cerebral I/R injury with strict temporal resolution. Rather than using the traditional subjective and qualitative methodologies, mitochondrial dynamics will be probed using novel computational and quantitative tools. Utilizing advances in computational science and artificial intelligence, a semi-automated machine learning-based classification pipeline will be constructed for the analysis of mitochondrial morphology, the product of mitochondrial dynamics. The classification model will be developed and validated using conditional knockout models of known molecular players in mitochondrial dynamics (e.g. Opa1, Drp1). Additionally, an agent-based computational model of single cell mitochondrial dynamics will be constructed and optimized using live cell imaging from primary neurons and novel genetic reporters (e.g. MitoTimer). This computational model will be used to test hypotheses regarding the basal and pathological rates of mitochondrial dynamics and quality control operations, as well as, inform future experiments as a method of reducing the required number of experimental groups, timepoints, and animals. Utilizing the newly developed and validated computational tools, live cell imaging will be performed in an in vitro model of cerebral I/R, oxygen glucose deprivation and reoxygenation (OGD/R). Tracking mitochondrial morphology over time during OGD/R will allow for the identification of distinct phases of mitochondrial dynamics in cerebral I/R injury. Additionally, the specific mechanisms involved with the identified phases will be probed using conditional knockout of key dynamics proteins. The above-mentioned procedures will further be scaled to 3D analysis of mitochondrial morphology in the middle cerebral artery occlusion (MCAO) model of focal ischemic stroke. This move to in vivo experimentation will provide knowledge of dynamics in a larger biological system and allow for future translational work regarding cerebral I/R injury. This research, along with advanced academic study and strong scientific mentorship, provide a tremendous amount of opportunities for growth and professional development. The proposed work is uniquely positioned at the intersection of computer science, mathematics, and neurobiology, and thus creates a remarkable and distinctive environment for the development of young scientist.