PROJECT SUMMARY Mitochondrial dysfunction is a prominent element of many leading causes of disability, namely cerebrovascular and neurodegenerative diseases. The function and stability of mitochondria are tightly regulated by the mechanisms of mitochondrial dynamics and quality control (QC). The dynamic nature of mitochondria is maintained by the balancing forces of fission and fusion. These processes of mitochondrial dynamics operate to preserve the functional architecture of the mitochondrial network. The mechanisms of mitochondrial QC, including mitophagy, proteostasis, and biogenesis, work to regulate the components of the mitochondrial network through synthesis and degradation. These forces actively control the functionality of the mitochondrial network to ensure efficient energy production. In cerebral ischemia/reperfusion (I/R) injury, the processes of mitochondrial dynamics and QC become dysregulated, contributing to metabolic dysfunction and neurological damage. The F99 phase of this proposal aims to identify the phases of disrupted mitochondrial dynamics and QC in cerebral I/R injury, and their respective molecular mechanisms. Utilizing advanced technologies related to machine learning, computational modeling, and live cell imaging, I have created an agent-based model of mitochondrial dynamics for these investigations. This model allows for the simulation of the dynamic actions of individual mitochondrial units to culminate in the complex patterns normally observed in mammalian cells. Live cell imaging of mouse primary cortical neurons from novel transgenic reporter lines (i.e., MitoTimer, MitoQC) and conditional knockout lines will be utilized to observe the respective contributions of individual dynamics proteins to the patterns of mitochondrial morphology. Knockout neurons will be exposed to oxygen glucose deprivation (OGD), an in vitro model of I/R injury, and mitochondrial parameters (i.e., morphology, oxidation) will be imaged in real time to generate a mechanistic timeline of mitochondrial dynamics. These live cell recordings will be used to optimize and expand our agent-based model to allow for in silico experimental manipulation of mitochondrial proteins. Our expanded model will have the ability to test hypotheses regarding the basal and pathological rates of mitochondrial dynamics and quality control, as well as inform future experiments with decreased costs and increased efficiency. In the K00 phase of this proposal, I will transition from studying mitochondrial quality control in I/R to its study in neurodegeneration. Utilizing the technical skills acquired in the predoctoral phase, I will investigate age-related changes in mitochondrial proteostasis and critical long-lived mitochondrial proteins at the synapse. The K00 phase aims to determine how aging affects the turnover of synaptic mitochondrial proteins, with specific emphasis on the roles of intramitochondrial proteostasis and the integrated stress response. I intend t...