Investigating the Interplay of Mitochondrial Dynamics and Proteostasis during Neuronal Ischemia/Reperfusion Injury using Integrated Imaging and Modeling Approaches

Abstract

Cerebral ischemia/reperfusion (I/R) injuries, including stroke and cardiac arrest can lead to irreversible neuronal damage and neurological complications. Mitochondrial damage and dysfunction are critical components of both ischemia and reperfusion-induced injury. Protecting and restoring mitochondrial function during reperfusion is a promising therapeutic strategy for limiting potentiation of neuronal damage. Mitochondria are highly dynamic organelles that exist in size and shape ranging from small punctate to large sprawling networks. These morphologies are dictated by the mechanisms of mitochondrial dynamics and quality control. Mitochondrial dynamics, consisting of fission and fusion, generate the complex architecture of mitochondrial networks. Recycling of old and damaged mitochondrial components is mediated by the quality control pathways of intramitochondrial proteostasis and mitophagy, the autophagic degradation of mitochondria. These pathways collectively work to remodel the mitochondrial proteome. The processes of mitochondrial dynamics and quality control have been demonstrated to be active in neurons during I/R injury. The aim of my thesis was to characterize the patterns and interactions of mitochondrial dynamics and proteostasis during the temporal window of acute reperfusion. In a primary neuron model of in vitro I/R injury, I imaged mitochondrial fission and fusion patterns, along with changes in mitochondrial proteostasis. Mitochondrial dynamics were analyzed by a novel machine learning-based morphological classification pipeline with single mitochondrion resolution. Proteostasis was evaluated using the fluorescent reporter MitoTimer, which allows for visualization of new and aged proteins. Combining these imaging techniques, I detailed the temporal patterns and partial mechanisms of mitochondrial dynamics and protein turnover. I found that mitochondrial fission is highly active during the ischemic-like phase of injury, whereas fusion is active during early reoxygenation. However, secondary fragmentation events and fusion inhibition were found during the latter stages of reoxygenation. At the same time as changes in dynamics occurred during reoxygenation, protein turnover via LonP1 proteolysis and Parkin-dependent mitophagy were increased to clear older/oxidized proteins. Additionally, I found that fission (Drp1) and fusion (Opa1) machinery alter mitochondrial protein turnover during physiological and pathological states. Utilizing live cell recordings of mitochondrial dynamics, I identified distinct properties of individual mitochondria performing either fission or fusion. Finally, I summarized my data into an agent-based model of mitochondrial dynamics for predictive in silico experimentation. Together, my findings and computational tools greatly enhance our understanding of mitochondrial homeostatic mechanisms in response to I/R injury in neurons.