Neurons are highly polarized cells with high energy demands. The mitochondria meet those demands by generating 90% of energy in neurons, thus, it is unsurprising that mitochondrial dysfunction has been linked to age-related neurodegenerative disorders. However, the mitochondrial molecular cascade leading to age-dependent energy failure remains to be elucidated. Prominent aging theories hypothesize that accumulation of mitochondrial DNA (mtDNA) mutations plays a role in age-dependent decline in neuronal metabolism. Heteroplasmy of pathogenic mtDNA variants has been shown to cause alterations in ETC protein function, TCA metabolite levels, and mitochondrial dynamics. Still, the mechanism through which mtDNA mutations instigate changes in neuronal metabolism remains to be explored. Due to the importance of mtDNA in producing key mitochondrial proteins required for neuronal energy homeostasis, we hypothesize that accumulated mtDNA mutations alter neuronal metabolism locally, which then leads to age-dependent neuronal dysfunction. By systematically inducing mtDNA mutations through a Cre-loxP system in Tfamfl/fl mouse neurons we propose to accomplish three aims that will (1) characterize mtDNA distribution and motility throughout wild-type and TFAM-mutant neurons, (2) investigate the neuronal metabolic decline pathways in TFAM-mutant neurons, and (3) elucidate the impact of accumulating mtDNA mutations in neuronal metabolism and identify compensatory metabolic pathways. This work will reveal subcellular mechanisms that contribute to the mitochondrial molecular cascade that leads to neurodegeneration and provide insights into age-dependent energy failure.