Project Summary Mitochondria are dynamic organelles which support the metabolic needs of neurons across their entire lifetime. Previous research has sought to define the regulation of mitochondrial transport as dependent on the activity of the neuron, including changes in energy demand. However, previous studies and unpublished data from the Barnhart lab show mitochondrial motility is not dependent on neuronal activity. Given defects in mitochondrial transport have been implicated with neurodegeneration, the goal of this research is to elucidate the fundamental role of mitochondrial transport in maintaining healthy and functional neurons. I propose to define both in vivo mitochondrial localization across the neuron and contributions of mitochondrial motility in the function of the mitochondria in neurons. To define mitochondrial transport and function in vivo, I will use the Drosophila melanogaster visual system with live cell imaging using genetically encoded reporters of mitochondrial health and neuronal function. In Aim 1, I will use confocal microscopy, immunostaining and EM reconstructions to define the relationship between mitochondrial motility, mitochondrial distribution patterns and neuronal architecture. This aim will evaluate the relationship between neuronal structure and overall mitochondrial localization. In Aim 2, I will use mitochondrial perturbations to assess how mitochondrial motility affects mitochondrial health and neuronal function in vivo. These studies will define the regulation and functional relevance of mitochondrial motility in neurons, which paves the way towards future studies on the role of mitochondrial transport defects in disease, including in neurodegeneration.