Parkinson’s disease (PD) is a neurodegenerative disorder characterized by dopaminergic neuron loss in the substantia nigra. Loss-of-function mutations in PARK2 are the leading cause of autosomal recessive juvenile parkinsonism (AR-JP). An AR-JP Drosophila melanogaster model exhibits pathologies associated with loss-of-function mutations of the parkin (park, the Drosophila PARK2 ortholog) gene including swollen mitochondria and mitochondrial dysfunction. Preliminary results from our lab show that mitochondrial networks have smaller volumes and more fragmentation in dopaminergic neurons in heterozygous Drosophila park25 (loss-of-function allele) flies, suggesting that park25 causes pre-mitophagic fragmentation. Disruptions in oxidative phosphorylation increase reactive oxygen species (ROS) production, which can lead to oxidative stress, mitochondrial fragmentation and aberrant mitochondrial morphology. Disruptions in mitochondrial respiration have been observed in Parkin loss-of-function models as well as in patients. Although Parkin-mediated mitophagy has been described in a number of cell lines and in different physiological conditions, contradicting ideas about the relationship between Parkin and mitophagy persist. An in vivo assessment of mitophagy would help determine how Parkin affects mitochondrial function and thus provide further insight in the role of mitochondrial pathology in the etiology of PD. To assess ROS production, we are utilizing a MitoTimer construct that consists of a mitochondrial-targeted DsRed variant that shifts from green to red fluorescence upon oxidation. Using confocal microscopy we are measuring this fluorescent ratio in TH-Gal4, UAS-MitoTimer PPL1 dopaminergic neurons in control (park+/+), heterozygous (park+/-) and homozygous (park-/-) flies. To measure mitophagy in the PPL1 neurons, we are analyzing co-localization of mCherry-Atg8a and mitoGFP constructs driven by TH-Gal4 in these same park genotypes. We hypothesize that the decrease in mitochondrial network volume we have detected in Parkin loss-of-function flies is due to higher rates of oxidation and subsequent increases in mitophagy. Our results, which will be presented at the meeting, will provide insight on the dose-dependent effect of park mutation toxicity within the mitochondria of dopaminergic neurons and shed light on morphology and mitophagy. This will promote a better understanding of the mechanisms of degeneration caused by Parkin loss-of-function.