Abstract Mitochondrial dysfunction is a common contributing factor in neurodegenerative diseases including Alzheimer’s (AD), Parkinson’s (PD), Huntington’s (HD), and amyotrophic lateral sclerosis (ALS). Mechanistic studies in model systems are deficient because adequate tools are lacking. The proposal hypothesis is that strategies to regulate selectively the removal or accumulation of dysfunctional mitochondria can delay or prevent the onset of mitochondrial dysfunction that is an underpinning of neurodegenerative diseases, particularly in Alzheimer’s disease. The team has characterized a collection of small molecule probes that modulate mitochondrial protein translocation and strong preliminary evidence shows that manipulating these protein import pathways regulates a selective pathway for autophagy of mitochondria, referred to as mitophagy. The overalls goals of this supplement are (1) to characterize at a mechanistic level how this set of small molecule probes regulate and induce various mitophagy pathways in cultured cell models including a neuronal cell model for AD, and (2) to develop new zebrafish models for AD to determine how mitochondrial dysfunction contributes to AD. The probes will subsequently be applied to the zebrafish models to determine how altering mitophagy impacts neuronal function. This research will advance the field because (1) such mitochondrial- specific probes have not been previously available and (2) the use of probes allows for selective control in the induction and regulation of mitophagy pathways, which is difficult with current genetic models. To accomplish the proposal goals, two specific proposal aims will be undertaken: In Aim 1, cell models including neural cells will be treated with the small molecules and mitophagy induction and progression will be studied. In Aim 2, these experiments will be extended to zebrafish models that exhibit mitochondrial dysfunction or aggregate accumulation in the neural system and the small molecule modulators will be tested. An outcome of this application is that studies will be advanced to dissect how mitophagy pathways are regulated by modulation of mitochondrial protein import pathways. This supplement will have a broad impact on AD and other neurodegenerative diseases, because a new set of probes will be verified that can be applied to understand how mitochondrial dysfunction contributes to AD progression and/or attenuation.