Mechanisms of ATAD3A in Regulating the Mitochondrial Genome

ABSTRACT Mitochondria are dynamic organelles, with the ability to rapidly change their morphology in response to metabolic or environmental stimuli. These dynamics are particularly important for neuronal function, as mitochondria must maintain a robust capacity to respond to the physiological and pathological stresses associated with the high metabolic demands of these cells. Imbalances in mitochondrial morphology that result in excessive mitochondrial fission (fragmentation) are associated with a wide range of neurological and neurodegenerative diseases, and mitochondrial fragmentation is a hallmark of ischemia/reperfusion-associated neuronal death. However, we lack a molecular description of the complex signaling pathways responsible for the rampant fragmentation associated with disease states and neuronal death. Morphological rearrangements that occur in response to events such as DNA damage involve communication from the mitochondrial interior to the exterior for recruitment of the endoplasmic reticulum, we don’t know how these external interactions are communicated. We hypothesize that the mitochondrial AAA+ protein ATAD3A, which has been shown to play a critical role in regulating mitochondrial dynamics, serves as an inter-organelle signaling conduit, recognizing mitochondrial DNA damage to cytosolic proteins as a mediator of mitochondrial DNA stress response. The C-terminal AAA+ domain of ATAD3A is located within the mitochondrial interior, while its N-terminus is located at the mitochondrial surface, enabling ATAD3A to establish bilateral interactions with mitochondrial and cytosolic fission/fusion cofactors. Our hypothesis is further supported by the observation that ATAD3A has been shown to accumulate at mitochondrion-ER junction sites. Notably, variants of the ATAD3A gene have been identified in patients exhibiting optic atrophies, fatal congenital pontocerebellar hypoplasia, encephalopathy with cerebellar atrophy, ataxia, dystonia, and other neurodevelopmental delays and axonal neuropathies, although how these mutations perturb ATAD3A function or associated interactions is unknown. These studies will provide foundational insights into how ATAD3A functions as a cross-membrane communication complex, transferring molecular cues across the outer mitochondrial membrane (OMM) to coordinate mitochondrial fission. We will combine biochemical, cellular, and structural approaches to confirm the role of ATAD3A in regulating mitochondrial morphology and dynamics through two aims: In Aim 1 we will characterize the interactions between ATAD3A and mitochondrial DNA to understand the rules of engagement. Aim 2 will use structure-based experiments to define the specific molecular mechanisms by which ATAD3A interacts with DNA and allosterically signals DNA damage events to the cytosol. These studies will provide key insights into the role that ATAD3A plays in dictating morphological rearrangements, laying the groundwork for a further investigation of how genetic mutations perturb ATAD3A function and the interactions that drive the mitochondrial fragmentation.