The long-term goal of this project is to ameliorate neurotransmission defects due to mitochondrial dysfunction, as a way to stop disease progression to later degenerative stages, increasing healthspan in populations increasingly subject to age-related neurological diseases. Dynamin-related protein 1 (DRP1) acts to promote mitochondrial fission and has been identified as a therapeutic target for limiting aberrant mitochondrial fragmentation in Alzheimer’s and Huntington’s disease. The goal of this project is to determine how DRP1 interaction with mitochondrial fission adapters impacts presynaptic terminal function. We propose that the need for high levels of mitochondrial respiration to support synaptic transmission makes the presynaptic terminal a high cellular stress environment. Regulated mitochondrial fission is important for cell survival in response to cellular stressors, acting through DRP1, but the adapters utilized at the neuronal presynaptic terminal are unknown. In Specific Aim 1, we will examine how loss of the mitochondrial fission adapter proteins MFF and FIS1 affect mitochondria homeostasis and synaptic transmission. In Specific Aim 2, we will examine distinct parameters of mitochondrial function and ultrastructure when DRP1 is eliminated, and attempt to rescue function by targeting DRP1 re-expression to mitochondrial outer membrane. Phenotypic differences will be corelated with those in Aim 1, to generate a complete picture of the effect of regulated mitochondrial fission on synaptic function. DRP1 may also facilitate scission of plasma membrane at the synapse, but the impact of this additional function on synaptic transmission is unresolved. In Specific Aim 3, we will test the hypothesis that DRP1 facilitates synaptic vesicle retrieval and recycling, and determine whether membrane-associated DRP1 is sufficient to facilitate SV retrieval, and restore synaptic transmission. In collaboration, the PI and two other world-class investigators have developed novel approaches to allow dissection of the isoform-specific role(s) of DRP1, using the mouse calyx of Held as a model system. Using a combination of viral-mediated transgenesis, advanced electrophysiology, and high-resolution light and electron microscopy, the ability of specific DRP1 isoforms to support mitochondrial fission versus synaptic transmission and presynaptic SV retrieval will be systematically tested. In contrast to small conventional synapses, experimental accessibility of giant ‘calyx-like’ excitatory synapses allow recordings from the presynaptic terminal, permitting manipulation of presynaptic [ATP] and tracking membrane exo/endocytosis in real time. This approach is necessary to dissect the energy-supporting roles of synaptic mitochondria from mechanisms underlying synaptic vesicle recycling. Results from this project can be used to inform, predict, and test function and dysfunction at conventional glutamatergic synapses where disease-relevant neurodegeneration first appears. Knowledge generated from this project will identify viable routes of intervention for restoring function to synapses where DRP1 function is altered, which can be leveraged therapeutically to alleviate disease-related synaptic dysfunction and neurodegeneration.

The product of this research will immediately provide new, critical, and fundamental knowledge on the relative importance of mitochondrial fission adapter proteins for synaptic function. Furhter, this project will elucidate the reasons why dysfunction in the mitochondrial fission protein DRP1 is more severe than loss of mitochondrial fission adapters: we posit that DRP1 facilitates synaptic vesicle biogenesis, independent of its function in mitochondrial fission. Pursuit of this knowledge is important, as DRP1 has been identified as a therapeutic target to alleviate core causes of aging and neurodegenerative diseases such as Alzheimer’s Disease. The motivations for this project are to increase our basic science knowledge of synaptic function in the brain, and also enhance healthspan in populations increasingly subject to age-related neurological diseases.