Mitofusin 2 Sustains the Axonal Mitochondrial Network to Support Presynaptic Ca2+Homeostasis and the Synaptic Vesicle Cycle in Rat Hippocampal Axons

Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metab-olite biosynthesis. The trafficking and function of these organelles are particularly important in neurons, with impaired mito-chondrial function or altered morphology observed in every neurodegenerative disorder studied. While mitochondrial biosynthetic products play a crucial role in maintaining cellular function, their resulting byproducts can have negative conse-quences. Thus, organelle quality control (QC) mechanisms that maintain mitochondrial function are imperative to restrict de-structive signaling cascades in the cell. Axons are particularly sensitive to damage, and there is little consensus regarding the mechanisms that mediate mitochondrial QC in this compartment. Here, we first investigated the unstressed behavior of mito-chondria in rat hippocampal neurons of mixed sex, focusing on mitochondrial trafficking and fusion to better understand potential QC mechanisms. We observed size and redox asymmetry of mitochondrial traffic in axons, suggesting an active QC mechanism in this compartment. We also document biochemical complementation upon the fusion and fission of axonal mi-tochondria. Eliminating fusion by knocking down the neuronal mitochondrial fusion protein mitofusin 2 (MFN2) reduced the rates of axonal mitochondrial trafficking and fusion, decreased the levels of synaptic vesicle (SV) proteins, inhibited exocyto-sis, and impaired SV recruitment from the reserve pool during extended stimulation. MFN2 knockdown also resulted in pre -synaptic Ca2+ dyshomeostasis. Remarkably, upon MFN2 knockdown, presynaptic mitochondria sequestered Ca2+ more efficiently, effectively limiting presynaptic Ca2+ transients during stimulation. These results support an active mitochondrial trafficking and fusion-related QC process that supports presynaptic Ca2+ handling and the SV cycle.