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Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metabolite biosynthesis. The trafficking and function of these organelles are particularly important in neurons, with impaired mitochondrial 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 consequences. Thus, organelle quality control (QC) mechanisms that maintain mitochondrial function are imperative to restrict destructive 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 mitochondria 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 mitochondria. 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 exocytosis, and impaired SV recruitment from the reserve pool during extended stimulation. MFN2 knockdown also resulted in presynaptic 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.SIGNIFICANCE STATEMENTDecreased or altered mitochondrial function is observed in many disease states. All neurodegenerative diseases co-present with some sort of mitochondrial abnormality. Therefore, identifying quality control mechanisms that sustain the mitochondrial network in neurons, and particularly in axons, is of significant interest. The response of axonal mitochondria to acutely applied toxins or injury has been studied in detail. Although informative, the response of neurons to these insults might not be physiologically relevant, so it is crucial to also study the basal behavior of axonal mitochondria. Here, we use fluorescent biosensors to investigate the mitochondrial network in neurons and examine the role of mitofusin 2 in maintaining the axonal mitochondrial network and in supporting the synaptic vesicle cycle.

Neurotransmitter-filled synaptic vesicles (SVs) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading model posits that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH (retention using selective hooks) system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct transmembrane SV proteins, synaptotagmin 1 and synaptobrevin 2, from the soma of mature cultured rat and mouse neurons. For these studies, the SV reporter constructs were expressed at carefully controlled, very low levels. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons with minimal entry into dendrites. However, even moderate overexpression resulted in the spillover of SV proteins into dendrites, potentially explaining the origin of previous non-polarized transport models, revealing the limited, saturable nature of the direct axonal trafficking pathway. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway, for SV proteins, in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.

Mitochondria exert powerful control over cellular physiology, contributing to ion homeostasis, energy production, and metabolite biosynthesis. Mitochondrial trafficking and function are vital to neurons, with organelle impairment or altered morphology observed in every neurodegenerative disorder studied. While this organelle and its biosynthetic products are critical for cellular function, decreased output and/or byproducts (e.g., free radicals), can be harmful, and organelle quality control (QC) mechanisms are required to maintain function and prevent a cascade of damage. Owing to its length and general lack of biosynthetic machinery, the axon is particularly sensitive to damage and there is little consensus regarding the details of mitochondrial QC mechanisms in this compartment. Here we investigate the basal, unstressed behavior of axonal mitochondria, focusing on mitochondrial trafficking and fusion to better understand potential QC mechanisms. We observed size and redox asymmetry of mitochondrial traffic in the axon, suggestive of an active QC mechanism. Importantly, we demonstrate, in detail, biochemical complementation of axonal mitochondria. Upon disruption of mitochondrial fusion, we observed an altered synaptic proteome, presynaptic calcium dyshomeostasis, decreased levels of exocytosis, and a reduction in synaptic vesicle recruitment from the reserve pool during extended stimulation. These results support an active mitochondrial trafficking and fusion related QC process that supports presynaptic physiology.

The development of enzyme-based self-labeling tags allow the labeling of proteins in living cells with synthetic small-molecules. Use of a fluorophore-containing ligand enables the visualization of protein location inside cells using fluorescence microscopy. Alternatively, deployment of a biotin-containing ligand allows purification of tagged protein using affinity resins. Despite these various applications of self-labeling tags, most ligands serve a single purpose. Here, we describe self-labeling tag ligands that allow both visualization and subsequent capture of a protein. A key design principle is exploiting the chemical properties and size of a rhodamine fluorophore to optimize cell-permeability of the ligand and the capture efficiency of the biotin conjugate. This work generates useful “multifunctional” fluorophores with generalizable design principles that will allow the construction of new tools for biology.

Neurotransmitter-filled synaptic vesicles (SV) mediate synaptic transmission and are a hallmark specialization in neuronal axons. Yet, how SV proteins are sorted to presynaptic nerve terminals remains the subject of debate. The leading models posit that these proteins are randomly trafficked throughout neurons and are selectively retained in presynaptic boutons. Here, we used the RUSH system, in conjunction with HaloTag labeling approaches, to study the egress of two distinct SV proteins from the soma of cultured neurons. In sharp contrast to the selective retention model, both proteins selectively and specifically entered axons and did not traffic through dendrites; only upon overexpression do SV proteins spillover into other compartments. Moreover, we observed that SV constituents were first delivered to the presynaptic plasma membrane before incorporation into SVs. These experiments reveal a new-found membrane trafficking pathway in classically polarized mammalian neurons and provide a glimpse at the first steps of SV biogenesis.