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 STATEMENT Decreased 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.

线粒体对细胞生理学发挥强大的控制作用，有助于离子稳态、能量产生和代谢物生物合成。这些细胞器的运输和功能在神经元中尤其重要，在研究的每种神经退行性疾病中都观察到线粒体功能受损或形态改变。虽然线粒体生物合成产物在维持细胞功能方面发挥着至关重要的作用，但其产生的副产物可能会产生负面后果。因此，维持线粒体功能的细胞器质量控制（QC）机制对于限制细胞中破坏性的信号级联至关重要。轴突对损伤特别敏感，并且对于介导该区室中线粒体QC的机制几乎没有达成共识。这里，我们首先研究了混合性别大鼠海马神经元中线粒体的无应激行为，重点关注线粒体运输和融合，以更好地了解潜在的QC机制。我们观察到轴突中线粒体运输的大小和氧化还原不对称性，表明该区室中存在活跃的QC机制。我们还记录了轴突线粒体融合和裂变的生化互补。通过敲低神经元线粒体融合蛋白 mitofusin 2 (MFN2) 来消除融合，降低了轴突线粒体运输和融合的速率，降低了突触小泡 (SV) 蛋白的水平，抑制了胞吐作用，并在长时间刺激期间损害了 SV 从储备库中的募集。MFN2 敲低也导致突触前 Ca 专注于线粒体运输和融合，以更好地了解潜在的 QC 机制。我们观察到轴突中线粒体运输的大小和氧化还原不对称性，表明该区室中存在活跃的QC机制。我们还记录了轴突线粒体融合和裂变的生化互补。通过敲低神经元线粒体融合蛋白线粒体融合蛋白 2 (MFN2) 来消除融合，降低了轴突线粒体运输和融合的速率，降低了突触小泡 (SV) 蛋白的水平，抑制了胞吐作用，并在长时间刺激期间损害了 SV 从储备库中的募集。MFN2 敲低也导致突触前 Ca 专注于线粒体运输和融合，以更好地了解潜在的 QC 机制。我们观察到轴突中线粒体运输的大小和氧化还原不对称性，表明该区室中存在活跃的QC机制。我们还记录了轴突线粒体融合和裂变的生化互补。通过敲低神经元线粒体融合蛋白线粒体融合蛋白 2 (MFN2) 来消除融合，降低了轴突线粒体运输和融合的速率，降低了突触小泡 (SV) 蛋白的水平，抑制了胞吐作用，并在长时间刺激期间损害了 SV 从储备库中的募集。MFN2 敲低也导致突触前 Ca 表明该隔室中存在活跃的 QC 机制。我们还记录了轴突线粒体融合和裂变的生化互补。通过敲低神经元线粒体融合蛋白 mitofusin 2 (MFN2) 来消除融合，降低了轴突线粒体运输和融合的速率，降低了突触小泡 (SV) 蛋白的水平，抑制了胞吐作用，并在长时间刺激期间损害了 SV 从储备库中的募集。MFN2 敲低也导致突触前 Ca 表明该隔室中存在活跃的 QC 机制。我们还记录了轴突线粒体融合和裂变的生化互补。通过敲低神经元线粒体融合蛋白线粒体融合蛋白 2 (MFN2) 来消除融合，降低了轴突线粒体运输和融合的速率，降低了突触小泡 (SV) 蛋白的水平，抑制了胞吐作用，并在长时间刺激期间损害了 SV 从储备库中的募集。MFN2 敲低也导致突触前 Ca 降低突触小泡 (SV) 蛋白的水平，抑制胞吐作用，并在长时间刺激期间损害 SV 从储备库中的募集。MFN2 敲低也导致突触前 Ca 降低突触小泡 (SV) 蛋白的水平，抑制胞吐作用，并在长时间刺激期间损害 SV 从储备库中的募集。MFN2 敲低也导致突触前 Ca²⁺体内平衡失调。值得注意的是，在 MFN2 敲低后，突触前线粒体更有效地隔离 Ca ^{2+ ，有效限制刺激期间的突触前 Ca 2+}瞬态。这些结果支持活跃的线粒体运输和融合相关的 QC 过程，支持突触前 Ca ²⁺处理和 SV 循环。

意义声明在许多疾病状态下都观察到线粒体功能降低或改变。所有神经退行性疾病都与某种线粒体异常同时存在。因此，确定维持神经元（尤其是轴突）线粒体网络的质量控制机制具有重要意义。轴突线粒体对急性毒素或损伤的反应已被详细研究。尽管信息丰富，但神经元对这些损伤的反应可能与生理无关，因此研究轴突线粒体的基本行为也至关重要。在这里，我们使用荧光生物传感器来研究神经元中的线粒体网络，并检查线粒体融合蛋白 2 在维持轴突线粒体网络和支持突触小泡循环中的作用。