Chaperone-mediated autophagy controls the turnover of E3 ubiquitin ligase MARCHF5 and regulates mitochondrial dynamics

As a highly dynamic organelle, mitochondria undergo constant fission and fusion to change their morphology and function, coping with various stress conditions. Loss of the balance between fission and fusion leads to impaired mitochondria function, which plays a critical role in the pathogenesis of Parkinson disease (PD). Yet the mechanisms behind mitochondria dynamics regulation remain to be fully illustrated. Chaperone-mediated autophagy (CMA) is a lysosome-dependent process that selectively degrades proteins to maintain cellular proteostasis. In this study, we demonstrated that MARCHF5, an E3 ubiquitin ligase required for mitochondria fission, is a CMA substrate. MARCHF5 interacted with key CMA regulators and was degraded by lysosomes. Severe oxidative stress compromised CMA activity and stabilized MARCHF5, which facilitated DNM1L translocation and led to excessive fission. Increase of CMA activity promoted MARCHF5 turnover, attenuated DNM1L translocation, and reduced mitochondria fragmentation, which alleviated mitochondrial dysfunction under oxidative stress. Furthermore, we showed that conditional expression of LAMP2A, the key CMA regulator, in dopaminergic (DA) neurons helped maintain mitochondria morphology and protected DA neuronal viability in a rodent PD model. Our work uncovers a critical role of CMA in maintaining proper mitochondria dynamics, and loss of this regulatory control may occur in PD and underlie its pathogenic process. Abbreviations: CMA: chaperone-mediated autophagy; DA: dopaminergic; DNM1L: dynamin 1 like; FCCP: carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone; HSPA8: heat shock protein family A (Hsp70) member 8; LAMP2A: lysosomal associated membrane protein 2A; MARCHF5: membrane-associated ring-CH-type finger 5; MMP: mitochondria membrane potential; OCR: oxygen consumption rate; 6-OHDA: 6-hydroxydopamine; PD: Parkinson disease; SNc: substantia nigra pars compacta; TEM: transmission electron microscopy; TH: tyrosine hydroxylase; TMRE: tetramethylrhodamine ethyl ester perchlorate; WT: wild type.作为一种高度动态的细胞器，线粒体（mitochondria）会持续进行分裂与融合以改变自身形态与功能，从而应对各类应激条件。分裂与融合的平衡失衡会导致线粒体功能受损，这在帕金森病（Parkinson disease, PD）的发病机制中发挥关键作用。然而，线粒体动态调控的背后机制仍有待全面阐明。 伴侣蛋白介导的自噬（chaperone-mediated autophagy, CMA）是一种依赖溶酶体的过程，可选择性降解蛋白质以维持细胞蛋白质稳态。本研究证实，线粒体分裂所需的E3泛素连接酶（E3 ubiquitin ligase）MARCHF5（membrane-associated ring-CH-type finger 5）是一种CMA底物。MARCHF5可与关键CMA调控因子相互作用，并被溶酶体降解。严重的氧化应激会削弱CMA活性并稳定MARCHF5，这会促进动力蛋白1样（dynamin 1 like, DNM1L）的转位，进而引发过度的线粒体分裂。增强CMA活性可促进MARCHF5的周转、减弱DNM1L转位并减少线粒体碎片化，从而缓解氧化应激下的线粒体功能障碍。此外，本研究发现，在啮齿类PD模型中，多巴胺能（dopaminergic, DA）神经元中条件性表达关键CMA调控因子溶酶体相关膜蛋白2A（lysosomal associated membrane protein 2A, LAMP2A），有助于维持线粒体形态，并保护多巴胺能神经元的存活。本研究揭示了CMA在维持正常线粒体动态平衡中的关键作用，而这种调控机制的缺失可能发生在帕金森病中，并成为其致病过程的基础。 缩写说明： CMA：伴侣蛋白介导的自噬； DA：多巴胺能； DNM1L：动力蛋白1样； FCCP：羰基氰化物4-(三氟甲氧基)苯腙； HSPA8：热休克蛋白家族A（Hsp70）成员8； LAMP2A：溶酶体相关膜蛋白2A； MARCHF5：膜相关环-CH型指蛋白5； MMP：线粒体膜电位； OCR：耗氧率； 6-OHDA：6-羟基多巴胺； PD：帕金森病； SNc：黑质致密部； TEM：透射电子显微镜； TH：酪氨酸羟化酶； TMRE：四甲基罗丹明乙酯高氯酸盐； WT：野生型。