Parkinson's disease-associated kinase PINK1 regulates Miro protein level and axonal transport of mitochondria

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.     

VPS13D bridges the ER to mitochondria and peroxisomes via Miro

Mitochondria, which are excluded from the secretory pathway, depend on lipid transport proteins for their lipid supply from the ER, where most lipids are synthesized. In yeast, the outer mitochondrial membrane GTPase Gem1 is an accessory factor of ERMES, an ER–mitochondria tethering complex that contains lipid transport domains and that functions, partially redundantly with Vps13, in lipid transfer between the two organelles. In metazoa, where VPS13, but not ERMES, is present, the Gem1 orthologue Miro was linked to mitochondrial dynamics but not to lipid transport. Here we show that Miro, including its peroxisome-enriched splice variant, recruits the lipid transport protein VPS13D, which in turn binds the ER in a VAP-dependent way and thus could provide a lipid conduit between the ER and mitochondria. These findings reveal a so far missing link between function(s) of Gem1/Miro in yeast and higher eukaryotes, where Miro is a Parkin substrate, with potential implications for Parkinson’s disease pathogenesis.     

PINK1 and Parkin target Miro for phosphorylation and degradation to arrest mitochondrial motility

Cells keep their energy balance and avoid oxidative stress by regulating mitochondrial movement, distribution, and clearance. We report here that two Parkinson's disease proteins, the Ser/Thr kinase PINK1 and ubiquitin ligase Parkin, participate in this regulation by arresting mitochondrial movement. PINK1 phosphorylates Miro, a component of the primary motor/adaptor complex that anchors kinesin to the mitochondrial surface. The phosphorylation of Miro activates proteasomal degradation of Miro in a Parkin-dependent manner. Removal of Miro from the mitochondrion also detaches kinesin from its surface. By preventing mitochondrial movement, the PINK1/Parkin pathway may quarantine damaged mitochondria prior to their clearance. PINK1 has been shown to act upstream of Parkin, but the mechanism corresponding to this relationship has not been known. We propose that PINK1 phosphorylation of substrates triggers the subsequent action of Parkin and the proteasome.     

PINK1 and Parkin flag Miro to direct mitochondrial traffic

The Parkinson's disease proteins PINK1 and Parkin are proposed guardians of mitochondrial fidelity, targeting damaged mitochondria for degradation by mitophagy. In this issue of Cell, Wang et al. (2011) now show that PINK1 and Parkin also regulate mitochondrial trafficking and quarantine damaged mitochondria by severing their connection to the microtubule network.     

Msp1: patrolling mitochondria for lost proteins

Intracellular protein localization is critical for establishing organelle identity, avoiding inappropriate interactions, and maintaining cellular function. An emerging mechanism for ensuring high‐fidelity protein localization is the selective destruction of mislocalized copies. Two new papers find that the mitochondrial outer membrane is kept free of mislocalized proteins by Msp1 (Chen et al, 2014 ; Okreglak & Walter, 2014 ).     

A Highlights from MBoC Selection: APC binds the Miro/Milton motor complex to stimulate transport of mitochondria to the plasma membrane

Mutations in adenomatous polyposis coli (APC) disrupt regulation of Wnt signaling, mitosis, and the cytoskeleton. We describe a new role for APC in the transport of mitochondria. Silencing of wild-type APC by small interfering RNA caused mitochondria to redistribute from the cell periphery to the perinuclear region. We identified novel APC interactions with the mitochondrial kinesin-motor complex Miro/Milton that were mediated by the APC C-terminus. Truncating mutations in APC abolished its ability to bind Miro/Milton and reduced formation of the Miro/Milton complex, correlating with disrupted mitochondrial distribution in colorectal cancer cells that could be recovered by reconstitution of wild-type APC. Using proximity ligation assays, we identified endogenous APC-Miro/Milton complexes at mitochondria, and live-cell imaging showed that loss of APC slowed the frequency of anterograde mitochondrial transport to the membrane. We propose that APC helps drive mitochondria to the membrane to supply energy for cellular processes such as directed cell migration, a process disrupted by cancer mutations.     

帕金森病相关蛋白PINK1和Parkin通过降解Miro影响线粒体运动

帕金森病是一种常见的神经退行性疾病,发病机制尚不清楚,线粒体功能障碍是可能的原因之一。帕金森病相关蛋白PINK1和Parkin均被证明影响线粒体功能和形态,并参与线粒体质量监控。2011年11月《细胞》杂志 （Cell）147期 发表了题为《PINK1和Parkin导致Miro磷酸化降解和线粒体运动阻滞》的文章,发现PINK1 / Parkin 通路可以作用于定位在线粒体外膜的线粒体移动相关蛋白Miro,PINK1直接磷酸化Miro,Parkin参与Miro降解,使受损线粒体脱离微管,从而阻滞线粒体运动。作者猜测这一过程能够隔离受损线粒体,避免了受损线粒体在细胞中的扩散。该研究深入探讨了PINK1和Parkin相互作用机制,揭示了线粒体质量控制系统如何直接调控线粒体运输,提出了受损线粒体的不正常运输可能是PD的致病原因。     

Miro1 regulates intercellular mitochondrial transport & enhances mesenchymal stem cell rescue efficacy

There is emerging evidence that stem cells can rejuvenate damaged cells by mitochondrial transfer. Earlier studies show that epithelial mitochondrial dysfunction is critical in asthma pathogenesis. Here we show for the first time that Miro1, a mitochondrial Rho‐GTPase, regulates intercellular mitochondrial movement from mesenchymal stem cells (MSC) to epithelial cells (EC). We demonstrate that overexpression of Miro1 in MSC (MSCmiro^Hi) leads to enhanced mitochondrial transfer and rescue of epithelial injury, while Miro1 knockdown (MSCmiro^Lo) leads to loss of efficacy. Treatment with MSCmiro^Hi was associated with greater therapeutic efficacy, when compared to control MSC, in mouse models of rotenone (Rot) induced airway injury and allergic airway inflammation (AAI). Notably, airway hyperresponsiveness and remodeling were reversed by MSCmiro^Hi in three separate allergen‐induced asthma models. In a human in vitro system, MSCmiro^Hi reversed mitochondrial dysfunction in bronchial epithelial cells treated with pro‐inflammatory supernatant of IL‐13‐induced macrophages. Anti‐inflammatory MSC products like NO, TGF‐β, IL‐10 and PGE2, were unchanged by Miro1 overexpression, excluding non‐specific paracrine effects. In summary, Miro1 overexpression leads to increased stem cell repair.     

The Miro GTPases: At the heart of the mitochondrial transport machinery

Mitochondria are organelles of elaborate structure that in addition to supplying cellular energy, have significant roles in calcium homeostasis and apoptosis. Failure to maintain mitochondrial dynamics results in neurodegenerative diseases and neuromuscular pathologies. The Miro GTPases, which constitute a unique subgroup of the Ras superfamily, have emerged as essential regulators of mitochondrial morphogenesis and trafficking along microtubules. Miro GTPases function as calcium-dependent sensors in the control of mitochondrial motility. Increased awareness of the biological function of Miro GTPases can contribute to elucidate the molecular mechanisms underlying diseases caused by deregulated mitochondrial dynamics.     

Miro: A molecular switch at the center of mitochondrial regulation

The orchestration of mitochondria within the cell represents a critical aspect of cell biology. At the center of this process is the outer mitochondrial membrane protein, Miro. Miro coordinates diverse cellular processes by regulating connections between organelles and the cytoskeleton that range from mediating contacts between the endoplasmic reticulum and mitochondria to the regulation of both actin and microtubule motor proteins. Recently, a number of cell biological, biochemical, and protein structure studies have helped to characterize the myriad roles played by Miro. In addition to answering questions regarding Miro's function, these studies have opened the door to new avenues in the study of Miro in the cell. This review will focus on summarizing recent findings for Miro's structure, function, and activity while highlighting key questions that remain unanswered.     

Understanding Miro GTPases: Implications in the Treatment of Neurodegenerative Disorders

The Miro GTPases represent an unusual subgroup of the Ras superfamily and have recently emerged as important mediators of mitochondrial dynamics and for maintaining neuronal health. It is now well-established that these enzymes act as essential components of a Ca²⁺-sensitive motor complex, facilitating the transport of mitochondria along microtubules in several cell types, including dopaminergic neurons. The Miros appear to be critical for both anterograde and retrograde mitochondrial transport in axons and dendrites, both of which are considered essential for neuronal health. Furthermore, the Miros may be significantly involved in the development of several serious pathological processes, including the development of neurodegenerative and psychiatric disorders. In this review, we discuss the molecular structure and known mitochondrial functions of the Miro GTPases in humans and other organisms, in the context of neurodegenerative disease. Finally, we consider the potential human Miros hold as novel therapeutic targets for the treatment of such disease.     

Carborane derivatives of 1,2,3-triazole depolarize mitochondria by transferring protons through the lipid part of membranes

Boron containing polyhedra (carboranes) are three-dimensional delocalized aromatic systems. These structures have been shown to transport protons through lipid membranes and mitochondria. Conjugation of carboranes to various organic moieties is aimed at obtaining biologically active compounds with novel properties. Taking advantage of 1,2,3-triazoles as the scaffolds valuable in medicinal chemistry, we synthesized 1-(o-carboranylmethyl)-4-pentyl-1,2,3-triazole (c-triazole) and 1-(o-carboranylmethyl)-4-pentyl-1,2,3-triazolium iodide (c-triazolium). Both compounds interacted with model lipid membranes and exhibited a proton carrying activity in planar bilayers and liposomes in a concentration- and pH-dependent manner. Importantly, mechanisms of the protonophoric activity differed; namely, protonation-deprotonation reactions of the triazole and the o-carborane moieties were involved in the transport cycles of c-triazole and c-triazolium, respectively. At micromolar concentrations, c-triazole and c-triazolium stimulated respiration of isolated rat liver mitochondria and depolarized their membrane potential, with c-triazole being more potent. In living K562 (human chronic myelogenous leukemia) cells, both c-triazolium and c-triazole altered the mitochondrial membrane potential as determined by a decreased intracellular accumulation of the potential-dependent dye tetramethylrhodamine ethyl ester. Finally, cell viability testing demonstrated a cytotoxic potency of c-triazolium and, to a lesser extent, of c-triazole against K562 cells, whereas non-malignant fibroblasts were much less sensitive. In all tests, the reference boron-free benzyl-4-pentyl-1,2,3-triazole showed little-to-no effects. These results demonstrated that carboranyltriazoles carry protons across biological membranes, a property potentially important in anticancer drug design.     

Old dog,new tricks: Arf1 required for mitochondria homeostasis

The small GTPase Arf1 that is classically required for the budding of COPI‐coated vesicles from the Golgi membrane is now proposed to have novel and conserved roles in the morphological and functional maintenance of mitochondria: It functionally localizes to ER/mitochondria contact sites; it allows for the recruitment of a degradation machinery to mitochondria to remove toxic mitofusin/Fzo1 clusters; and it allows the extension of autophagy sequestration membranes needed for mitophagy to clear damaged mitochondria.     

Loss of neuronal Miro1 disrupts mitophagy and induces hyperactivation of the integrated stress response

Clearance of mitochondria following damage is critical for neuronal homeostasis. Here, we investigate the role of Miro proteins in mitochondrial turnover by the PINK1/Parkin mitochondrial quality control system in vitro and in vivo. We find that upon mitochondrial damage, Miro is promiscuously ubiquitinated on multiple lysine residues. Genetic deletion of Miro or block of Miro1 ubiquitination and subsequent degradation lead to delayed translocation of the E3 ubiquitin ligase Parkin onto damaged mitochondria and reduced mitochondrial clearance in both fibroblasts and cultured neurons. Disrupted mitophagy in vivo, upon post‐natal knockout of Miro1 in hippocampus and cortex, leads to a dramatic increase in mitofusin levels, the appearance of enlarged and hyperfused mitochondria and hyperactivation of the integrated stress response (ISR). Altogether, our results provide new insights into the central role of Miro1 in the regulation of mitochondrial homeostasis and further implicate Miro1 dysfunction in the pathogenesis of human neurodegenerative disease.