Impaired mitochondrial–endoplasmic reticulum interaction and mitophagy in Miro1-mutant neurons in Parkinson’s disease

Abstract

Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson’s disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.

The Emerging Role of RHOT1/Miro1 in the Pathogenesis of Parkinson's Disease

“…In our studies, PD patient-derived Miro1-mutant fibroblasts and iPSC-derived Miro1-R272Q neurons demonstrated an increased mitochondrial fragmentation after treatment compared to control cells. This finding was not surprising given the delayed buffering of calcium transients and resulting retained high levels of calcium in the cytosol (13,14,67). Our findings imply that mutations in Miro1 cause an impairment of calcium homeostasis, resulting in decreased ATP production and increased calcium-dependent mitochondrial fragmentation, thereby contributing to the pathogenesis of PD.…”

“…Impaired cellular calcium homeostasis is a shared phenotype observed in different models of PD (105)(106)(107)(108)(109)(110). In line with this, iPSC-derived neurons with heterozygous Miro1-R272Q display a significantly higher peak of cytosolic calcium and delayed buffering capacity after ionomycin treatment compared to control neurons (67). Hence, one might speculate that mutations in Miro1 drive neurodegeneration by impairing calcium homeostasis, subsequently affecting mitochondrial function and energy production in the pathogenesis of PD.…”

“…Specific inhibition of Polo kinase with BI2536 reduced the localization of Miro1 to MERCs and also caused a destabilization of MERCs (84). This finding is especially interesting in the light of increased numbers of MERCs and enhanced localization of Miro1 to MERCs observed in iPSC-derived Miro1-R272Q neurons (67). Thus, pharmacologically targeting regulators of Miro1 function such as Polo kinase offers another promising approach for personalized medicine but also bears the risk of unwanted side effects.…”

“…Association of Gem1p to ERMES controls phospholipid exchange for lipid biosynthesis between mitochondria and ER (15) and regulates mitochondrial division and morphology (81)(82)(83). The localization of dMiro at MERCs was also demonstrated in Drosophila neural stem cells and dopaminergic neurons (17,84), as well as mammalian Miro1 in COS-7 cells, HeLa cells, MEFs, human fibroblasts, and human iPSC-derived neurons (14,15,67,84,85).…”

“…Moreover, based on the increased amount of overall MERCs and impaired CCCP-induced mitochondrial clearance observed in iPSC-derived Miro1-R272Q neurons, we speculate that damaged mitochondria may not uncouple from the ER, consequently hampering the initiation and flux of mitophagy (67).…”