Mutations in <i>RHOT1</i> Disrupt Endoplasmic Reticulum–Mitochondria Contact Sites Interfering with Calcium Homeostasis and Mitochondrial Dynamics in Parkinson's Disease

Großmann, Dajana; Berenguer-Escuder, Clara; Bellet, Marie E.; Scheibner, David; Bohler, Jill; Massart, François; Rapaport, Doron; Skupin, Alexander; d’Hérouël, Aymeric Fouquier; Sharma, Manu; Ghelfi, Jenny; Raković, Aleksandar; Lichtner, Peter; Antony, Paul; Glaab, Enrico; May, Patrick; Dimmer, Kai Stefan; Fitzgerald, Julia C.; Grünewald, Anne; Krüger, Rejko

doi:10.1089/ars.2018.7718

Cited by 61 publications

(88 citation statements)

References 51 publications

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“…In this study, we provide further evidence for a role of rare genetic variants in RHOT1 in PD, now presenting a wider spectrum of Miro1 point mutations in a total of 4 independent patients. In agreement with our previous findings in Miro1-R272Q and Miro1-R450C mutant fibroblasts [10], fibroblasts with the newly identified heterozygous mutations T351A and T610A also display a reduction of MERCs as well as impaired calcium homeostasis. Furthermore, we analyzed the MERC composition in all four Miro1-mutant cultures and found differences in the recruitment of Miro1 protein to MERCs and in the amounts of different types of MERCs compared to control fibroblasts.…”

Section: Introductionsupporting

confidence: 92%

“…In Lübeck as well as in Tübingen, skin biopsies to establish fibroblast cultures are taken from the arm of the individuals. Cell culture conditions and immortalization of native fibroblasts were explained in our previous study [10].…”

Section: Fibroblast Cell Culturementioning

confidence: 99%

“…The protocol for live cell imaging of cytosolic calcium levels using Fluo4-AM staining and treatment with thapsigargin and Ru360 was described in detail before [10]. Live cell imaging for the analysis of calcium-induced fragmentation using MitoTracker green FM staining and ionomycin treatment was likewise described in detail [10]. The aspect ratio was determined to assess mitochondrial fragmentation.…”

Section: Live Cell Imaging For Calcium Analysismentioning

confidence: 99%

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Variants in Miro1 Cause Alterations of ER-Mitochondria Contact Sites in Fibroblasts from Parkinson’s Disease Patients

Berenguer-Escuder

Großmann

Massart

et al. 2019

JCM

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Background: Although most cases of Parkinson´s disease (PD) are idiopathic with unknown cause, an increasing number of genes and genetic risk factors have been discovered that play a role in PD pathogenesis. Many of the PD-associated proteins are involved in mitochondrial quality control, e.g., PINK1, Parkin, and LRRK2, which were recently identified as regulators of mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs) linking mitochondrial homeostasis to intracellular calcium handling. In this context, Miro1 is increasingly recognized to play a role in PD pathology. Recently, we identified the first PD patients carrying mutations in RHOT1, the gene coding for Miro1. Here, we describe two novel RHOT1 mutations identified in two PD patients and the characterization of the cellular phenotypes. Methods: Using whole exome sequencing we identified two PD patients carrying heterozygous mutations leading to the amino acid exchanges T351A and T610A in Miro1. We analyzed calcium homeostasis and MERCs in detail by live cell imaging and immunocytochemistry in patient-derived fibroblasts. Results: We show that fibroblasts expressing mutant T351A or T610A Miro1 display impaired calcium homeostasis and a reduced amount of MERCs. All fibroblast lines from patients with pathogenic variants in Miro1, revealed alterations of the structure of MERCs. Conclusion: Our data suggest that Miro1 is important for the regulation of the structure and function of MERCs. Moreover, our study supports the role of MERCs in the pathogenesis of PD and further establishes variants in RHOT1 as rare genetic risk factors for neurodegeneration.

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Section: Introductionsupporting

confidence: 92%

Section: Fibroblast Cell Culturementioning

confidence: 99%

Section: Live Cell Imaging For Calcium Analysismentioning

confidence: 99%

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Variants in Miro1 Cause Alterations of ER-Mitochondria Contact Sites in Fibroblasts from Parkinson’s Disease Patients

Berenguer-Escuder

Großmann

Massart

et al. 2019

JCM

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“…Recently, a pair of heterozygous mutations in Miro were identified in a cohort of Parkinson's patients . Patient fibroblasts bearing R272Q and R450C mutations were treated with thapsigargin, which depletes ER calcium into the cytosol.…”

Section: Miro Facilitates Inter‐organelle Interactions With Cytoplasmmentioning

confidence: 99%

Miro: A molecular switch at the center of mitochondrial regulation

et al. 2020

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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.

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“…4 Although autophagosomes are generated at ER-contact sites 47 PINK1 may play important roles in addition to mitophagy at mitochondrial-ER contact sites and in ER stress [48][49][50] . Proteins such as Miro1, VDAC and mitofusins have been strongly linked to ER and PINK1 function relevant to PD 21,[51][52][53][54] . Mitochondrial-ER contact sites are regulatory hubs for calcium, iron, lipids and dopamine.…”

Section: Introductionmentioning

confidence: 99%

PINK1 Regulates Dopamine and Lipids at Mitochondria to Maintain Synapses and Neuronal Function

Buș

Geisler

Feldkaemper

et al. 2019

Preprint

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word count: 266 2 AbstractMitochondrial dysfunction contributes to the pathogenesis of Parkinson's disease but it is not clear why inherent mitochondrial defects lead specifically to the death of dopaminergic neurons of the mid brain. PINK1 is mitochondrial kinase and PINK1 mutations cause early onset Parkinson's disease.We found that in neuronal progenitors, PINK1 regulates mitochondrial morphology, mitochondrial contact to the endoplasmic reticulum (ER) and the phosphorylation of Miro1. A compensatory metabolic shift towards lipid synthesis provides mitochondria with the components needed for membrane renewal and oxidative phosphorylation, maintaining the mitochondrial network once mature.Cholesterol is increased by loss of PINK1, promoting overall membrane rigidity. This alters the distribution of phosphorylated DAT at synapses and impairs dopamine uptake. PINK1 is required for the phosphorylation of tyrosine hydroxylase at Ser19, dopamine and calcium homeostasis and dopaminergic pacemaking.We suggest a novel mechanism for PINK1 pathogenicity in Parkinson's disease in addition to but not exclusive of mitophagy. We also provide a basis for potential therapeutics by showing that low doses of the cholesterol depleting drug ßcyclodextrin reverse PINK1-specific phenotypes.Gurion University of the Negev, Beer-Sheva, 8410501 Israel who developed the constructs for performing the BRET experiments for measurement of mitochondrial-ER contacts and kindly shared them with HFR and AG. Gerrit Machetanz of the

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Mutations in RHOT1 Disrupt Endoplasmic Reticulum–Mitochondria Contact Sites Interfering with Calcium Homeostasis and Mitochondrial Dynamics in Parkinson's Disease

Cited by 61 publications

References 51 publications

Variants in Miro1 Cause Alterations of ER-Mitochondria Contact Sites in Fibroblasts from Parkinson’s Disease Patients

Variants in Miro1 Cause Alterations of ER-Mitochondria Contact Sites in Fibroblasts from Parkinson’s Disease Patients

Miro: A molecular switch at the center of mitochondrial regulation

PINK1 Regulates Dopamine and Lipids at Mitochondria to Maintain Synapses and Neuronal Function

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