Functional characterization of novel RhoT1 variants, which are associated with Parkinson's disease

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Dokumentart: PhDThesis
Date: 2017-06-15
Language: English
Faculty: 4 Medizinische Fakultät
Department: Interdisziplinäre Einrichtungen
Advisor: Krüger, Rejko (Prof. Dr.)
Day of Oral Examination: 2016-05-13
DDC Classifikation: 570 - Life sciences; biology
610 - Medicine and health
Keywords: Mitochondrium , Parkinson-Krankheit
Other Keywords:
Parkinson's disease
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Parkinson’s disease (PD) is a common neurodegenerative disease affecting up to 2 % of the population older than 65 years. Most PD cases are sporadic with unknown cause, and about 10 % are familial inherited. PD is a progressive neurodegenerative disease characterized by loss of predominantly dopaminergic neurons, leading to typical symptoms like rigidity and tremor. Commonly involved pathogenic pathways are linked to mitochondrial dysfunction, e.g. increased oxidative stress, disruption of calcium homeostasis, decreased energy supply and mitochondrial-controlled apoptosis. The mitochondrial outer membrane protein Miro1 is important for mitochondrial distribution, quality control and maintenance. To date Miro1 is not established as risk factor for PD. Using a comprehensive mutation screening of RhoT1 in German PD patients we dissected the role of the first PD-associated mutations in RhoT1, the gene encoding for Miro1. Three mutations in RhoT1 have been identified in three PD patients with positive family history for PD. For analysis of mitochondrial phenotypes patient-derived fibroblasts from two of the three patients were available. As independent cell model served the neuroblastoma cell line M17 with stable knockdown of endogenous RhoT1 and transiently overexpression of the RhoT1 mutant variants. Investigation of yeast with knockout of endogenous Gem1 (the yeast orthologue of Miro1) and overexpression of Gem1-R298Q (the orthologue of Miro1-R272Q) revealed that growth on non-fermentable carbon source was impaired. These findings suggest that Miro1-R272Q is a loss of function mutation. Interestingly, the Miro1 protein amount was significantly reduced in Miro1-R272Q and Miro1-R450C mutant fibroblast lines compared to controls. Functional analysis revealed that mitochondrial mass was decreased in Miro1-R450C, but not in Miro1-R272Q fibroblasts, whereas mitochondrial biogenesis was increased in Miro1-R450C fibroblasts, as indicated by elevation of PGC1α. A similar phenotype with reduction of mitochondrial mass was also observed in M17 cells overexpressing Miro1-R272Q or Miro1-R450C. Additionally, spare respiratory capacity was reduced in Miro1-R272Q fibroblasts compared to Ctrl 1 fibroblasts. In contrast, Miro1-R450C fibroblasts showed increased respiratory activity compared to Ctrl 1, despite citrate synthase activity was significantly reduced. Both alterations of respiratory activity lead to mitochondrial membrane hyperpolarization in Miro1-R272Q and Miro1-R450C fibroblasts, a phenotype which was also found in M17 cells with knockdown of RhoT1. Both Miro1 mutant fibroblasts lines displayed different problems with cytosolic calcium buffering: in Miro1-R272Q fibroblasts histamine treatment increased cytosolic calcium concentration significantly compared to Ctrl 1 fibroblasts, indicating that calcium homeostasis was impaired, whereas in Miro1-R450C fibroblasts the buffering capacity for cytosolic calcium was impaired. The results indicate that mutations in Miro1 cause significant mitochondrial dysfunction, which are likely contributing to neurodegeneration in PD and underline the importance of Miro1 for mitochondrial maintenance.

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