Reference : Functional characterization of novel RhoT1 variants, which are associated with Parkin...
Dissertations and theses : Doctoral thesis
Life sciences : Biochemistry, biophysics & molecular biology
Systems Biomedicine
Functional characterization of novel RhoT1 variants, which are associated with Parkinson's disease.
Grossmann, Dajana mailto [University of Luxembourg > Luxembourg Centre for Systems Biomedicine (LCSB) > >]
University of Luxembourg, ​Luxembourg, ​​Luxembourg
Docteur en Biologie
xx, 180 +4
Krüger, Rejko mailto
Rieß, Olaf mailto
Valente, Enza-Maria mailto
Skupin, Alexander mailto
Wissinger, Bernd mailto
Vogt-Weisenhorn, Daniela mailto
[en] Parkinson's disease ; mitochondria ; Miro1 ; RhoT1
[en] 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 mutant Gem1 revealed that growth on non-fermentable carbon source was impaired. These findings suggest that Miro1-mutant1 is a loss of function mutation. Interestingly, the Miro1 protein amount was significantly reduced in Miro1-mutant1 and Miro1-mutant2 fibroblast lines compared to controls. Functional analysis revealed that mitochondrial mass was decreased in Miro1-mutant2, but not in Miro1-mutant1 fibroblasts, whereas mitochondrial biogenesis was increased in Miro1-mutant2 fibroblasts, as indicated by elevation of PGC1α. A similar phenotype with reduction of mitochondrial mass was also observed in M17 cells overexpressing Miro1-mutant1 or Miro1-mutant2. Additionally, spare respiratory capacity was reduced in Miro1-mutant1 fibroblasts compared to Ctrl 1 fibroblasts. In contrast, Miro1-mutant2 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-mutant1 and Miro1-mutant2 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-mutant1 fibroblasts histamine treatment increased cytosolic calcium concentration significantly compared to Ctrl 1 fibroblasts, indicating that calcium homeostasis was impaired, whereas in Miro1-mutant2 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.
Luxembourg Centre for Systems Biomedicine (LCSB) - Clinical and Experimental neuroscience (Krüger Group) ; Hertie Institute for Clinical Brain Research, University of Tübingen, Germany
Fortüne programe, Medical Faculty, University of Tübingen, Germany ; Fonds National de la Recherche - FnR
Researchers ; Students
FnR ; FNR6682797 > Rejko Kruger > Endophenotypes in Neurodegeneration > Comprehensive assessment of endophenotypes in neurodegenerative diseases - translating impaired molecular signalling pathways into novel therapeutic strategies for Parkinson’s disease > 01/06/2014 > > 2013

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