Parkinson's disease (PD) is an age-related neurodegenerative disease characterized by the selective loss of dopaminergic neurons in the substantia nigra of the brain. The pathogenesis and etiology of PD are unclear. Mitochondrial dysfunction occurs in PD, causing a decrease in complex I activity in postmortem brain, and exacerbating reactive oxygen species production and ATP deficiency contributing to neuronal cell death. Mutation of leucine-rich-repeat kinase 2 (LRRK2) gene is the most common genetic factor identified in both familial and sporadic PD cases. Several mutations in LRRK2 have been linked to PD, in which R1441G is the second commonest mutation after G2019S. LRRK2 protein is ubiquitously expressed in human body, in which a portion is localized to the mitochondria. Mutations of LRRK2 directly or indirectly cause mitochondria dysfunction. Dysfunction of mitochondrial respiratory complexes has been described in skin fibroblasts and skeletal muscle of PD patients. Therefore, these clinically accessible tissues are good for monitoring disease progression. The objectives of this study were to investigate how LRRK2 R1441G mutation affects normal mitochondrial function, and whether this specific LRRK2 mutation potentiates age-dependent deterioration of mitochondrial function.
To achieve these aims, colonies of skin fibroblast carrying LRRK2 R1441G mutation or wild-type LRRK2 were derived from a novel LRRK2 R1441G knock-in (KI) mouse model and its wild-type (WT) littermates. Skeletal muscles were dissected from the hind legs of WT and KI mice. The effects of aging and LRRK2 R1441G mutation on mitochondrial function were investigated in vitro using these derived skin fibroblast cultures, and ex vivo using skeletal muscle obtained from young (3-month-old) and aged (18-month-old) WT and KI mice. Reduction-oxidation activities of mitochondrial complex I and complex II in skin fibroblasts and skeletal muscle were measured spectrophotometrically. Intracellular ATP levels in skin fibroblasts were determined by bioluminescent assay.
Phase-contrast microscopy showed that aging and LRRK2 R1441G mutation did not affect cell morphology of the derived skin fibroblast cultures. Complex I activity determined in skin fibroblasts and skeletal muscle derived from KI and their WT littermates revealed that, aging caused a significant increase in complex I activity in WT but not KI skin fibroblasts. Conversely, a significant decrease in complex I activity was observed in both WT and KI skeletal muscle, demonstrating an aging effect ex vivo. LRRK2 R1441G mutation did not affect complex I activity in WT and KI skin fibroblasts and skeletal muscle. Moreover, complex II activity in these two tissues was neither affected by aging nor R1441G LRRK2 mutation. Intracellular ATP levels in the skin fibroblast cultures were also unaltered by aging and LRRK2 R1441G mutation.
In conclusion, my current findings indicated a significant aging effect on mitochondrial complex I activity ex vivo, supporting the role of age-dependent deterioration of complex I activity in mitochondrial dysfunction of PD. LRRK2 R1441G mutation did not affect complex I and II activities in both skin fibroblasts and skeletal muscle. Also, this mutation did not potentiate the age-dependent deterioration of complex I activities as observed in skin fibroblasts and skeletal muscle of the LRRK2 R1441G knock-in mice. / published_or_final_version / Medicine / Master / Master of Philosophy
| Identifer | oai:union.ndltd.org:HKU/oai:hub.hku.hk:10722/183070 |
| Date | January 2013 |
| Creators | So, Hon-fai., 蘇漢暉. |
| Contributors | Ho, WL, Ho, SL |
| Publisher | The University of Hong Kong (Pokfulam, Hong Kong) |
| Source Sets | Hong Kong University Theses |
| Language | English |
| Detected Language | English |
| Type | PG_Thesis |
| Source | http://hub.hku.hk/bib/B50162846 |
| Rights | The author retains all proprietary rights, (such as patent rights) and the right to use in future works., Creative Commons: Attribution 3.0 Hong Kong License |
| Relation | HKU Theses Online (HKUTO) |
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