| Abstract No.: | B-C2078 |
| Country: | Canada |
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| Title: | THE ROLE OF PARKIN IN REGULATING MITOCHONDRIAL MORPHOLOGY AND ITS IMPORTANCE IN PARKINSON’S DISEASE |
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| Authors/Affiliations: | 1 Pierre Robinson*; 1 Edward A. Fon;
1 Center for Neuronal Survival, Montreal Neurological Institute, McGill University, QC, Canada
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| Content: | Objectives: Mitochondria are very dynamic organelles that continuously undergo fusion and fission processes in many physiological situations. The proteins required for fusion in mammalian cells include Mitofusin1 (Mfn1), Mitofusin2 (Mfn2) and OPA1 (Optic Dominant Atrophy-1) which are all members of the dynamin-family (GTPases). On the other hand, fission requires the action Drp1 (dynamin-related GTPase-1). Recent studies have suggested that fragmentation of mitochondria is closely related to the regulation of cell death progression by various stimuli. It is also well documented that fission is associated with most form of cell death. Mitochondrial dysfunction has long been linked to Parkinson’s disease (PD). Recently, several genes responsible for monogenic forms of PD have been discovered (parkin, DJ-1, PINK-1, LRRK2 and Omi/HtrA2). Interestingly, the product of these genes have been associated with mitochondrial dysfunction, which was demonstrated either by association with mitochondrial membranes or by altering mitochondrial functions. Studies from parkin knock-out (KO) flies, have indicated that parkin might have a role in regulating mitochondrial integrity and that the loss of parkin leads to reduced lifespan, muscle degeneration and disintegration of the mitochondrial matrix. However, parkin KO mice have showed only modest effects on protein levels and activity of the mitochondrial respiratory complex I, and IV. Therefore, we will investigate mitochondrial morphology (size and length) in mouse embryonic fibroblasts (MEFs), in order to determine if the loss of parkin expression induces dysfunctions in mitochondrial dynamics. We also verified if the absence of parkin affect mitochondrial fusion or fission protein levels.
Material and methods: MEFs were prepared from E13.5 wild type (C57BL/6) and Parkin KO mice. Cells (5.0 x 104/mL) were plated on poly-D-lysine coated coverslips in 12-well plates. Cells were grown in DMEM to 50-60% confluence and incubated with Mitotracker Deep Red (100 nM) for 40 min. After, cells were rinsed with PBS, fixed with PFA (4%) at RT for 10 min. and rinsed with ice-cold PBS. Cells were then rinsed and counterstained with Hoechst-33342 (1:10,000) for 2 minutes. Microscopic analysis was performed with the fluorescence microscope (Nikon/Eclipse880) at 60X and measurements using the Volocity software (Improvision) and predetermined ROIs within each cell. For each genotype, 15 cells were analyzed by measuring the length of 25 mitochondria per cell.
Results and conclusion: We observed that mitochondrial length is reduced in parkin KO MEFs and mitochondria displayed abnormal morphology compared to wild type MEFs. Western blot analyses revealed that levels of fusion and fission proteins were not altered in parkin KO MEFs. Interestingly, higher levels of the fission protein Drp1 was found in the brain of parkin KO mice. Since normal mitochondria morphology results from equal balance between fusion and fission mechanisms, it is therefore possible that mitochondria length is decreased in the brain of parkin KO mice due to higher fission rate. |
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