Annual Meeting of the Society for Neuroscience edition:38 location:Washington DC, USA date:15-19 November 2008
PTEN-induced putative kinase (PINK1) is a serine/threonine kinase with a predicted mitochondrial targeting signal. A number of mutations in PINK1 have been linked to early-onset familial PARK6 parkinsonism. Both the autosomal-recessive form of inheritance and further experimental evidence indicate that these mutations induce a loss-of-function. However, how loss of PINK1 function leads to Parkinson’s disease is still unclear.
To mimic the loss-of-function mutations, we investigated both stable PINK1 knock-down Neuro2A cell lines generated by lentiviral vector-mediated RNA interference and fibroblasts derived from PINK1 knock-out mice. We focused our analysis to mitochondrial Ca2+-metabolism and mitochondrial morphology. To study the role of mitochondrial Ca2+ handling, we used the photoprotein aequorin which emits light in the presence of Ca2+. Different modifications of aequorin targeted to specific subcellular compartments allow quantitative monitoring of organellar Ca2+. In the PINK1 knock-down Neuro2A cell lines, we observed a modest but significant reduction in mitochondrial Ca2+ uptake compared to control cells. In addition, incubation of the cells with Rhodamine 123 was used to visualize the mitochondrial network. Quantification of the mitochondrial morphology revealed a more fragmented mitochondrial network in the PINK1 knock-down cell lines compared to control cells. This suggests that knock-down of PINK1 affects mitochondrial shape and function in neuronal cells. In the PINK1 knock-out fibroblasts, a similar, but more pronounced alteration in mitochondrial Ca2+ response was observed. This phenotype could be rescued by reintroduction of mouse and human wild-type PINK1. These results thus indicate that defects in mitochondrial Ca2+ homeostasis and morphology may be relevant events in PINK1-related Parkinson pathogenesis.