Recessive parkinsonism and mitochondrial function

National Institute On Aging

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Abstract

PINK1 is a mitochondrially-directed Serine/Threonine kinase that is genetically associated with inherited parkinsonism. Loss of function mutations in this gene cause, when homozygous, an early onset disease with loss of specific groups of neurons in the substantia nigra pars compacta. We have previously shown that some mutations are very simple and result in loss of function by making the protein unstable. Some forms of mutant PINK1 are degraded by the ubiquitin-proteasome system in cells. In the course of these experiments we noticed that there are two pools of PINK1 in cells, including the predicted mitochondrial protein but also a distinct amount in the cytosol. Interestingly, the mature protein form, which lacks the amino-terminal mitochondrial targeting sequence, is enriched in the cytoplasm.[unreadable] This result made us question whether the mitochondrial targeting sequence is actually required for PINK1s reported neuroprotective ability. In collaboration with the Park group at the University of Ottawa, Canada, we have found that an artificial mutant of PINK1 lacking the N-terminus is functional in vitro and in vivo at protecting neurons. This suggests that the cytoplasmic pool of PINK1 is most critical for the kinases protective function. We also know from these experiments that the kinase activity is important as well, as artificial kinase dead versions of PINK1 are non-functional. We have confirmed these observations in our own lab using quantitative measures of mitochondrial function and again see that the amino-terminal peptide is dispensible for function but kinase activity is not. In our hands, the (delta-N) PINK1 mutant is more stable than the full length protein and slightly less efficient, suggesting that localization of the protein to mitochondria is still important even if it is not strictly required. Our interpretation is that the targeting peptide is used to direct PINK1 to the cytoplasmic face of mitochondria and that it has one or more critical substrates at that location.[unreadable] We have also addressed the cellular expression of endogenous PINK1 in rodent and human brains as this is a potentially important clue in addressing which cells might be most sensitive to loss of PINK1 function. We find that the mRNA for PINK1 is broadly expressed throughout the central nervous system in both species. The in situ hybridization signals are consistent with a predominantly neuronal expression pattern as there was only background level of signal in glial cells. We infer that PINK1 has critical roles in neuronal mitochondria.