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Evolutionarily conserved developmental pathways
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta and the accumulation of proteinaceous intraneuronal inclusions known as Lewy bodies. Little is known of the molecular mechanisms responsible for loss of dopaminergic neurons in PD; however, evidence suggests that environmental and genetic factors both play contributing roles.
Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila Pink1 and Parkin indicate that PINK1, a mitochondrially targeted serine/threonine kinase, acts upstream of Parkin, a cytosolic ubiquitin-protein ligase, to promote mitochondrial fragmentation, although the molecular mechanisms by which the PINK1/Parkin pathway promotes mitochondrial fragmentation are unknown. This study tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. The steady-state abundance of the conserved mitochondrial fusion-promoting factor Drosophila Mitofusin (Mitochondrial assembly regulatory factor, Marf or dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and dMfn co-immunoprecipitates with Parkin. By contrast, perturbations of PINK1 or Parkin did not influence the steady-state abundance of the mitochondrial fission-promoting factor Drp1 or the mitochondrial fusion-promoting factor Opa1, or the subcellular distribution of Drp1. These findings suggest that dMfn is a direct substrate of the PINK1/Parkin pathway and that the mitochondrial morphological alterations and tissue degeneration phenotypes that derive from mutations in PINK1 and parkin result at least in part from reduced ubiquitin-mediated turnover of dMfn (Poole, 2010).
In previous work, it has been shown that genetic manipulations that promote mitochondrial fragmentation, including increased drp1 gene dosage and decreased opa1 or dmfn gene dosage, dramatically suppress the PINK1 and parkin mutant phenotypes in Drosophila. These findings, coupled with previous work demonstrating that PINK1 acts upstream of Parkin in a common pathway, led to a hypothesis that PINK1 and Parkin influence mitochondrial integrity by regulating core components of the mitochondrial morphogenesis machinery through ubiquitination. The current results provide direct support for this hypothesis by demonstrating that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that the steady-state abundance of dMfn is increased in PINK1 and parkin mutants and decreased in PINK1- and Parkin-overexpressing flies. These findings suggest a model in which PINK1 phosphorylates either dMfn or Parkin and thereby increases the efficiency with which Parkin is able to ubiquitinate dMfn. The subsequent ubiquitin-mediated turnover of dMfn would then inhibit mitochondrial fusion, and thus promote mitochondrial fragmentation (Poole, 2010).
The finding that the PINK1/Parkin pathway promotes mitochondrial fragmentation led to a proposal that this pathway may act to segregate damaged portions of the mitochondrial reticulum for turnover through an autophagic mechanism (Poole, 2008). Several recent studies provide compelling support for this hypothesis by demonstrating that treatment of cultured vertebrate cells with mitochondrial damaging agents triggers PINK1 to selectively recruit Parkin to damaged mitochondria, where Parkin acts to promote the autophagic turnover of these mitochondria, presumably by ubiquitinating particular mitochondrial targets. These studies, together with the current findings raise the possibility that the selective Parkin-mediated ubiquitination and subsequent degradation of dMfn on damaged portions of the mitochondrial reticulum, coupled with ongoing mitochondrial fission serves to sequester the mitochondrial damage to small fusion-incompetent mitochondria that are subsequently eliminated through autophagy. However, the size of ubiquitinated dMfn suggests that it is triply ubiquitinated and previous work indicates that a chain of four or more ubiquitins is required for efficient targeting to the proteasome. Thus, alternative interpretations of the findings, although not mutually exclusive, are that ubiquitination of dMfn inactivates the fusion-promoting activity of dMfn, or serves as a tag marking the damaged mitochondria for destruction by autophagy. The finding that the ubiquitination of a peroxisomal surface protein is sufficient to signal the autophagic degradation of this organelle is consistent with the latter model. Experiments are currently underway to distinguish these possibilities (Poole, 2010).
While a model in which the PINK1/Parkin pathway promotes mitochondrial fragmentation through the ubiquitination of dMfn is completely consistent with previous work on PINK1 and Parkin in Drosophila, recent findings from vertebrate cell culture studies challenge this model. In particular, several of the studies of PINK1 in vertebrate systems have found that reduced PINK1 activity results in mitochondrial fragmentation, suggesting that PINK1 may promote mitochondrial fusion—exactly the opposite of the conclusion drawn from studies of the PINK1/Parkin pathway in flies. While additional work will be required to resolve these apparent conflicts, it is important to point out that the findings from studies of PINK1 and Parkin in flies have involved tissues that are profoundly affected by loss of PINK1 and Parkin activity, whereas the tissue sources of the cells that have been used in at least some of the conflicting vertebrate studies are largely unaffected by mutations in PINK1 and parkin. Thus, a possible explanation for these apparently discordant findings is that the mitochondrial fragmentation resulting from reduced PINK1 activity that has been observed in vertebrate systems involves a compensatory induction of mitochondrial fragmentation in these cells, which perhaps also explains their relative insensitivity to the loss of PINK1 activity. In potential support of this model is the finding that while the mitochondrial fragmentation seen in PINK1-deficient vertebrate cells can be rescued by inactivating Drp1, this manipulation enhances the cell death associated with PINK1 deficiency, a finding that is entirely consistent with work in flies. Future work should resolve these apparent conflicts and further clarify the influence of PINK1- and Parkin-dependent ubiquitination of dMfn on mitochondrial integrity (Poole, 2010).
Poole, A. C., et al. (2010). The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway. PLoS One. 2010 Apr 7;5(4):e10054. PubMed Citation: 20383334
date revised: 10 February 2012
Developmental Pathways conserved in Evolution
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