PINK1/Parkin mitophagy and neurodegeneration-what do we really know in vivo?

Mitochondria are essential organelles that provide cellular energy and buffer cytoplasmic calcium. At the same time they produce damaging reactive oxygen species and sequester pro-apoptotic factors. Hence, eukaryotes have evolved exquisite homeostatic processes that maintain mitochondrial integrity, or ultimately remove damaged organelles. This subject has garnered intense interest recently following the discovery that two Parkinson's disease genes, PINK1 and parkin, regulate mitochondrial degradation (mitophagy). The molecular details of PINK1/Parkin-induced mitophagy are emerging but much of our insight derives from work using cultured cells and potent mitochondrial toxins, raising questions about the physiological significance of these findings. Here we review the evidence supporting PINK1/Parkin mitophagy in vivo and its causative role in neurodegeneration, and outline outstanding questions for future investigations.AJW acknowledges funding from the MRC (MC-A070-5PSB0) and ERC (StG 309742), and LJP acknowledges funding from the NIH (5R01GM104990)

Drosophila DJ-1 Mutants Are Selectively Sensitive to Environmental Toxins Associated with Parkinson’s Disease

SummaryParkinson’s disease (PD) is a common neurodegenerative disorder that displays both sporadic and inherited forms [1]. Exposure to several common environmental toxins acting through oxidative stress has been shown to be associated with PD [2]. One recently identified inherited PD gene, DJ-1, may have a role in protection from oxidative stress [3–10], thus potentially linking a genetic cause with critical environmental risk factors. To develop an animal model that would allow integrative study of genetic and environmental influences, we have generated Drosophila lacking DJ-1 function. Fly DJ-1 homologs exhibit differential expression: DJ-1β is ubiquitous, while DJ-1α is predominantly expressed in the male germline. DJ-1α and DJ-1β double knockout flies are viable, fertile, and have a normal lifespan; however, they display a striking selective sensitivity to those environmental agents, including paraquat and rotenone, linked to PD in humans. This sensitivity results primarily from loss of DJ-1β protein, which also becomes modified upon oxidative stress. These studies demonstrate that fly DJ-1 activity is selectively involved in protection from environmental oxidative insult in vivo and that the DJ-1β protein is biochemically responsive to oxidative stress. Study of these flies will provide insight into the critical interplay of genetics and environment in PD

Culling sick mitochondria from the herd

The PINK1–Parkin pathway plays a critical role in mitochondrial quality control by selectively targeting damaged mitochondria for autophagy. In this issue, Tanaka et al. (2010. J. Cell Biol. doi: 10.1083/jcb.201007013) demonstrate that the AAA-type ATPase p97 acts downstream of PINK1 and Parkin to segregate fusion-incompetent mitochondria for turnover. p97 acts by targeting the mitochondrial fusion-promoting factor mitofusin for degradation through an endoplasmic reticulum–associated degradation (ERAD)-like mechanism

Mitochondrially-targeted APOBEC1 is a potent mtDNA mutator affecting mitochondrial function and organismal fitness in Drosophila

Abstract: Somatic mutations in the mitochondrial genome (mtDNA) have been linked to multiple disease conditions and to ageing itself. In Drosophila, knock-in of a proofreading deficient mtDNA polymerase (POLG) generates high levels of somatic point mutations and also small indels, but surprisingly limited impact on organismal longevity or fitness. Here we describe a new mtDNA mutator model based on a mitochondrially-targeted cytidine deaminase, APOBEC1. mito-APOBEC1 acts as a potent mutagen which exclusively induces C:G>T:A transitions with no indels or mtDNA depletion. In these flies, the presence of multiple non-synonymous substitutions, even at modest heteroplasmy, disrupts mitochondrial function and dramatically impacts organismal fitness. A detailed analysis of the mutation profile in the POLG and mito-APOBEC1 models reveals that mutation type (quality) rather than quantity is a critical factor in impacting organismal fitness. The specificity for transition mutations and the severe phenotypes make mito-APOBEC1 an excellent mtDNA mutator model for ageing research

The Mitochondrial Fusion-Promoting Factor Mitofusin Is a Substrate of the PINK1/Parkin Pathway

Loss-of-function mutations in the PINK1 or parkin genes result in recessive heritable forms of parkinsonism. Genetic studies of Drosophila orthologs of 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. We tested the hypothesis that PINK1 and Parkin promote mitochondrial fragmentation by targeting core components of the mitochondrial morphogenesis machinery for ubiquitination. We report that the steady-state abundance of the mitochondrial fusion-promoting factor Mitofusin (dMfn) is inversely correlated with the activity of PINK1 and Parkin in Drosophila. We further report that dMfn is ubiquitinated in a PINK1- and Parkin-dependent fashion and that 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. Our 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

Inactivation of the mitochondrial protease Afg3l2 results in severely diminished respiratory chain activity and widespread defects in mitochondrial gene expression.

The m-AAA proteases play a critical role in the proteostasis of inner mitochondrial membrane proteins, and mutations in the genes encoding these proteases cause severe incurable neurological diseases. To further explore the biological role of the m-AAA proteases and the pathological consequences of their deficiency, we used a genetic approach in the fruit fly Drosophila melanogaster to inactivate the ATPase family gene 3-like 2 (AFG3L2) gene, which encodes a critical component of the m-AAA proteases. We found that null alleles of Drosophila AFG3L2 die early in development, but partial inactivation of AFG3L2 using RNAi allowed survival to the late pupal and adult stages of development. Flies with partial inactivation of AFG3L2 exhibited behavioral defects, neurodegeneration, accumulation of unfolded mitochondrial proteins, and diminished respiratory chain (RC) activity. Further work revealed that the reduced RC activity was primarily a consequence of severely diminished mitochondrial transcription and translation. These defects were accompanied by activation of the mitochondrial unfolded protein response (mito-UPR) and autophagy. Overexpression of mito-UPR components partially rescued the AFG3L2-deficient phenotypes, indicating that protein aggregation partly accounts for the defects of AFG3L2-deficient animals. Our work suggests that strategies designed to activate mitochondrial stress pathways and mitochondrial gene expression could be therapeutic in the diseases caused by mutations in AFG3L2