The PINK1/Parkin pathway: a mitochondrial quality control system?

Significant insight into the mechanisms that contribute to dopaminergic neurodegeneration in Parkinson disease has been gained from the analysis of genes linked to rare heritable forms of parkinsonism such as PINK1 and parkin, loss-of-function mutations of which cause autosomal recessive parkinsonism. PINK1 encodes a mitochondrially targeted Ser/Thr kinase and parkin encodes a ubiquitin-protein ligase. Functional studies of PINK1 and Parkin in animal and cellular model systems have shown that both proteins play important roles in maintaining mitochondrial integrity. Genetic studies of PINK1 and Parkin orthologs in flies have shown that PINK1 acts upstream from Parkin in a common pathway that appears to regulate mitochondrial morphology. Mitochondrial morphology is regulated by mitochondrial fission and fusion-promoting proteins, and is important in a variety of contexts, including mitochondrial trafficking and mitochondrial quality control. In particular, mitochondrial fission appears to promote the segregation of terminally dysfunctional mitochondria for degradation in the lysosome through a process termed mitophagy. Recent work has shown that Parkin promotes the degradation of dysfunctional mitochondria in vertebrate cell culture. Here we postulate a model whereby the PINK1/Parkin pathway regulates mitochondrial dynamics in an effort to promote the turnover of damaged mitochondria

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

Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy

Parkin, an E3 ubiquitin ligase implicated in Parkinson's disease, promotes degradation of dysfunctional mitochondria by autophagy. Using proteomic and cellular approaches, we show that upon translocation to mitochondria, Parkin activates the ubiquitin–proteasome system (UPS) for widespread degradation of outer membrane proteins. This is evidenced by an increase in K48-linked polyubiquitin on mitochondria, recruitment of the 26S proteasome and rapid degradation of multiple outer membrane proteins. The degradation of proteins by the UPS occurs independently of the autophagy pathway, and inhibition of the 26S proteasome completely abrogates Parkin-mediated mitophagy in HeLa, SH-SY5Y and mouse cells. Although the mitofusins Mfn1 and Mfn2 are rapid degradation targets of Parkin, we find that degradation of additional targets is essential for mitophagy. These results indicate that remodeling of the mitochondrial outer membrane proteome is important for mitophagy, and reveal a causal link between the UPS and autophagy, the major pathways for degradation of intracellular substrates

Characterization of the Adaptor-related Protein Complex, AP-3

We have recently shown that two proteins related to two of the adaptor subunits of clathrincoated vesicles, p47 (μ3) and β-NAP (β3B), are part of an adaptor-like complex not associated with clathrin (Simpson, F., N.A. Bright, M.A. West, L.S. Newman, R.B. Darnell, and M.S. Robinson, 1996. J. Cell Biol. 133:749–760). In the present study we have searched the EST database and have identified, cloned, and sequenced a ubiquitously expressed homologue of β-NAP, β3A, as well as homologues of the α/γ and σ adaptor subunits, δ and σ3, which are also ubiquitously expressed. Antibodies raised against recombinant δ and σ3 show that they are the other two subunits of the adaptor-like complex. We are calling this complex AP-3, a name that has also been used for the neuronalspecific phosphoprotein AP180, but we feel that it is a more appropriate designation for an adaptor-related heterotetramer. Immunofluorescence using anti-δ antibodies reveals that the AP-3 complex is associated with the Golgi region of the cell as well as with more peripheral structures. These peripheral structures show only limited colocalization with endosomal markers and may correspond to a postTGN biosynthetic compartment. The δ subunit is closely related to the protein product of the Drosophila garnet gene, which when mutated results in reduced pigmentation of the eyes and other tissues. Because pigment granules are believed to be similar to lysosomes, this suggests either that the AP-3 complex may be directly involved in trafficking to lysosomes or alternatively that it may be involved in another pathway, but that missorting in that pathway may indirectly lead to defects in pigment granules

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 β Subunit Increases the Ca2+ Sensitivity of Large Conductance Ca2+-activated Potassium Channels by Retaining the Gating in the Bursting States

Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+ (BK) channels greatly increases the apparent Ca2+ sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+ in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+ binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+ sensitivity of the channel. In the presence of the β subunit, each burst of openings is greatly amplified in duration through increases in both the numbers of openings per burst and in the mean open times. Native BK channels from cultured rat skeletal muscle were found to have bursting kinetics similar to channels expressed from alpha subunits alone

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

Auditory Function in the Tc1 Mouse Model of Down Syndrome Suggests a Limited Region of Human Chromosome 21 Involved in Otitis Media

Down syndrome is one of the most common congenital disorders leading to a wide range of health problems in humans, including frequent otitis media. The Tc1 mouse carries a significant part of human chromosome 21 (Hsa21) in addition to the full set of mouse chromosomes and shares many phenotypes observed in humans affected by Down syndrome with trisomy of chromosome 21. However, it is unknown whether Tc1 mice exhibit a hearing phenotype and might thus represent a good model for understanding the hearing loss that is common in Down syndrome. In this study we carried out a structural and functional assessment of hearing in Tc1 mice. Auditory brainstem response (ABR) measurements in Tc1 mice showed normal thresholds compared to littermate controls and ABR waveform latencies and amplitudes were equivalent to controls. The gross anatomy of the middle and inner ears was also similar between Tc1 and control mice. The physiological properties of cochlear sensory receptors (inner and outer hair cells: IHCs and OHCs) were investigated using single-cell patch clamp recordings from the acutely dissected cochleae. Adult Tc1 IHCs exhibited normal resting membrane potentials and expressed all K+ currents characteristic of control hair cells. However, the size of the large conductance (BK) Ca2+ activated K+ current (IK,f), which enables rapid voltage responses essential for accurate sound encoding, was increased in Tc1 IHCs. All physiological properties investigated in OHCs were indistinguishable between the two genotypes. The normal functional hearing and the gross structural anatomy of the middle and inner ears in the Tc1 mouse contrast to that observed in the Ts65Dn model of Down syndrome which shows otitis media. Genes that are trisomic in Ts65Dn but disomic in Tc1 may predispose to otitis media when an additional copy is active