Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinson's disease (FPD). PINK1 has previously been implicated in mitochondrial fission/fusion dynamics, quality control, and electron transport chain function. However, it is not clear how these processes are interconnected and whether they are sufficient to explain all aspects of PINK1 pathogenesis. Here we show that PINK1 also controls mitochondrial motility. In Drosophila, downregulation of dMiro or other components of the mitochondrial transport machinery rescued dPINK1 mutant phenotypes in the muscle and dopaminergic (DA) neurons, whereas dMiro overexpression alone caused DA neuron loss. dMiro protein level was increased in dPINK1 mutant but decreased in dPINK1 or dParkin overexpression conditions. In Drosophila larval motor neurons, overexpression of dPINK1 inhibited axonal mitochondria transport in both anterograde and retrograde directions, whereas dPINK1 knockdown promoted anterograde transport. In HeLa cells, overexpressed hPINK1 worked together with hParkin, another FPD gene, to regulate the ubiquitination and degradation of hMiro1 and hMiro2, apparently in a Ser-156 phosphorylation-independent manner. Also in HeLa cells, loss of hMiro promoted the perinuclear clustering of mitochondria and facilitated autophagy of damaged mitochondria, effects previously associated with activation of the PINK1/Parkin pathway. These newly identified functions of PINK1/Parkin and Miro in mitochondrial transport and mitophagy contribute to our understanding of the complex interplays in mitochondrial quality control that are critically involved in PD pathogenesis, and they may explain the peripheral neuropathy symptoms seen in some PD patients carrying particular PINK1 or Parkin mutations. Moreover, the different effects of loss of PINK1 function on Miro protein level in Drosophila and mouse cells may offer one explanation of the distinct phenotypic manifestations of PINK1 mutants in these two species.

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinson's disease (PD). Another early onset PD gene product, Parkin, has been suggested to function downstream of the PINK1 signalling pathway based on genetic studies in Drosophila. PINK1 is a serine/threonine kinase with a predicted mitochondrial target sequence and a probable transmembrane domain at the N-terminus, while Parkin is a RING-finger protein with ubiquitin-ligase (E3) activity. However, how PINK1 and Parkin regulate mitochondrial activity is largely unknown. To explore the molecular mechanism underlying the interaction between PINK1 and Parkin, we biochemically purified PINK1-binding proteins from human cultured cells and screened the genes encoding these binding proteins using Drosophila PINK1 (dPINK1) models to isolate a molecule(s) involved in the PINK1 pathology. Here we report that a PINK1-binding mitochondrial protein, PGAM5, modulates the PINK1 pathway. Loss of Drosophila PGAM5 (dPGAM5) can suppress the muscle degeneration, motor defects, and shorter lifespan that result from dPINK1 inactivation and that can be attributed to mitochondrial degeneration. However, dPGAM5 inactivation fails to modulate the phenotypes of parkin mutant flies. Conversely, ectopic expression of dPGAM5 exacerbated the dPINK1 and Drosophila parkin (dParkin) phenotypes. These results suggest that PGAM5 negatively regulates the PINK1 pathway related to maintenance of the mitochondria and, furthermore, that PGAM5 acts between PINK1 and Parkin, or functions independently of Parkin downstream of PINK1.

Parkin, a ubiquitin E3 ligase of the ring between ring fingers family, has been implicated in mitochondrial quality control. A series of recent reports have suggested that the recruitment of parkin is regulated by phosphorylation. However, the molecular mechanism that activates parkin to induce mitochondrial degradation is not well understood. Here, and in contrast to previous reports that S-nitrosylation of parkin is exclusively inhibitory, we identify a previously unrecognized site of S-nitrosylation in parkin (Cys323) that induces mitochondrial degradation. We demonstrate that endogenous S-nitrosylation of parkin is in fact responsible for activation of its E3 ligase activity to induce aggregation and degradation. We further demonstrate that mitochondrial uncoupling agents result in denitrosylation of parkin, and that prevention of denitrosylation restores mitochondrial degradation. Our data indicates that NO both positive effects on mitochondrial quality control, and suggest that targeted S-nitrosylation could provide a novel therapeutic strategy against Parkinson's disease.

Eukaryotes employ elaborate mitochondrial quality control (MQC) to maintain the function of the power-generating organelle. Parkinson's disease-associated PINK1 and Parkin actively participate in MQC. However, the signaling events involved are largely unknown. Here we show that mechanistic target of rapamycin 2 (mTORC2) and Tricornered (Trc) kinases act downstream from PINK1 to regulate MQC. Trc is phosphorylated in mTORC2-dependent and mTORC2-independent manners and is specifically localized to mitochondria in response to PINK1, which regulates mTORC2 through mitochondrial complex-I activity. Genetically, mTORC2 and Trc act upstream of Parkin. Thus, multiplex kinase signaling is acting between PINK1 and Parkin to regulate MQC, a process highly conserved in mammals.Supplemental material is available for this article. We continued using the powerful tools available in Drosophila to dissect the genetic program underlying PINK1/Parkin-directed MQC. Here we identify mechanistic target of rapamycin 2 (mTORC2) and tricornered (Trc) kinase signaling as intermediate steps between PINK1 and Parkin in the MQC process in Drosophila and further verify the findings in mammalian cells. Our results reveal a previously unanticipated complexity of signaling events involved in PINK1/Parkin-directed MQC and tissue maintenance and shed new light on how mTORC2 and Trc, two important kinases critically involved in cellular morphogenesis, development, growth, and disease, are regulated in an in vivo setting. Results and DiscussionTo elucidate the signaling events acting downstream from PINK1, we performed genetic screens as described before (Liu and Lu 2010;Liu et al. 2012) to identify genes that can modify PINK1 loss-of-function (LOF)-induced wing posture and flight ability defects. This led to the identification of mTORC2 components as strong modifiers of PINK1. While overexpression of the mTORC2 components Rictor and Sin1 (Hietakangas and Cohen 2007), either individually or in combination, had no obvious effects on their own (Supplemental Fig. S1), they effectively suppressed dPINK1 LOF phenotypes with respect to the integrity and function of flight muscle, as measured by thoracic ATP level and wing posture (Fig. 1A,B;Supplemental Fig. S1D) and the loss of dopaminergic neurons (DNs) in the PPL1 cluster (Fig. 1E). Conversely, rictor or sin1 mutations significantly enhanced dPINK1 LOF phenotypes ( Fig. 1A-E; Supplemental Fig. S1D), although the rictor or sin1 mutant alone did not show obvious phenotypes in these assays (Supplemental Fig. S1A-C). The effects of mTORC2 components on tissue integrity correlated well with the effects on mitochondrial morphology, with mTORC2 gain of function (GOF) rescuing the mitochondrial aggregation phenotype caused by dPINK1 LOF, whereas mTORC2 LOF had opposite effects (Fig. 1C,D).We next explored the molecular mechanisms underlying the strong genetic interaction between PINK1 and mTORC2. Using phosphorylation of Drosophila AKT at S505 as readout of mTORC2 activity (Sarbassov et al.