Tomoyo Sawada

Parkinson's Disease-Associated Kinase PINK1 Regulates Miro Protein Level and Axonal Transport of Mitochondria

Mutations in Pten-induced kinase 1 (PINK1) are linked to early-onset familial Parkinsons 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.

The Loss of PGAM5 Suppresses the Mitochondrial Degeneration Caused by Inactivation of PINK1 in Drosophila

PTEN-induced kinase 1 (PINK1), which is required for mitochondrial homeostasis, is a gene product responsible for early-onset Parkinsons 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.

S-nitrosylation regulates mitochondrial quality control via activation of parkin

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 Parkinsons disease.

Tricornered/NDR kinase signaling mediates PINK1-directed mitochondrial quality control and tissue maintenance

Eukaryotes employ elaborate mitochondrial quality control (MQC) to maintain the function of the power-generating organelle. Parkinsons 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.

Parkin interacts with Klokin1 for mitochondrial import and maintenance of membrane potential

Parkin is a multifunctional protein, including maintaining mitochondrial homeostasis. Recent evidence suggests that Parkin is recruited from the cytoplasm to damaged mitochondria with low membrane potential. We found that intracellular localization of Parkin changed with cellular growth phase. Parkin was preferentially localized in the mitochondria of cultured cells. The mitochondria with large amounts of Parkin showed preserved membrane potentials even during treatment with carbonyl cyanide m-chlorophenylhydrazone. Here we report a novel protein named Klokin 1 that transports Parkin to the mitochondria. Klokin 1 was localized to the mitochondria and enhanced mitochondrial expression of Parkin. Klokin 1 enhanced cell viability in Parkin-silenced cells. Klokin 1 expression was enhanced in the brains of Parkin-deficient mice but not in an autopsied PARK2 brain. Our findings indicate that mitochondrial Parkin prevents mitochondrial depolarization and that Klokin 1 may compensate for Parkin deficiency.

The Nitric Oxide-Cyclic GMP Pathway Regulates FoxO and Alters Dopaminergic Neuron Survival in Drosophila

Activation of the forkhead box transcription factor FoxO is suggested to be involved in dopaminergic (DA) neurodegeneration in a Drosophila model of Parkinsons disease (PD), in which a PD gene product LRRK2 activates FoxO through phosphorylation. In the current study that combines Drosophila genetics and biochemical analysis, we show that cyclic guanosine monophosphate (cGMP)-dependent kinase II (cGKII) also phosphorylates FoxO at the same residue as LRRK2, and Drosophila orthologues of cGKII and LRRK2, DG2/For and dLRRK, respectively, enhance the neurotoxic activity of FoxO in an additive manner. Biochemical assays using mammalian cGKII and FoxO1 reveal that cGKII enhances the transcriptional activity of FoxO1 through phosphorylation of the FoxO1 S319 site in the same manner as LRRK2. A Drosophila FoxO mutant resistant to phosphorylation by DG2 and dLRRK (dFoxO S259A corresponding to human FoxO1 S319A) suppressed the neurotoxicity and improved motor dysfunction caused by co-expression of FoxO and DG2. Nitric oxide synthase (NOS) and soluble guanylyl cyclase (sGC) also increased FoxOs activity, whereas the administration of a NOS inhibitor L-NAME suppressed the loss of DA neurons in aged flies co-expressing FoxO and DG2. These results strongly suggest that the NO-FoxO axis contributes to DA neurodegeneration in LRRK2-linked PD.

PINK1 promotes the clearance of unfolded Pael-R in mammalian cell cultures

Activated microglia play important roles in the inflammatory process in neurodegenerative diseases. We demonstrated the effects of neonatal microglia activated with lipopolysaccharide (LPS) on the nigro-striatal dopamine neurons in mice treated with MPTP, the decreased cell viability of dopamine neurons in mice with MPTP is recovered by LPS administration. On the other hand, we examined the phenotype of activated neonatal microglia under severe damage produced by injection of ethanol into the striatum. Administration of LPS increased the number of activated microglia around the necrosis by ethanol injection, and the volumes of necrotic and degenerative neurons in the striatum were further increased. Our results indicate the neuroprotective potential in the MPTP-treated mice and the neurotoxic potential in the ethanol-injected mice. Activated neonatal microglia may have a neurotoxic effect on neurons depending on the severity of the injury.