Mutations in Pink1, a gene encoding a Ser͞Thr kinase with a mitochondrial-targeting signal, are associated with Parkinson's disease (PD), the most common movement disorder characterized by selective loss of dopaminergic neurons. The mechanism by which loss of Pink1 leads to neurodegeneration is not understood. Here we show that inhibition of Drosophila Pink1 (dPink1) function results in energy depletion, shortened lifespan, and degeneration of select indirect flight muscles and dopaminergic neurons. The muscle pathology was preceded by mitochondrial enlargement and disintegration. These phenotypes could be rescued by the wild type but not the pathogenic C-terminal deleted form of human Pink1 (hPink1). The muscle and dopaminergic phenotypes associated with dPink1 inactivation show similarity to that seen in parkin mutant flies and could be suppressed by the overexpression of Parkin but not DJ-1. Consistent with the genetic rescue results, we find that, in dPink1 RNA interference (RNAi) animals, the level of Parkin protein is significantly reduced. Together, these results implicate Pink1 and Parkin in a common pathway that regulates mitochondrial physiology and cell survival in Drosophila.mitochondria ͉ Parkinson's disease ͉ Pten-induced kinase 1 ͉ indirect flight muscle P arkinson's disease (PD) is the most common movement disorder characterized pathologically by the deficiency of brain dopamine content and the selective degeneration of dopaminergic neurons in the substantia nigra. The most common forms of PD are sporadic with no known cause. Nevertheless, postmortem studies have identified common features associated with sporadic PD, such as mitochondrial complex I dysfunction, oxidative stress, and aggregation of abnormal proteins (1, 2).Although initial studies on the etiology of PD have focused on environmental factors, recent genetic studies have firmly established the contribution of inheritable factors in PD pathogenesis (2, 3). At least ten distinct loci have been associated with rare familial forms of PD (FPD). It is anticipated that understanding the molecular lesions associated with these FPD genes will shed light on the pathogenesis of the more common forms of the disease. Dominant mutations in ␣-Synuclein (␣-Syn) and LRRK2͞dardarin and recessive mutations in parkin, DJ-1, and Pink1 have been associated with FPD (4-10). Of these five genes, ␣-Syn, parkin, and DJ-1 have been most intensively studied. Studies using in vivo animal models and in vitro cell culture have linked mutations of these genes to impairments of mitochondrial structure and function and oxidative stress response, reinforcing the general involvement of mitochondrial dysfunction and oxidative stress in PD pathogenesis (11-21). Consistent with this notion, these proteins have been shown to be present in mitochondria or interact with mitochondrial proteins (8,(22)(23)(24), suggesting that they may directly regulate mitochondria function.A further link between mitochondria and PD was supported by the fact that Pink1 encodes a predicted Se...

Dominant mutations in leucine-rich repeat kinase 2 (LRRK2) are the most frequent molecular lesions so far found in Parkinson's disease (PD), an age-dependent neurodegenerative disorder affecting dopaminergic (DA) neuron. The molecular mechanisms by which mutations in LRRK2 cause DA degeneration in PD are not understood. Here, we show that both human LRRK2 and the Drosophila orthologue of LRRK2 phosphorylate eukaryotic initiation factor 4E (eIF4E)-binding protein (4E-BP), a negative regulator of eIF4E-mediated protein translation and a key mediator of various stress responses. Although modulation of the eIF4E/4E-BP pathway by LRRK2 stimulates eIF4E-mediated protein translation both in vivo and in vitro, it attenuates resistance to oxidative stress and survival of DA neuron in Drosophila. Our results suggest that chronic inactivation of 4E-BP by LRRK2 with pathogenic mutations deregulates protein translation, eventually resulting in age-dependent loss of DA neurons.

Gain-of-function mutations in leucine-rich repeat kinase 2 (LRRK2) cause familial as well as sporadic Parkinson's disease (PD) characterized by age-dependent dopaminergic neuron (DN) degeneration1 , 2. The molecular mechanism of LRRK2 action is not known. Here we show that LRRK2 interacts with the microRNA (miRNA) pathway to regulate protein synthesis. Drosophila e2f1 and dp mRNAs are translationally repressed by let-7 and miR-184*, respectively. Pathogenic LRRK2 antagonizes these miRNAs, leading to overproduction of E2F1/DP previously implicated in cell cycle and survival control 3 , and shown here to be critical for LRRK2 pathogenesis. Genetic deletion of let-7, antagomir-mediated blockage of let-7 and miR-184* action, transgenic expression of dp target protector, or replacing endogenous dp with a dp transgene non-responsive to let-7 all had similar toxic effects as pathogenic LRRK2. Conversely, increasing let-7 or miR-184* level attenuates pathogenic LRRK2 effects. LRRK2 associates with Drosophila Argonaute-1 (dAgo1) or human Argonaute-2 (hAgo2) of the RNA-induced silencing complex (RISC). In aged fly brain, dAgo1 protein level is negatively regulated by LRRK2. Further, pathogenic LRRK2 promotes the association of phospho-4E-BP1 with hAgo2. Our results implicate deregulated synthesis of E2F1/DP caused by miRNA pathway impairment as a key event in LRRK2 pathogenesis and suggest novel miRNA-based therapeutic strategies.Analyses of Dicer knockout mice have implicated the miRNA pathway in maintaining postmitotic neurons 4,5 . To test whether LRRK2 might affect miRNA function, we generated an in vivo EGFP reporter with let-7-binding sites in the 3′UTR (EGFP-let-7-3′UTR). Loss of one copy of Drosophila dicer-1 or argonaute 1 (ago1) genes of the miRNA pathway enhanced reporter expression (Fig.1a), suggesting that this reporter faithfully monitors endogenous miRNA activity. Da-Gal4-directed ubiquitous or TH-Gal4-directed DN-specific co-expression of pathogenic dLRRK(I1915T) or hLRRK2(G2019S) all stimulated reporter expression, more so than WT proteins (Fig.1a, 1b). This effect correlated with differential toxicity 6 , rather than expression levels of the proteins, since the pathogenic proteins were actually expressed at lower levels (Fig.2a). dLRRK RNAi led to decreased reporter expression, indicating that endogenous dLRRK also impacts miRNA function (Fig.1a). The kinase-dead dLRRK(3KD) did not affect reporter expression (Fig.1a , s1a). This result, together with the observation that I1915T and G2019S mutations augmented kinase activity 7,8 , suggests that the effect of LRRK2 is kinase-dependent. gfp mRNA levels were not affected (Fig.s1b,c). Moreover, the expression of an EGFP transgene without let-7-

Parkinson's disease ͉ PI3K͞PTEN͞Akt signaling ͉ reactive oxygen species P arkinson's disease (PD) is the most common movement disorder and the second most common neurodegenerative disease. The movement abnormality in PD arises from deficiency of brain dopamine (DA) contents and the degeneration of dopaminergic neurons in the substantia nigra. The most common forms of PD are sporadic with no known cause. Nevertheless, postmortem studies have identified common features associated with sporadic PD, including defects in mitochondrial complex I function, oxidative damage, and abnormal protein aggregation (1).The contribution of genetic factors in the pathogenesis of PD, although initially controversial, has been firmly established by recent human genetic studies. At least 10 distinct loci (PARK1 to -11) have been linked to rare familial forms of PD (2). It is anticipated that understanding the molecular lesions associated with these familial PD (FPD) genes will shed light on the pathogenesis of the sporadic forms of the disease. To date, five unequivocal FPD genes have been molecularly cloned. These include ␣-Synuclein (␣-Syn), Parkin, DJ-1, PINK-1, and dardarin. Biochemical and biophysical studies of ␣-Syn and Parkin have primarily linked dysfunction of these genes to aberrant protein folding and ubiquitin-proteasome dysfunction. Intriguingly, in vivo genetic and in vitro cell culture studies have revealed their connection to mitochondrial dysfunction and oxidative stress, reinforcing the involvement of these processes in PD pathogenesis in general (3).DJ-1 encodes a conserved protein belonging to the ThiJ͞PfpI͞ DJ-1 superfamily. The exact molecular function of DJ-1 is still unclear. Human DJ-1 was initially discovered as a candidate oncoprotein that could transform cells in cooperation with activated ras (4), and it was later found as a component of an RNA-binding protein complex and was associated with male infertility (4-6). Under oxidative stress conditions, DJ-1 was modified by oxidation, and the modified form associated with mitochondria in cultured cells (7-10). Knocking down DJ-1 expression with small interfering RNA (siRNA) resulted in susceptibility to oxidative stress, endoplasmic reticulum stress, and proteasome inhibition (11). Recent analyses of DJ-1 knockout mice have shed light on the physiological function of DJ-1 in mammals. DJ-1-deficient mice were found to have nigrostriatal dopaminergic dysfunction, motor deficits, and hypersensitivity to the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyrindine (MPTP) and oxidative stress stimuli (12)(13)(14). In mammalian cells, DJ-1 was found to regulate the phosphorylation status of protein kinase B (PKB)͞Akt through the tumor suppressor PTEN (15). The relevance of this novel finding of DJ-1 function to PD pathogenesis remains to be explored.As an alternative approach to understanding the role of DJ-1 dysfunction in PD pathogenesis, we have used Drosophila as a model system. We inhibited the function of a Drosophila DJ-1 homologue (DJ-1A) by transgenic RN...