Parkinson's disease is a common neurodegenerative disease with complex clinical features. Autosomal recessive juvenile parkinsonism (AR-JP) maps to the long arm of chromosome 6 (6q25.2-q27) and is linked strongly to the markers D6S305 and D6S253; the former is deleted in one Japanese AR-JP patient. By positional cloning within this microdeletion, we have now isolated a complementary DNA done of 2,960 base pairs with a 1,395-base-pair open reading frame, encoding a protein of 465 amino acids with moderate similarity to ubiquitin at the amino terminus and a RING-finger motif at the carboxy terminus. The gene spans more than 500 kilobases and has 12 exons, five of which (exons 3-7) are deleted in the patient. Four other AR-JP patients from three unrelated families have a deletion affecting exon 4 alone. A 4.5-kilobase transcript that is expressed in many human tissues but is abundant in the brain, including the substantia nigra, is shorter in brain tissue from one of the groups of exon-4-deleted patients. Mutations in the newly identified gene appear to be responsible for the pathogenesis of AR-JP, and we have therefore named the protein product 'Parkin'.

Mitochondria/cytosol fractionation in vitro. Mitochondria and cytosol were fractionated using the Mitochondria/Cytosol Fractionation Kit according to the manufacturer's protocol (Enzo Life Sciences).Cytotoxicity assays. Cytotoxicity was assessed by measuring the release of LDH into the media (LDH-Cytotoxicity Colorimetric Assay Kit II; BioVision) according to the manufacturer's protocol.Flow cytometry. To discriminate live and dead cells, cells were simultaneously stained with green fluorescent calcein-AM to indicate intracellular esterase activity and red fluorescent ethidium homodimer-1 to indicate loss of plasma membrane integrity using the LIVE/DEAD Viability/Cytotoxicity Kit (Molecular Probes). To assess the functional mitochondrial pool, cells were stained for 20 minutes at 37°C with 100 nM TMRE (Abcam), followed by CSE treatment. mtROS was measured in cells by MitoSOX (Invitrogen) staining (2.5 μM for 10 minutes at 37°C). Data were acquired with aIn vivo CS and chemical treatments. Age-matched mice (6-12 weeks old) were exposed to RA or CS in whole-body exposure chambers as described (5) Human lung bronchial epithelial Beas-2B cells were purchased from ATCC and maintained in DMEM containing 10% FBS and gentamicin (100 μg/ml). The primary alveolar epithelial cells of mouse lung were obtained as previously described and used for experiments before passage (70, 71). CSE was prepared and added to culture media as previously described (5, 6). , and Drp1 in human lung homogenate samples from control subjects and COPD patients. β-Actin served as the standard. PINK1, RIP3, and Drp1 expression was assessed by densitometry of immunoblots. Band intensities were normalized to β-actin. n = 9 samples/group. Representative immunohistochemical study (original magnification, ×200) for PINK1 (B) or RIP3 (C) in human lung sections from never-smokers (n = 3 patients, 5 images/patient) or COPD patients (n = 6 patients, 5 images/patient). Scale bar: 100 μm. Outlined areas are shown enlarged at right (scale bar: 20 μm). (D) Immunofluorescence staining (original magnification, ×40) for PINK1 (green), RIP3 (red), and nuclear (blue) in human lung tissue from never-smokers (n = 2 patients, 3 images/patient) and COPD patients (n = 2 patients, 3 images/patient). Scale bar: 50 μm. Yellow-outlined areas are shown enlarged in bottom panels (scale bar: 10 μm). Data represent the mean ± SEM (A). **P < 0.01 by unpaired, 2-tailed Student's t test (A). The Journal of Clinical Investigation R e s e a R c h a R t i c l e4 0 0 1

Parkinson's disease (PD) is a common neurodegenerative disorder thought to be associated with mitochondrial dysfunction. Loss of function mutations in the putative mitochondrial protein PINK1 (PTEN-induced kinase 1) have been linked to familial forms of PD, but the relation of PINK1 to mammalian mitochondrial function remains unclear. Here, we report that germline deletion of the PINK1 gene in mice significantly impairs mitochondrial functions. Quantitative electron microscopic studies of the striatum in PINK1 ؊/؊ mice at 3-4 and 24 months revealed no gross changes in the ultrastructure or the total number of mitochondria, although the number of larger mitochondria is selectively increased. Functional assays showed impaired mitochondrial respiration in the striatum but not in the cerebral cortex at 3-4 months of age, suggesting specificity of this defect for dopaminergic circuitry. Aconitase activity associated with the Krebs cycle is also reduced in the striatum of PINK1 ؊/؊ mice. Interestingly, mitochondrial respiration activities in the cerebral cortex are decreased in PINK1 ؊/؊ mice at 2 years compared with control mice, indicating that aging can exacerbate mitochondrial dysfunction in these mice. Furthermore, mitochondrial respiration defects can be induced in the cerebral cortex of PINK1 ؊/؊ mice by cellular stress, such as exposure to H2O2 or mild heat shock. Together, our findings demonstrate that mammalian PINK1 is important for mitochondrial function and provides critical protection against both intrinsic and environmental stress, suggesting a pathogenic mechanism by which loss of PINK1 may lead to nigrostriatal degeneration in PD.neurodegeneration ͉ PARK6 ͉ genetic Parkinson's disease ͉ knockout ͉ parkinsonism

The manifestations of Parkinson's disease are caused by reduced dopaminergic innervation of the striatum. Loss-of-function mutations in the DJ-1 gene cause early-onset familial parkinsonism. To investigate a possible role for DJ-1 in the dopaminergic system, we generated a mouse model bearing a germline disruption of DJ-1. Although DJ-1(-/-) mice had normal numbers of dopaminergic neurons in the substantia nigra, evoked dopamine overflow in the striatum was markedly reduced, primarily as a result of increased reuptake. Nigral neurons lacking DJ-1 were less sensitive to the inhibitory effects of D2 autoreceptor stimulation. Corticostriatal long-term potentiation was normal in medium spiny neurons of DJ-1(-/-) mice, but long-term depression (LTD) was absent. The LTD deficit was reversed by treatment with D2 but not D1 receptor agonists. Furthermore, DJ-1(-/-) mice displayed hypoactivity in the open field. Collectively, our findings suggest an essential role for DJ-1 in dopaminergic physiology and D2 receptor-mediated functions.

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PTEN-induced putative kinase 1 (Pink1) is a recently identified gene linked to a recessive form of familial Parkinson's disease (PD). The kinase contains a mitochondrial localization sequence and is reported to reside, at least in part, in mitochondria. However, neither the manner by which the loss of Pink1 contributes to dopamine neuron loss nor its impact on mitochondrial function and relevance to death is clear. Here, we report that depletion of Pink1 by RNAi increased neuronal toxicity induced by MPP ؉ . Moreover, wild-type Pink1, but not the G309D mutant linked to familial PD or an engineered kinase-dead mutant K219M, protects neurons against MPTP both in vitro and in vivo. Intriguingly, a mutant that contains a deletion of the putative mitochondrial-targeting motif was targeted to the cytoplasm but still provided protection against 1-methyl-4-phenylpyridine (MPP ؉ )/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced toxicity. In addition, we also show that endogenous Pink1 is localized to cytosolic as well as mitochondrial fractions. Thus, our findings indicate that Pink1 plays a functional role in the survival of neurons and that cytoplasmic targets, in addition to its other actions in the mitochondria, may be important for this protective effect.Parkinson's disease ͉ neurodegeneration ͉ neuroprotection P arkinson's disease (PD) is a movement disorder with progressive loss of dopamine neurons in the substantia nigra pars compacta (SNc). The molecular events responsible for the loss of dopaminergic neurons in PD remain poorly understood. One common feature of PD is the dysfunction of mitochondria, which results in reduced complex I activity in the SNc (1, 2). Experimentally, inhibitors of complex I of the mitochondrial respiratory chain can recapitulate this selective dopaminergic neuronal loss and consequent behavioral deficits (1, 3-5). These observations support the hypothesis that nigral dopamine neurons are highly vulnerable to stress arising from mitochondrial dysfunction.Recently, several genes have been identified that cause PD (6). These genes include ␣-synuclein, parkin, PTEN-induced putative kinase 1 (Pink1), DJ-1, and LRRK2. Although several of the genes have been partially localized to the mitochondria, Pink1 is the only gene with a putative mitochondrial targeting motif. Several studies have shown that the mitochondrial targeting motif at the Nterminal region of Pink1 is sufficient to direct proteins to the mitochondria (7). Pink1 was initially identified as a PTEN-inducible transcript and contains a serine/threonine kinase domain (8). Interestingly, the G309D mutation of the kinase domain leads to a mild decrease in mitochondrial complex I activity, elevation of superoxide radicals, and increased lipid peroxidation (9). Studies with Drosophila lacking Pink1 showed mitochondrial pathology with the similar phenotype as seen in Parkin knockout flies (10, 11). The above observations suggest that mitochondrial dysfunction may be linked to the Pink1 PD phenotype.The mechanisms by which Pi...

Autosomal recessive juvenile parkinsonism (AR‐JP) is a distinct clinical entity characterized by a selective degeneration of nigral neurons. Recently, the parkin gene responsible for AR‐JP has been identified. Now, we report the subcellular localization of Parkin protein in patients with AR‐JP or Parkinson's disease (PD) and in controls by immunoblotting and immunohistochemistry using antibodies raised against the Parkin molecule. Parkin protein was absent in all regions of the brains of patients with AR‐JP. Parkin protein was not decreased in the brains of sporadic PD patients. Immunoreactivity was detected in a few Lewy bodies. Parkin protein was located in both the Golgi complex and cytosol. Ann Neurol 1999;45:668–672

Recessively inherited loss‐of‐function mutations in the parkin, DJ‐1, or PINK1 gene are linked to familial cases of early‐onset Parkinson’s diseases (PD), and heterozygous mutations are associated with increased incidence of late‐onset PD. We previously reported that single knockout mice lacking Parkin, DJ‐1, or PINK1 exhibited no nigral degeneration, even though evoked dopamine release from nigrostriatal terminals was reduced and striatal synaptic plasticity was impaired. In this study, we tested whether inactivation of all three recessive PD genes, each of which was required for nigral neuron survival in the aging human brain, resulted in nigral degeneration during the lifespan of mice. Surprisingly, we found that triple knockout mice lacking Parkin, DJ‐1, and PINK1 have normal morphology and numbers of dopaminergic and noradrenergic neurons in the substantia nigra and locus coeruleus, respectively, at the ages of 3, 16, and 24 months. Interestingly, levels of striatal dopamine in triple knockout mice were normal at 16 months of age but increased at 24 months. These results demonstrate that inactivation of all three recessive PD genes is insufficient to cause significant nigral degeneration within the lifespan of mice, suggesting that these genes may be protective rather than essential for the survival of dopaminergic neurons during the aging process. These findings also support the notion that mammalian Parkin and PINK1 may function in the same genetic pathway as in Drosophila.