Mutations in the leucine-rich repeat kinase 2 gene (LRRK2) cause late-onset Parkinson's disease (PD) with a clinical appearance indistinguishable from idiopathic PD. Initial studies suggest that LRRK2 mutations are the most common yet identified determinant of PD susceptibility, transmitted in an autosomal-dominant mode of inheritance. Herein, we characterize the LRRK2 gene and transcript in human brain and subclone the predominant ORF. Exogenously expressed LRRK2 protein migrates at Ϸ280 kDa and is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Familial-linked mutations G2019S or R1441C do not have an obvious effect on protein steady-state levels, turnover, or localization. However, in vitro kinase assays using full-length recombinant LRRK2 reveal an increase in activity caused by familial-linked mutations in both autophosphorylation and the phosphorylation of a generic substrate. These results suggest a gain-of-function mechanism for LRRK2-linked disease with a central role for kinase activity in the development of PD.dardarin ͉ parkinsonism ͉ PARK8

Mutations in the the leucine-rich repeat kinase-2 (LRRK2) gene cause autosomal-dominant Parkinson disease and some cases of sporadic Parkinson disease. Here we found that LRRK2 kinase activity was regulated by GTP via the intrinsic GTPase Roc domain, and alterations of LRRK2 protein that reduced kinase activity of mutant LRRK2 correspondingly reduced neuronal toxicity. These data elucidate the pathogenesis of LRRK2-linked Parkinson disease, potentially illuminate mechanisms of sporadic Parkinson disease and suggest therapeutic targets.

It is widely accepted that the familial Parkinson's disease (PD)-linked gene product, parkin, functions as a ubiquitin ligase involved in protein turnover via the ubiquitin-proteasome system. Substrates ubiquitinated by parkin are hence thought to be destined for proteasomal degradation. Because we demonstrated previously that parkin interacts with and ubiquitinates synphilin-1, we initially expected synphilin-1 degradation to be enhanced in the presence of parkin. Contrary to our expectation, we found that synphilin-1 is normally ubiquitinated by parkin in a nonclassical, proteasomal-independent manner that involves lysine 63 (K63)-linked polyubiquitin chain formation. Parkin-mediated degradation of synphilin-1 occurs appreciably only at an unusually high parkin to synphilin-1 expression ratio or when primed for lysine 48 (K48)-linked ubiquitination. In addition we found that parkin-mediated ubiquitination of proteins within Lewy-body-like inclusions formed by the coexpression of synphilin-1, ␣-synuclein, and parkin occurs predominantly via K63 linkages and that the formation of these inclusions is enhanced by K63-linked ubiquitination. Our results suggest that parkin is a dual-function ubiquitin ligase and that K63-linked ubiquitination of synphilin-1 by parkin may be involved in the formation of Lewy body inclusions associated with PD.

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Parkinson's disease (PD) is a neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Alpha-synuclein is a major component of Lewy bodies in sporadic PD, and mutations in alpha-synuclein cause autosomal-dominant hereditary PD. Here, we generated A53T mutant alpha-synuclein-inducible PC12 cell lines using the Tet-off regulatory system. Inducing expression of A53T alpha-synuclein in differentiated PC12 cells decreased proteasome activity, increased the intracellular ROS level and caused up to approximately 40% cell death, which was accompanied by mitochondrial cytochrome C release and elevation of caspase-9 and -3 activities. Cell death was partially blocked by cyclosporine A [an inhibitor of the mitochondrial permeability transition (MPT) process], z-VAD (a pan-caspase inhibitor) and inhibitors of caspase-9 and -3 but not by a caspase-8 inhibitor. Furthermore, induction of A53T alpha-synuclein increased endoplasmic reticulum (ER) stress and elevated caspase-12 activity. RNA interference to knock down caspase-12 levels or salubrinal (an ER stress inhibitor) partially protected against cell death and further reduced A53T toxicity after treatment with z-VAD. Our results indicate that both ER stress and mitochondrial dysfunction contribute to A53T alpha-synuclein-induced cell death. This study sheds light into the pathogenesis of alpha-synuclein cellular toxicity in PD and provides a cell model for screening PD therapeutic agents.

Mutations in the leucine-rich repeat kinase (LRRK2) gene cause lateonset autosomal dominant Parkinson's disease (PD) with pleiomorphic pathology. Previously, we and others found that expression of mutant LRRK2 causes neuronal degeneration in cell culture. Here we used the GAL4/UAS system to generate transgenic Drosophila expressing either wild-type human LRRK2 or LRRK2-G2019S, the most common mutation associated with PD. Expression of either wild-type human LRRK2 or LRRK2-G2019S in the photoreceptor cells caused retinal degeneration. Expression of LRRK2 or LRRK2-G2019S in neurons produced adult-onset selective loss of dopaminergic neurons, locomotor dysfunction, and early mortality. Expression of mutant G2019S-LRRK2 caused a more severe parkinsonism-like phenotype than expression of equivalent levels of wild-type LRRK2. Treatment with L-DOPA improved mutant LRRK2-induced locomotor impairment but did not prevent the loss of tyrosine hydroxylase-positive neurons. To our knowledge, this is the first in vivo''gain-of-function'' model which recapitulates several key features of LRRK2-linked human parkinsonism. These flies may provide a useful model for studying LRRK2-linked pathogenesis and for future therapeutic screens for PD intervention.dopaminergic neuron ͉ Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that ␣-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of ␣-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of ␣-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type ␣-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A ␣-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with ␣-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A ␣-synuclein instead of wild-type ␣-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A ␣-synuclein with synphilin-1 compared with wild-type ␣-synuclein. Expression of S129A ␣-synuclein instead of wild-type ␣-synuclein also decreased the association of synphilin-1 and parkin and subsequently reduced the parkin-mediated ubiquitination of synphilin-1 and the formation of ubiquitinated inclusions. Treatment of SH-SY5Y cells with H 2 O 2 increased ␣-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of ␣-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-␤-D-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of ␣-synuclein at S129 may be important for the formation of inclusions in PD and related ␣ synucleinopathies.

Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinson's disease (PD). Although most cases of PD are sporadic, some are inherited, including those caused by LRRK2 mutations. Because these mutations may be associated with a toxic gain of function, controlling the expression of LRRK2 may decrease its cytotoxicity. Here we show that the carboxyl terminus of HSP70-interacting protein (CHIP) binds, ubiquitinates, and promotes the ubiquitin proteasomal degradation of LRRK2. Overexpression of CHIP protects against and knockdown of CHIP exacerbates toxicity mediated by mutant LRRK2. Moreover, HSP90 forms a complex with LRRK2, and inhibition of HSP90 chaperone activity by 17AAG leads to proteasomal degradation of LRRK2, resulting in increased cell viability. Thus, increasing CHIP E3 ligase activity and blocking HSP90 chaperone activity can prevent the deleterious effects of LRRK2. These findings point to potential treatment options for LRRK2-associated PD.is a progressive neurodegenerative disorder pathologically characterized by loss of dopaminergic neurons from the substantia nigra and the presence of Lewy bodies (1-3). The etiology of PD is incompletely understood but appears to involve both genetic and environmental factors. To date, 5 genes (␣-synuclein, parkin, DJ-1, PINK-1, and LRRK2) are associated with genetic forms of PD that closely resemble idiopathic PD (4-10). Mutation in LRRK2 is the most frequent genetic cause of PD (11). Patients with LRRK2 mutations exhibit clinical and neurochemical phenotypes that are indistinguishable from sporadic PD (9, 10). These patients suffer neuronal loss and gliosis in the substantia nigra and development of Lewy bodies, and also exhibit pleomorphic neuropathology, including ␣-synuclein and tau pathology (9, 12, 13). Thus, LRRK2 is important for the pathogenesis of several major neurodegenerative disorders associated with parkinsonism.LRRK2, a member of the ROCO protein family, contains a guanosine triphosphatase (GTPase), a C-terminal of Ras domain with a kinase effector domain (14), repeat sequences beginning at the N terminus, and a leucine-rich repeat structure near its GTPase domain (15). LRRK2 is localized to membranous structures, where it may be in involved in neuronal polarity (16)(17)(18). Mutations in LRRK2 are frequent in autosomaldominant PD as well as sporadic PD (19-23). PD-associated LRRK2 mutants seem to enhance kinase activity, and mutant LRRK2-mediated neuronal toxicity requires GTP-binding and kinase activity (17,(24)(25)(26).The ubiquitin proteosomal system (UPS) appears to regulate LRRK2 level, with little influence from the autophagic and lysosomal degradation pathways (17). LRRK2 also dimerizes and interacts with HSP90 (18, 27, 28), which is somehow involved in controlling LRRK2 levels. The identity of the E3 ligase and the mechanisms that regulate the stability of LRRK2 via HSP90 are not known. Whether the levels of LRRK2 are linked to toxicity also is unclear.The carboxyl terminus of HSP70-interacting protei...