Wanli W. Smith

Kinase activity of mutant LRRK2 mediates neuronal toxicity

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.

Parkin Mediates Nonclassical, Proteasomal-Independent Ubiquitination of Synphilin-1: Implications for Lewy Body Formation

It is widely accepted that the familial Parkinsons 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, α-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.

A Drosophila model for LRRK2-linked parkinsonism

Mutations in the leucine-rich repeat kinase (LRRK2) gene cause late-onset autosomal dominant Parkinsons 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.

α-Synuclein Phosphorylation Enhances Eosinophilic Cytoplasmic Inclusion Formation in SH-SY5Y Cells

Parkinsons disease (PD) is a neurodegenerative disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. Previous reports have shown that α-synuclein deposited in brain tissue from individuals with synucleinopathy is extensively phosphorylated at Ser-129. Here, we investigate the role of phosphorylation of α-synuclein in the formation of inclusions involving synphilin-1 and parkin using site-directed mutagenesis to change Ser-129 of α-synuclein to alanine (S129A) to abolish phosphorylation at this site. Coexpression of wild-type α-synuclein and synphilin-1 in human neuroblastoma SH-SY5Y cells yielded cytoplasmic eosinophilic inclusions with some features resembling Lewy bodies, whereas coexpression of S129A α-synuclein and synphlin-1 formed few or no inclusions. Moreover, coexpression of parkin with α-synuclein and synphilin-1 formed more ubiquitinated inclusions, but these inclusions decreased with expression of S129A α-synuclein instead of wild-type α-synuclein. Coimmunoprecipitation assays revealed a decreased interaction of S129A α-synuclein with synphilin-1 compared with wild-type α-synuclein. Expression of S129A α-synuclein instead of wild-type α-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 H2O2 increased α-synuclein phosphorylation and enhanced the formation of inclusions formed by coexpression of α-synuclein, synphilin-1, and parkin, whereas treatment with the casein kinase 2 inhibitor 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole had the opposite affect. These results indicate that phosphorylation of α-synuclein at S129 may be important for the formation of inclusions in PD and related α synucleinopathies.

Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptins Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptins anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptin-mediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptins signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptins anorectic signaling cascade.

CHIP regulates leucine-rich repeat kinase-2 ubiquitination, degradation, and toxicity

Mutation in leucine-rich repeat kinase-2 (LRRK2) is the most common cause of late-onset Parkinsons 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.

Inhibitors of LRRK2 kinase attenuate neurodegeneration and Parkinson-like phenotypes in Caenorhabditis elegans and Drosophila Parkinson's disease models

Mutations in leucine-rich repeat kinase 2 (LRRK2) have been identified as a genetic cause of familial Parkinsons disease (PD) and have also been found in the more common sporadic form of PD, thus positioning LRRK2 as important in the pathogenesis of PD. Biochemical studies of the disease-causing mutants of LRRK2 implicates an enhancement of kinase activity as the basis of neuronal toxicity and thus possibly the pathogenesis of PD due to LRRK2 mutations. Previously, a chemical library screen identified inhibitors of LRRK2 kinase activity. Here, two of these inhibitors, GW5074 and sorafenib, are shown to protect against G2019S LRRK2-induced neurodegeneration in vivo in Caenorhabditis elegans and in Drosophila. These findings indicate that increased kinase activity of LRRK2 is neurotoxic and that inhibition of LRRK2 activity can have a disease-modifying effect. This suggests that inhibition of LRRK2 holds promise as a treatment for PD.

Gene–environment interactions in Parkinson's disease

PD is the second most common neurodegenerative disease, and affects 5% of the population by the age of 85. PD is a multi-factorial disease with a complex etiology including genetic risk factors, environmental exposure and aging. The pathogenesis is not fully understood. Here we review research on the genetic and environmental causes of PD and the current research models. None of the single models replicate all the features of PD. Genetic models (possibly including more than one mutation) in combination with toxins or other environmental manipulation may provide better models of PD pathogenesis.

Curcumin protects against A53T alpha-synuclein-induced toxicity in a PC12 inducible cell model for Parkinsonism

Parkinsons disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Treatment for PD that prevents neuronal death in the dopaminergic system and abnormal protein deposition in the brain is not yet available. Evidence from human and animal studies has suggested that oxidative damage critically contributes to neuronal loss in PD. Here we test whether curcumin, a potent antioxidant compound, derived from the curry spice turmeric, can protect against mutant A53T α-synuclein-induced cell death. We used PC12 cells that inducibly express A53T α-synuclein. We found that curcumin protected against A53T α-synuclein-induced cell death in a dose-dependent manner. We further found that curcumin can reduce mutant α- synuclein-induced intracellular reactive oxygen species (ROS) levels, mitochondrial depolarization, cytochrome c release, and caspase-9 and caspase-3 activation. This study demonstrate that curcumin protected against A53T mutant α-synuclein-induced cell death via inhibition of oxidative stress and the mitochondrial cell death pathway, suggesting that curcumin may be a candidate neuroprotective agent for A53T α-synuclein-linked Parkinsonism, and possibly for other genetic or sporadic forms of PD.

Parkinson's disease genetic mutations increase cell susceptibility to stress: Mutant α-synuclein enhances H2O2- and Sin-1-induced cell death

Abstract Parkinsons disease (PD) is a progressive neurodegenerative movement disorder characterized by selective loss of dopaminergic neurons and the presence of Lewy bodies. α-Synuclein is a major component of Lewy bodies in sporadic PD, and genetic alterations in α-synuclein cause autosomal-dominant hereditary PD. The pathogenesis of PD remains incompletely understood, but it appears to involve both genetic susceptibility and environmental factors. Here we investigated the effect of α-synuclein expression on cell susceptibility to proteasome inhibition, oxidative and nitrative stresses by using a PC 12-Tet-off regulatory system. We found that inducible expression of A30P or A53T mutant α-synuclein decreased the proteasome activity, increased intracellular ROS levels, and enhanced lactacystin- and H2O2-induced cell death. Furthermore, 3-nitrotyrosine levels increased in cells expressing α-synuclein, and further increased after Sin-1 (a NO donor) treatment compared with untreated or treated non-induced cells. Expression of α-synuclein (mutant more than wild type) significantly enhances Sin-1 toxicity. These results indicate that genetic mutations in α-synuclein may increase neuronal vulnerability to cellular stress in aging and PD pathogenesis.