Jean-Christophe Rochet

Purdue University, ウェストラファイエット, Indiana, United States

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Publications (50)317.97 Total impact

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    ABSTRACT: The Saccharomyces cerevisiae heat shock protein Hsp31 is a stress-inducible homodimeric protein that is involved in diauxic shift reprogramming and has glyoxalase activity. We show that substoichiometric concentrations of Hsp31 can abrogate aggregation of a broad array of substrates in vitro. Hsp31 also modulates the aggregation of α-synuclein (αSyn), a target of the chaperone activity of human DJ-1, an Hsp31 homolog. We demonstrate that Hsp31 is able to suppress the in vitro fibrillization of αSyn, aggregation of citrate synthase and insulin. Chaperone activity was also observed in vivo because constitutive overexpression of Hsp31 reduced the incidence of αSyn cytoplasmic foci, and yeast cells are rescued from αSyn-generated proteotoxicity upon Hsp31 overexpression. Moreover, we showed that Hsp31 protein levels are increased by H2O2, in the diauxic phase of normal growth conditions, and in cells under αSyn mediated proteotoxic stress. We show that Hsp31 chaperone activity and not the methylglyoxalase activity or the autophagy pathway drives the protective effects. We also demonstrate reduced aggregation of the Sup35 prion domain, PrD-Sup35, as visualized by fluorescent protein fusions. In addition, Hsp31 acts on its substrates prior to the formation of large aggregates because Hsp31 does not mutually localize with prion aggregates, and it prevents formation of detectable in vitro αSyn fibrils. These studies establish that the protective role of Hsp31 against cellular stress is achieved by chaperone activity that intervenes early in the protein misfolding process and is effective on a wide spectrum of substrate proteins, including αSyn and prion proteins. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Full-text · Article · Aug 2015 · Journal of Biological Chemistry
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    Hangyu Zhang · Jean-Christophe Rochet · Lia A Stanciu
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    ABSTRACT: The aggregation of α-synuclein is associated with dopamine neuron death in Parkinson's disease. There is controversy in the field over the question of which species of the aggregates, fibrils or protofibrils, are toxic. Moreover, compelling evidence suggested the exposure to heavy metals to be a risk of PD. Nevertheless, the mechanism of metal ions in promoting PD remains unclear. In this research, we investigated the structural basis of Cu(II) induced aggregation of α-synuclein. Using transmission electron microscopy experiments, Cu(II) was found to promote in vitro aggregation of α-synuclein by facilitating annular protofibril formation rather than fibril formation. Furthermore, neuroprotective baicalein disaggregated annular protofibrils accompanied by considerable decrease of β-sheet content. These results strongly support the hypothesis that annular protofibrils are the toxic species, rather than fibrils, thereby inspiring us to search novel therapeutic strategies for the suppression of the toxic annular protofibril formation. Copyright © 2015. Published by Elsevier Inc.
    Full-text · Article · Jun 2015 · Biochemical and Biophysical Research Communications
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    ABSTRACT: The post-mortem brains of individuals with Parkinson's disease (PD) and other synucleinopathy disorders are characterized by the presence of aggregated forms of the presynaptic protein α-synuclein (aSyn). Understanding the molecular mechanism of aSyn aggregation is essential for the development of neuroprotective strategies to treat these diseases. In this study, we examined how interactions between aSyn and phospholipid vesicles influence the protein's aggregation and toxicity to dopaminergic neurons. Two-dimensional NMR data revealed that two familial aSyn mutants, A30P and G51D, populated an exposed, membrane-bound conformer in which the central hydrophobic region was dissociated from the bilayer to a greater extent than in the case of wild-type aSyn. A30P and G51D had a greater propensity to undergo membrane-induced aggregation and elicited greater toxicity to primary dopaminergic neurons compared to the wild-type protein. In contrast, the non-familial aSyn mutant A29E exhibited a weak propensity to aggregate in the presence of phospholipid vesicles or to elicit neurotoxicity, despite adopting a relatively exposed membrane-bound conformation. Our findings suggest that the aggregation of exposed, membrane-bound aSyn conformers plays a key role in the protein's neurotoxicity in PD and other synucleinopathy disorders. Copyright © 2015. Published by Elsevier Inc.
    No preview · Article · Apr 2015 · Neurobiology of Disease
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    ABSTRACT: The aggregation of α-synuclein (α-Syn) is linked to Parkinson's disease. The mechanism of early aggregation steps and the effect of pathogenic single-point mutations remain elusive. We report here a single-molecule fluorescence study of α-Syn dimerization and the effect of mutations. Specific interactions between tethered fluorophore-free α-Syn monomers on a substrate and fluorophore-labeled monomers diffusing freely in solution were observed using total internal reflection fluorescence microscopy. The results showed that wild-type (WT) α-Syn dimers adopt two types of dimers. The lifetimes of type 1 and type 2 dimers were determined to be 197 ± 3 ms and 3334 ± 145 ms, respectively. All three of the mutations used, A30P, E46K, and A53T, increased the lifetime of type 1 dimer and enhanced the relative population of type 2 dimer, with type 1 dimer constituting the major fraction. The kinetic stability of type 1 dimers (expressed in terms of lifetime) followed the order A30P (693 ± 14 ms) > E46K (292 ± 5 ms) > A53T (226 ± 6 ms) > WT (197 ± 3 ms). Type 2 dimers, which are more stable, had lifetimes in the range of several seconds. The strongest effect, observed for the A30P mutant, resulted in a lifetime 3.5 times higher than observed for the WT type 1 dimer. This mutation also doubled the relative fraction of type 2 dimer. These data show that single-point mutations promote dimerization, and they suggest that the structural heterogeneity of α-Syn dimers could lead to different aggregation pathways. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All rights reserved.
    Full-text · Article · Apr 2015 · Biophysical Journal
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    Christopher A Bates · Sherleen Fu · Daniel Ysselstein · Jean-Christophe Rochet · Wei Zheng
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    ABSTRACT: The choroid plexus maintains the homeostasis of critical molecules in the brain by regulating their transport between the blood and cerebrospinal fluid (CSF). The current study was designed to investigate the potential role of the blood-CSF barrier (BCSFB) in α-synuclein (a-Syn) transport in the brain as affected by exposure to manganese (Mn), the toxic metal implicated in Parkinsonian disorders. Immunohistochemistry was used to identify intracellular a-Syn expression at the BCSFB. Quantitative real-time PCR was used to quantify the change in a-Syn mRNA expression following Mn treatments at the BCSFB in vitro. ELISA was used to quantify a-Syn levels following in vivo and in vitro treatments of Mn, copper (Cu), and/or external a-Syn. Thioflavin-T assay was used to investigate a-Syn aggregation after incubating with Mn and/or Cu in vitro. A two-chamber Transwell system was used to study a-Syn transport by BCSFB monolayer.Data revealed the expression of endogenous a-Syn in rat choroid plexus tissue and immortalized choroidal epithelial Z310 cells. The cultured primary choroidal epithelia from rats showed the ability to take up a-Syn from extracellular medium and transport a-Syn across the cellular monolayer from the donor to receiver chamber. Exposure of cells with Mn induced intracellular a-Syn accumulation without causing any significant changes in a-Syn mRNA expression. A significant increase in a-Syn aggregation in a cell-free system was observed with the presence of Mn. Moreover, Mn exposure resulted in a significant uptake of a-Syn by primary cells.These data indicate that the BCSFB expresses a-Syn endogenously and is capable of transporting a-Syn across the BCSFB monolayer; Mn exposure apparently increases a-Syn accumulation in the BCSFB by facilitating its uptake and intracellular aggregation.
    Full-text · Article · Apr 2015
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    Full-text · Article · Jan 2015 · Biophysical Journal
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    ABSTRACT: Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of WT or A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic cells and yeast in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways. Copyright © 2014. Published by Elsevier Inc.
    Full-text · Article · Dec 2014 · Neurobiology of Disease
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    ABSTRACT: Parkinson's disease (PD) is the second most common neurodegenerative disease. Much data has linked the etiology of PD to a variety of environmental factors. The majority of cases are thought to arise from a combination of genetic susceptibility and environmental factors. Chronic exposures to dietary factors, including meat, have been identified as potential risk factors. Although heterocyclic amines that are produced during high-temperature meat cooking are known to be carcinogenic, their effect on the nervous system has yet to be studied in depth. In this study, we investigated neurotoxic effects of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), a highly abundant heterocyclic amine in cooked meat, in vitro. We tested toxicity of PhIP and the two major Phase I metabolites, N-OH-PhIP and 4'-OH-PhIP, using primary mesencephalic cultures from rat embryos. This culture system contains both dopaminergic and non-dopaminergic neurons, which allows specificity of neurotoxicity to be readily examined. We find that exposure to PhIP or N-OH-PhIP is selectively toxic to dopaminergic neurons in primary cultures, resulting in a decreased percentage of dopaminergic neurons. Neurite length is decreased in surviving dopaminergic neurons. Exposure to 4'-OH-PhIP did not produce significant neurotoxicity. PhIP treatment also increased formation of oxidative damage markers, 4-hydroxy-2-nonenal (HNE) and 3-nitrotyrosine in dopaminergic neurons. Pre-treatment with N-acetylcysteine was protective. Finally, treatment with blueberry extract, a dietary factor with known antioxidant and other protective mechanisms, prevented PhIP-induced toxicity. Collectively, our study suggests, for the first time, that PhIP is selectively toxic to dopaminergic neurons likely through inducing oxidative stress.
    Preview · Article · Apr 2014 · Toxicological Sciences
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    ABSTRACT: Neuropathological evidence indicates that dopaminergic cell death in Parkinson's disease (PD) involves impairment of mitochondrial complex I, oxidative stress, microglial activation, and the formation of Lewy bodies. Epidemiological findings suggest that the consumption of berries rich in anthocyanins and proanthocyanidins may reduce PD risk. In this study, we investigated whether extracts rich in anthocyanins, proanthocyanidins, or other polyphenols suppress the neurotoxic effects of rotenone in a primary cell culture model of PD. Dopaminergic cell death elicited by rotenone was suppressed by extracts prepared from blueberries, grape seed, hibiscus, blackcurrant, and Chinese mulberry. Extracts rich in anthocyanins and proanthocyanidins exhibited greater neuroprotective activity than extracts rich in other polyphenols, and a number of individual anthocyanins interfered with rotenone neurotoxicity. The blueberry and grape seed extracts rescued rotenone-induced defects in mitochondrial respiration in a dopaminergic cell line, and a purple basal extract attenuated nitrite release from microglial cells stimulated by lipopolysaccharide. These findings suggest that anthocyanin- and proanthocyanidin-rich botanical extracts may alleviate neurodegeneration in PD via enhancement of mitochondrial function.
    No preview · Article · Mar 2014 · Brain research
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    Aurelie de Rus Jacquet · Rupa Subedi · Suresh K Ghimire · Jean-Christophe Rochet
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    ABSTRACT: Nepal is a hotspot for cultural and biological diversities. The tremendous diversity of ecosystems and climates and the blend of medicinal practices inherited from Ayurvedic and Traditional Tibetan Medicine are well suited to a study aimed at discovering information about medicinal plants to treat Parkinson’s disease (PD). In addition, this study across Nepal’s altitudinal range is relevant to understanding how cultural and ecological environments influence local traditional medicines. The aim of the study is to document the uses of medicinal plants in three different eco-geographical areas of Nepal (Chitwan–Panchase–Mustang) to treat symptoms related to PD. A second goal is to analyze the impact of culture and environment on the evolution of traditional medicine.
    Full-text · Article · Feb 2014 · Journal of ethnopharmacology
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    ABSTRACT: The misfolding of intrinsically disordered proteins such as α-synuclein, tau and the Aβ peptide has been associated with many highly debilitating neurodegenerative syndromes including Parkinson's and Alzheimer's diseases. Therapeutic targeting of the monomeric state of such intrinsically disordered proteins by small molecules has, however, been a major challenge because of their heterogeneous conformational properties. We show here that a combination of computational and experimental techniques has led to the identification of a drug-like phenyl-sulfonamide compound (ELN484228), that targets α-synuclein, a key protein in Parkinson's disease. We found that this compound has substantial biological activity in cellular models of α-synuclein-mediated dysfunction, including rescue of α-synuclein-induced disruption of vesicle trafficking and dopaminergic neuronal loss and neurite retraction most likely by reducing the amount of α-synuclein targeted to sites of vesicle mobilization such as the synapse in neurons or the site of bead engulfment in microglial cells. These results indicate that targeting α-synuclein by small molecules represents a promising approach to the development of therapeutic treatments of Parkinson's disease and related conditions.
    Full-text · Article · Feb 2014 · PLoS ONE
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    Full-text · Article · Jan 2014 · Biophysical Journal
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    ABSTRACT: α-Synuclein (α-syn) is a small lipid-binding protein implicated in several neurodegenerative diseases, including Parkinson’s disease, whose pathobiology is conserved from yeast to man. There are no therapies targeting these underlying cellular pathologies, or indeed those of any major neurodegenerative disease. Using unbiased phenotypic screens as an alternative to target-based approaches, we discovered an N-aryl benzimidazole (NAB) that strongly and selectively protected diverse cell types from α-syn toxicity. Three chemical genetic screens in wild-type yeast cells established that NAB promoted endosomal transport events dependent on the E3 ubiquitin ligase Rsp5/Nedd4. These same steps were perturbed by α-syn itself. Thus, NAB identifies a druggable node in the biology of α-syn that can correct multiple aspects of its underlying pathology, including dysfunctional endosomal and endoplasmic reticulum–to-Golgi vesicle trafficking.
    Full-text · Article · Oct 2013 · Science
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    ABSTRACT: Misfolding and subsequent aggregation of alpha-synuclein (α-Syn) protein are critically involved in the development of several neurodegenerative diseases, including Parkinson's disease (PD). Three familial single point mutations, A30P, E46K, and A53T, correlate with early-onset PD; however the molecular mechanism of the effects of these mutations on the structural properties of α-Syn and its propensity to misfold remains unclear. Here, we address this issue utilizing a single molecule AFM force spectroscopy approach in which structural details of dimers formed by all four variants of α-Syn are characterized. Analysis of the force spectroscopy data reflecting contour length distribution for α-Syn dimer dissociation suggests that multiple segments are involved in the assembly of the dimer. The interactions are not limited to the central non-amyloid-beta component (NAC) of the protein, but rather expand beyond this segment. All three mutations alter the protein's folding and interaction patterns affecting interactions far beyond their immediate locations. Implementation of these findings to our understanding of α-Syn aggregation pathways is discussed.
    Full-text · Article · Sep 2013 · Biochemistry
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    Hangyu Zhang · Amy Griggs · Jean-Christophe Rochet · Lia A Stanciu
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    ABSTRACT: The aggregation of α-synuclein is thought to play a role in the death of dopamine neurons in Parkinson's disease (PD). Alpha-synuclein transitions itself through an aggregation pathway consisting of pathogenic species referred to as protofibrils (or oligomer), which ultimately convert to mature fibrils. The structural heterogeneity and instability of protofibrils has significantly impeded advance related to the understanding of their structural characteristics and the amyloid aggregation mystery. Here, we report, to our knowledge for the first time, on α-synuclein protofibril structural characteristics with cryo-electron microscopy. Statistical analysis of annular protofibrils revealed a constant wall thickness as a common feature. The visualization of the assembly steps enabled us to propose a novel, to our knowledge, mechanisms for α-synuclein aggregation involving ring-opening and protofibril-protofibril interaction events. The ion channel-like protofibrils and their membrane permeability have also been found in other amyloid diseases, suggesting a common molecular mechanism of pathological aggregation. Our direct visualization of the aggregation pathway of α-synuclein opens up fresh opportunities to advance the understanding of protein aggregation mechanisms relevant to many amyloid diseases. In turn, this information would enable the development of additional therapeutic strategies aimed at suppressing toxic protofibrils of amyloid proteins involved in neurological disorders.
    Full-text · Article · Jun 2013 · Biophysical Journal
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    Alexandra Snyder · Zhenyu Bo · Robert Moon · Jean-Christophe Rochet · Lia Stanciu
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    ABSTRACT: Titanium dioxide (TiO2) is a well-studied photocatalyst that is known to break down organic molecules upon ultraviolet (UV) irradiation. Cellulose nanofibers (CNFs) act as an attractive matrix material for the suspension of photocatalytic particles due to their desirable mechanical and optical properties. In this work, TiO2-CNF composite films were fabricated and evaluated for photocatalytic activity under UV light and their potential to remove organic compounds from water. Subsequently, gold (Au) and silver (Ag) nanoclusters were formed on the film surfaces using simple reduction techniques. Au and Ag doped TiO2 films showed a wider spectral range for photocatalysis and enhanced mechanical properties. Scanning electron microscopy imaging and energy dispersive X-ray spectroscopy mapping were used to evaluate changes in microstructure of the films and monitor the dispersion of the TiO2, Au, and Ag particles. The ability of the films to degrade methylene blue (a model organic dye) in simulated sunlight has been demonstrated using UV-visible spectroscopy. Reusability and mechanical integrity of the films were also investigated.
    Full-text · Article · Mar 2013 · Journal of Colloid and Interface Science
  • Jean-Christophe Rochet · Bruce A Hay · Ming Guo
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    ABSTRACT: Mutations in SNCA, PINK1, parkin, and DJ-1 are associated with autosomal-dominant or autosomal-recessive forms of Parkinson's disease (PD), the second most common neurodegenerative disorder. Studies on the structural and functional properties of the corresponding gene products have provided significant insights into the molecular underpinnings of familial PD and the much more common sporadic forms of the disease. Here, we review recent advances in our understanding of four PD-related gene products: α-synuclein, parkin, PINK1, and DJ-1. In Part 1, we review new insights into the role of α-synuclein in PD. In Part 2, we summarize the latest developments in understanding the role of mitochondrial dysfunction in PD, emphasizing the role of the PINK1/parkin pathway in regulating mitochondrial dynamics and mitophagy. The role of DJ-1 is also discussed. In Part 3, we point out converging pathways and future directions.
    No preview · Article · Dec 2012 · Progress in molecular biology and translational science
  • Jean-Christophe Rochet

    No preview · Article · Aug 2012 · Movement Disorders
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    ABSTRACT: Alpha-synuclein (α-Syn) is a 140 aa presynaptic protein which belongs to a group of natively unfolded proteins that are unstructured in aqueous solutions. The aggregation rate of α-Syn is accelerated in the presence of physiological levels of cellular polyamines. Here we applied single molecule AFM force spectroscopy to characterize the effect of spermidine on the very first stages of α-Syn aggregation--misfolding and assembly into dimers. Two α-Syn variants, the wild-type (WT) protein and A30P, were studied. The two protein molecules were covalently immobilized at the C-terminus, one at the AFM tip and the other on the substrate, and intermolecular interactions between the two molecules were measured by multiple approach-retraction cycles. At conditions close to physiological ones at which α-Syn misfolding is a rare event, the addition of spermidine leads to a dramatic increase in the propensity of the WT and mutant proteins to misfold. Importantly, misfolding is characterized by a set of conformations, and A30P changes the misfolding pattern as well as the strength of the intermolecular interactions. Together with the fact that spermidine facilitates late stages of α-Syn aggregation, our data demonstrate that spermidine promotes the very early stages of protein aggregation including α-Syn misfolding and dimerization. This finding suggests that increased levels of spermidine and potentially other polyamines can initiate the disease-related process of α-Syn.
    Full-text · Article · May 2012 · PLoS ONE
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    ABSTRACT: Mutations in the gene encoding DJ-1 have been identified in patients with familial Parkinson's disease (PD) and are thought to inactivate a neuroprotective function. Oxidation of the sulfhydryl group to a sulfinic acid on cysteine residue C106 of DJ-1 yields the "2O " form, a variant of the protein with enhanced neuroprotective function. We hypothesized that some familial mutations disrupt DJ-1 activity by interfering with conversion of the protein to the 2O form. To address this hypothesis, we developed a novel quantitative mass spectrometry approach to measure relative changes in oxidation at specific sites in mutant DJ-1 as compared with the wild-type protein. Treatment of recombinant wild-type DJ-1 with a 10-fold molar excess of H(2)O(2) resulted in a robust oxidation of C106 to the sulfinic acid, whereas this modification was not detected in a sample of the familial PD mutant M26I exposed to identical conditions. Methionine oxidized isoforms of wild-type DJ-1 were depleted, presumably as a result of misfolding and aggregation, under conditions that normally promote conversion of the protein to the 2O form. These data suggest that the M26I familial substitution and methionine oxidation characteristic of sporadic PD may disrupt DJ-1 function by disfavoring a site-specific modification required for optimal neuroprotective activity. Our findings indicate that a single amino acid substitution can markedly alter a protein's ability to undergo oxidative modification, and they imply that stimulating the conversion of DJ-1 to the 2O form may be therapeutically beneficial in familial or sporadic PD.
    No preview · Article · Nov 2011 · Molecular & Cellular Proteomics

Publication Stats

4k Citations
317.97 Total Impact Points

Institutions

  • 2006-2015
    • Purdue University
      • Department of Medicinal Chemistry and Molecular Pharmacology (MCMP)
      ウェストラファイエット, Indiana, United States
  • 2007
    • University of Alberta
      Edmonton, Alberta, Canada
  • 2000-2004
    • Harvard Medical School
      • Department of Neurology
      Boston, MA, United States
  • 2002
    • Brigham and Women's Hospital
      • Center for Neurologic Diseases
      Boston, Massachusetts, United States