title: The effect of alpha-synuclein knockdown on MPP+ toxicity in models of human neurons.
abstract: The protein alpha-synuclein is central to the pathophysiology of Parkinson's disease (PD) but its role in the development of neurodegeneration remains unclear. alpha-Synuclein-knockout mice develop without gross abnormality and are resistant to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a mitochondrial inhibitor widely used to model parkinsonism. Here we show that differentiated human dopaminergic neuron-like cells also have increased resistance to 1-methyl-4-phenylpyridine (MPP+), the active metabolite of MPTP, when alpha-synuclein is knocked down using RNA interference. In attempting to understand how this occurred we found that lowering alpha-synuclein levels caused changes to intracellular vesicles, dopamine transporter (DAT) and vesicular monoamine transporter (VMAT2), each of which is known to be an important component of the early events leading to MPP+ toxicity. Knockdown of alpha-synuclein reduced the availability of DAT on the neuronal surface by 50%, decreased the total number of intracellular vesicles by 37% but increased the density of VMAT2 molecules per vesicle by 2.8-fold. However, these changes were not associated with any reduction in MPP+ -induced superoxide production, suggesting that alpha-synuclein knockdown may have other downstream effects which are important. We then showed that alpha-synuclein knockdown prevented MPP+ -induced activation of nitric oxide synthase (NOS). Activation of NOS is an essential step in MPTP toxicity and increasing evidence points to nitrosative stress as being important in neurodegeneration. Overall, these results show that as well as having a number of effects on cellular events upstream of mitochondrial dysfunction alpha-synuclein affects pathways downstream of superoxide production, possibly involving regulation of NOS activity.
authors.affiliation: Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford OX1 3QX, UK.
authors.forename: Timothy M
authors.initials: TM
authors.lastname: Fountaine
authors.forename: Lara Lourenco
authors.initials: LL
authors.lastname: Venda
authors.forename: Nicholas
authors.initials: N
authors.lastname: Warrick
authors.forename: Helen C
authors.initials: HC
authors.lastname: Christian
authors.forename: Patrik
authors.initials: P
authors.lastname: Brundin
authors.forename: Keith M
authors.initials: KM
authors.lastname: Channon
authors.forename: Richard
authors.initials: R
authors.lastname: Wade-Martins
mesh_terms: D015655:1-Methyl-4-phenylpyridinium / Q000378:metabolism / Q000633:toxicity*; D000818:Animals; D002460:Cell Line; D022162:Cytoplasmic Vesicles / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins / Q000378:metabolism; D004789:Enzyme Activation; D055785:Gene Knockdown Techniques; D006540:Herbicides / Q000378:metabolism / Q000633:toxicity; D006801:Humans; D051379:Mice; D009474:Neurons / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D019001:Nitric Oxide Synthase / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism; D050493:Vesicular Monoamine Transport Proteins / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism
chemical_list: D050483:Dopamine Plasma Membrane Transport Proteins; D006540:Herbicides; D034741:RNA, Small Interfering; C493452:SLC18A2 protein, human; D050493:Vesicular Monoamine Transport Proteins; D051844:alpha-Synuclein; D013481:Superoxides; D019001:Nitric Oxide Synthase; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine