title: Expression of mutant alpha-synuclein modulates microglial phenotype in vitro.
abstract: BACKGROUND
  Increased reactive microglia are a histological characteristic of Parkinson's disease (PD) brains, positively correlating with levels of deposited α-synuclein protein. This suggests that microglial-mediated inflammatory events may contribute to disease pathophysiology. Mutations in the gene coding for α-synuclein lead to a familial form of PD. Based upon our prior findings that α-synuclein expression regulates microglial phenotype we hypothesized that expression of mutant forms of the protein may contribute to the reactive microgliosis characteristic of PD brains.
  
  
  METHODS
  To quantify the effects of wild type and mutant α-synuclein over-expression on microglial phenotype a murine microglial cell line, BV2, was transiently transfected to express human wild type (WT), and mutant α-synuclein (A30P and A53T) proteins. Transfected cells were used to assess changes in microglia phenotype via Western blot analysis, ELISA, phagocytosis, and neurotoxicity assays.
  
  
  RESULTS
  As expected, over-expression of α-synuclein induced a reactive phenotype in the transfected cells. Expression of α-synuclein increased protein levels of cycloxygenase-2 (Cox-2). Transfected cells demonstrated increased secretion of the proinflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), as well as increased nitric oxide production. Transfected cells also had impaired phagocytic ability correlating with decreased protein levels of lysosomal-associated membrane protein 1 (LAMP-1). In spite of the increased cytokine secretion profile, the transfected cells did not exhibit increased neurotoxic ability above control non-transfected BV2 cells in neuron-microglia co-cultures.
  
  
  CONCLUSIONS
  These data demonstrated that over-expression of α-synuclein drives microglial cells into a form of reactive phenotype characterized by elevated levels of arachidonic acid metabolizing enzymes, cytokine secretion, and reactive nitrogen species secretion all superimposed upon impaired phagocytic potential.
authors.affiliation: Department of Pharmacology, Physiology, & Therapeutics, University of North Dakota School of Medicine and Health Sciences, 504 Hamline Street, Neuroscience Building, Grand Forks, ND 58203, USA.
authors.forename: Lalida
authors.initials: L
authors.lastname: Rojanathammanee
authors.forename: Eric J
authors.initials: EJ
authors.lastname: Murphy
authors.forename: Colin K
authors.initials: CK
authors.lastname: Combs
mesh_terms: D000818:Animals; D002460:Cell Line; D051545:Cyclooxygenase 1 / Q000235:genetics / Q000378:metabolism; D051546:Cyclooxygenase 2 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D052118:Lysosomal-Associated Membrane Protein 1 / Q000235:genetics / Q000378:metabolism; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D017628:Microglia / Q000166:cytology / Q000378:metabolism*; D009154:Mutation; D009573:Nitrites / Q000378:metabolism; D010300:Parkinson Disease / Q000235:genetics / Q000473:pathology / Q000503:physiopathology; D010587:Phagocytosis; D010641:Phenotype; D010739:Phospholipase D / Q000235:genetics / Q000378:metabolism; D026361:Reactive Nitrogen Species / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D051844:alpha-Synuclein / Q000235:genetics* / Q000378:metabolism*
chemical_list: D015850:Interleukin-6; D052118:Lysosomal-Associated Membrane Protein 1; D009573:Nitrites; D026361:Reactive Nitrogen Species; D014409:Tumor Necrosis Factor-alpha; D051844:alpha-Synuclein; D051545:Cyclooxygenase 1; D051546:Cyclooxygenase 2; C105606:phospholipase D2; D010739:Phospholipase D; C106801:phospholipase D1