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Expression of mutant alpha-synuclein modulates microglial phenotype in vitro.

Rojanathammanee L, Murphy EJ, Combs CK - J Neuroinflammation (2011)

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

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.

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.

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α-synuclein transfected BV2 cells increased Cox-2 protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Cells were lysed and Western blotted using anti-cPLA2, anti-Cox-1, anti-Cox-2, anti-PLD1, anti-PLD2, anti- α-synuclein, or anti-GAPDH (loading control) antibodies. Protein levels of B) cPLA2, C) Cox-1, D) Cox-2, E) PLD1, and F) PLD2 were quantified and normalized to GAPDH. Graphs are the average (± SD) of five independent experiments. * p < 0.05 compared to mock transfected cells.
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Figure 1: α-synuclein transfected BV2 cells increased Cox-2 protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Cells were lysed and Western blotted using anti-cPLA2, anti-Cox-1, anti-Cox-2, anti-PLD1, anti-PLD2, anti- α-synuclein, or anti-GAPDH (loading control) antibodies. Protein levels of B) cPLA2, C) Cox-1, D) Cox-2, E) PLD1, and F) PLD2 were quantified and normalized to GAPDH. Graphs are the average (± SD) of five independent experiments. * p < 0.05 compared to mock transfected cells.

Mentions: To address whether α-synuclein over-expression modulates microglial phenotype, the murine microglial cell line, BV2, [31] was selected as an in vitro model of microglia because it is amenable to transient transfection for exogenous gene over-expression. BV2 cells were transfected to express human WT, or missense mutation A30P [34] or A53T [14]. Following transfection, levels of WT and mutant α-synuclein as well as several protein associated with α-synuclein function were examined via Western blot and changes due to α-synuclein over-expression were quantified. Interestingly, over-expression of α-synuclein, both wild type and mutant forms, resulted in not only the monomeric species but also an SDS-stable oligomeric form migrating between 24 and 34 kDa. Because over-expression and interaction of α-synuclein with PLD attenuates PLD activity [25,35-37] we first examined transfected cells for changes in PLD1 or PLD2 levels. Over-expression of WT and the A30P and A53T mutants had no effect on PLD1/2 protein levels suggesting that although α-synuclein expression or function may regulate PLD activities it is not involved in regulating enzyme expression or turnover in these cells (Figure 1).


Expression of mutant alpha-synuclein modulates microglial phenotype in vitro.

Rojanathammanee L, Murphy EJ, Combs CK - J Neuroinflammation (2011)

α-synuclein transfected BV2 cells increased Cox-2 protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Cells were lysed and Western blotted using anti-cPLA2, anti-Cox-1, anti-Cox-2, anti-PLD1, anti-PLD2, anti- α-synuclein, or anti-GAPDH (loading control) antibodies. Protein levels of B) cPLA2, C) Cox-1, D) Cox-2, E) PLD1, and F) PLD2 were quantified and normalized to GAPDH. Graphs are the average (± SD) of five independent experiments. * p < 0.05 compared to mock transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3104357&req=5

Figure 1: α-synuclein transfected BV2 cells increased Cox-2 protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Cells were lysed and Western blotted using anti-cPLA2, anti-Cox-1, anti-Cox-2, anti-PLD1, anti-PLD2, anti- α-synuclein, or anti-GAPDH (loading control) antibodies. Protein levels of B) cPLA2, C) Cox-1, D) Cox-2, E) PLD1, and F) PLD2 were quantified and normalized to GAPDH. Graphs are the average (± SD) of five independent experiments. * p < 0.05 compared to mock transfected cells.
Mentions: To address whether α-synuclein over-expression modulates microglial phenotype, the murine microglial cell line, BV2, [31] was selected as an in vitro model of microglia because it is amenable to transient transfection for exogenous gene over-expression. BV2 cells were transfected to express human WT, or missense mutation A30P [34] or A53T [14]. Following transfection, levels of WT and mutant α-synuclein as well as several protein associated with α-synuclein function were examined via Western blot and changes due to α-synuclein over-expression were quantified. Interestingly, over-expression of α-synuclein, both wild type and mutant forms, resulted in not only the monomeric species but also an SDS-stable oligomeric form migrating between 24 and 34 kDa. Because over-expression and interaction of α-synuclein with PLD attenuates PLD activity [25,35-37] we first examined transfected cells for changes in PLD1 or PLD2 levels. Over-expression of WT and the A30P and A53T mutants had no effect on PLD1/2 protein levels suggesting that although α-synuclein expression or function may regulate PLD activities it is not involved in regulating enzyme expression or turnover in these cells (Figure 1).

Bottom Line: 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 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.

View Article: PubMed Central - HTML - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus