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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 over-expression was not toxic to BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. An LDH release assay was performed to determine cell viability by quantifying LDH release into the medium using commercial LDH assay reagents. Graphs are the average (± SD) of three independent experiments. Each experiment was performed with 8 replicates per condition.
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Figure 2: α-synuclein over-expression was not toxic to BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. An LDH release assay was performed to determine cell viability by quantifying LDH release into the medium using commercial LDH assay reagents. Graphs are the average (± SD) of three independent experiments. Each experiment was performed with 8 replicates per condition.

Mentions: Because α-synuclein over-expression can promote toxicity or increased vulnerability to stressors in some cells [47-49] it was necessary to validate that expression of the exogenous α-synuclein was not inducing cell death as a component of the acquisition of a proinflammatory phenotype in BV2 cells. This is particularly relevant for microglial cells which often include a programmed death response as a culmination of their activation pathways [50-52]. Cell viability was assessed by examining enzymatic activity of lactate dehydrogenase released into the media. Although the transfection procedure produced some expected toxicity of cells compared to non-transfected controls, expression of the exogenous human α-synuclein proteins was not toxic to the cells (Figure 2). This lack of toxicity correlated with increased detectable levels of monomeric α-synuclein as well as apparent higher molecular SDS-stable oligomers ranging from 24-34 kDa but no detectable detergent insoluble aggregates (Figure 1). This suggested that a higher molecular weight or aggregate form may be required for over-expression dependent toxicity in these cells. This data demonstrated that changes in proinflammatory Cox-2 levels were not indicative of a cell death pathway induced by mutant α-synuclein over-expression.


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

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

α-synuclein over-expression was not toxic to BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. An LDH release assay was performed to determine cell viability by quantifying LDH release into the medium using commercial LDH assay reagents. Graphs are the average (± SD) of three independent experiments. Each experiment was performed with 8 replicates per condition.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: α-synuclein over-expression was not toxic to BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. An LDH release assay was performed to determine cell viability by quantifying LDH release into the medium using commercial LDH assay reagents. Graphs are the average (± SD) of three independent experiments. Each experiment was performed with 8 replicates per condition.
Mentions: Because α-synuclein over-expression can promote toxicity or increased vulnerability to stressors in some cells [47-49] it was necessary to validate that expression of the exogenous α-synuclein was not inducing cell death as a component of the acquisition of a proinflammatory phenotype in BV2 cells. This is particularly relevant for microglial cells which often include a programmed death response as a culmination of their activation pathways [50-52]. Cell viability was assessed by examining enzymatic activity of lactate dehydrogenase released into the media. Although the transfection procedure produced some expected toxicity of cells compared to non-transfected controls, expression of the exogenous human α-synuclein proteins was not toxic to the cells (Figure 2). This lack of toxicity correlated with increased detectable levels of monomeric α-synuclein as well as apparent higher molecular SDS-stable oligomers ranging from 24-34 kDa but no detectable detergent insoluble aggregates (Figure 1). This suggested that a higher molecular weight or aggregate form may be required for over-expression dependent toxicity in these cells. This data demonstrated that changes in proinflammatory Cox-2 levels were not indicative of a cell death pathway induced by mutant α-synuclein over-expression.

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