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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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Over-expression of α-synuclein increased TNF-α, IL-6, and nitrite levels in medium from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T alpha-synuclein for 48 hours with or without 25 ng/ml LPS stimulation. Media were collected and used for quantifying concentrations of secreted A) and B) TNF-α, C) IL-6 using a commercial mouse TNF-α and IL-6 ELISA. D) Media was also used to perform Griess assay to detect the levels of nitrite secreted from BV2 cells. Each condition was performed with 8 replicates. Graphs are the average (± SD) of three independent experiments. ** p < 0.01, *** p < 0.001.
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Figure 4: Over-expression of α-synuclein increased TNF-α, IL-6, and nitrite levels in medium from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T alpha-synuclein for 48 hours with or without 25 ng/ml LPS stimulation. Media were collected and used for quantifying concentrations of secreted A) and B) TNF-α, C) IL-6 using a commercial mouse TNF-α and IL-6 ELISA. D) Media was also used to perform Griess assay to detect the levels of nitrite secreted from BV2 cells. Each condition was performed with 8 replicates. Graphs are the average (± SD) of three independent experiments. ** p < 0.01, *** p < 0.001.

Mentions: Based upon the fact that several reports have also demonstrated that select prostaglandins can negatively regulate cytokine secretion from microglia [58-60] we next examined whether secretion of the proinflammatory cytokine, TNF-α, was altered in transfected BV2 cells. Surprisingly, levels of secreted TNF-α were significantly higher in medium from cells over-expressing both wild type and mutant α-synuclein compared to mock transfected controls (Figure 4). In order to better examine the range of secretory phenotype change due to α-synuclein over-expression, cells over-expressing the A53T mutant as a representative over-expression phenotype were stimulated with and without the proinflammatory ligand, LPS, to quantify media concentrations of not only TNF-α, but also an additional cytokine, IL-6. As expected, stimulation with LPS significantly increased secretion of both cytokines from A53T transfected cells above the levels secreted from LPS stimulated mock transfected cells (Figure 4).


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

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

Over-expression of α-synuclein increased TNF-α, IL-6, and nitrite levels in medium from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T alpha-synuclein for 48 hours with or without 25 ng/ml LPS stimulation. Media were collected and used for quantifying concentrations of secreted A) and B) TNF-α, C) IL-6 using a commercial mouse TNF-α and IL-6 ELISA. D) Media was also used to perform Griess assay to detect the levels of nitrite secreted from BV2 cells. Each condition was performed with 8 replicates. Graphs are the average (± SD) of three independent experiments. ** p < 0.01, *** p < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Over-expression of α-synuclein increased TNF-α, IL-6, and nitrite levels in medium from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T alpha-synuclein for 48 hours with or without 25 ng/ml LPS stimulation. Media were collected and used for quantifying concentrations of secreted A) and B) TNF-α, C) IL-6 using a commercial mouse TNF-α and IL-6 ELISA. D) Media was also used to perform Griess assay to detect the levels of nitrite secreted from BV2 cells. Each condition was performed with 8 replicates. Graphs are the average (± SD) of three independent experiments. ** p < 0.01, *** p < 0.001.
Mentions: Based upon the fact that several reports have also demonstrated that select prostaglandins can negatively regulate cytokine secretion from microglia [58-60] we next examined whether secretion of the proinflammatory cytokine, TNF-α, was altered in transfected BV2 cells. Surprisingly, levels of secreted TNF-α were significantly higher in medium from cells over-expressing both wild type and mutant α-synuclein compared to mock transfected controls (Figure 4). In order to better examine the range of secretory phenotype change due to α-synuclein over-expression, cells over-expressing the A53T mutant as a representative over-expression phenotype were stimulated with and without the proinflammatory ligand, LPS, to quantify media concentrations of not only TNF-α, but also an additional cytokine, IL-6. As expected, stimulation with LPS significantly increased secretion of both cytokines from A53T transfected cells above the levels secreted from LPS stimulated mock transfected cells (Figure 4).

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