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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 attenuated the phagocytic ability of BV2 cells and lysosomal protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Transfected cells were incubated with FITC-labeled E. coli bioparticles (0.25 mg/mL) for 3 hours. After incubation, the media was removed and the signal from any unphagocytosed or membrane associated particles was quenched by incubating cells with a (0.25 mg/mL) trypan blue solution for 3 minutes. The fluorescence intensity of phagocytosed particles was measured via fluorescent plate reader (RFU). Each condition was performed with 8 replicates. B) Transfected cells were also lysed and Western blotted with anti-LAMP-1 and actin (loading control) antibodies. C) Optical density of LAMP-1 immunoreactive bands were normalized against their respective actin bands and averaged (± SD) from three independent experiments. * p < 0.05 compared to mock transfected cells, **p < 0.01 compared to mock transfected cells.
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Figure 3: Over-expression of α-synuclein attenuated the phagocytic ability of BV2 cells and lysosomal protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Transfected cells were incubated with FITC-labeled E. coli bioparticles (0.25 mg/mL) for 3 hours. After incubation, the media was removed and the signal from any unphagocytosed or membrane associated particles was quenched by incubating cells with a (0.25 mg/mL) trypan blue solution for 3 minutes. The fluorescence intensity of phagocytosed particles was measured via fluorescent plate reader (RFU). Each condition was performed with 8 replicates. B) Transfected cells were also lysed and Western blotted with anti-LAMP-1 and actin (loading control) antibodies. C) Optical density of LAMP-1 immunoreactive bands were normalized against their respective actin bands and averaged (± SD) from three independent experiments. * p < 0.05 compared to mock transfected cells, **p < 0.01 compared to mock transfected cells.

Mentions: Based upon several prior reports that prostaglandins can negatively regulate macrophage and microglial phagocytic ability [53-57] and our observation of increased Cox-2 protein levels in α-synuclein over-expressing cells we examined whether transfected cells would display an expected decrease in phagocytic ability. To examine changes in microglial phagocytic ability, uptake of FITC-labeled E. coli bioparticles was quantified from transfected cells. Consistent with the observed increase in Cox-2 protein levels, cells transfected to express WT, the A30P, or A53T mutants all displayed a significant decrease in ability to phagocytose the bioparticles compared to mock transfected cells (Figure 3A). Moreover, the decrease of phagocytic ability with over-expression correlated with a significant decrease in protein levels of the lysosomal marker protein, lysosome associated protein 1 (LAMP-1) (Figure 3B and 3C). These data again confirm that microglial over-expression of wild type or mutant α-synuclein results in an altered phenotype.


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

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

Over-expression of α-synuclein attenuated the phagocytic ability of BV2 cells and lysosomal protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Transfected cells were incubated with FITC-labeled E. coli bioparticles (0.25 mg/mL) for 3 hours. After incubation, the media was removed and the signal from any unphagocytosed or membrane associated particles was quenched by incubating cells with a (0.25 mg/mL) trypan blue solution for 3 minutes. The fluorescence intensity of phagocytosed particles was measured via fluorescent plate reader (RFU). Each condition was performed with 8 replicates. B) Transfected cells were also lysed and Western blotted with anti-LAMP-1 and actin (loading control) antibodies. C) Optical density of LAMP-1 immunoreactive bands were normalized against their respective actin bands and averaged (± SD) from three independent experiments. * p < 0.05 compared to mock transfected cells, **p < 0.01 compared to mock transfected cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Over-expression of α-synuclein attenuated the phagocytic ability of BV2 cells and lysosomal protein expression. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein for 48 hours. A) Transfected cells were incubated with FITC-labeled E. coli bioparticles (0.25 mg/mL) for 3 hours. After incubation, the media was removed and the signal from any unphagocytosed or membrane associated particles was quenched by incubating cells with a (0.25 mg/mL) trypan blue solution for 3 minutes. The fluorescence intensity of phagocytosed particles was measured via fluorescent plate reader (RFU). Each condition was performed with 8 replicates. B) Transfected cells were also lysed and Western blotted with anti-LAMP-1 and actin (loading control) antibodies. C) Optical density of LAMP-1 immunoreactive bands were normalized against their respective actin bands and averaged (± SD) from three independent experiments. * p < 0.05 compared to mock transfected cells, **p < 0.01 compared to mock transfected cells.
Mentions: Based upon several prior reports that prostaglandins can negatively regulate macrophage and microglial phagocytic ability [53-57] and our observation of increased Cox-2 protein levels in α-synuclein over-expressing cells we examined whether transfected cells would display an expected decrease in phagocytic ability. To examine changes in microglial phagocytic ability, uptake of FITC-labeled E. coli bioparticles was quantified from transfected cells. Consistent with the observed increase in Cox-2 protein levels, cells transfected to express WT, the A30P, or A53T mutants all displayed a significant decrease in ability to phagocytose the bioparticles compared to mock transfected cells (Figure 3A). Moreover, the decrease of phagocytic ability with over-expression correlated with a significant decrease in protein levels of the lysosomal marker protein, lysosome associated protein 1 (LAMP-1) (Figure 3B and 3C). These data again confirm that microglial over-expression of wild type or mutant α-synuclein results in an altered phenotype.

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