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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 did not increase neurotoxic secretion from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein then co-cultured onto a membrane insert with 7 days in vitro mouse cortical neurons for 72 hours in the A) absence or B) presence of 25 ng/ml LPS stimulation. After 72 hours, the inserts were removed and the neurons were fixed in 4% paraformaldehyde and immunostained with an anti-MAP2 antibody. MAP2 positive cells were counted to assess viability. Experiments were performed with 8 replicates per condition. Graphs are the average (± SD) of three independent experiments. *** p < 0.001 compared to neurons only.
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Figure 5: Over-expression of α-synuclein did not increase neurotoxic secretion from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein then co-cultured onto a membrane insert with 7 days in vitro mouse cortical neurons for 72 hours in the A) absence or B) presence of 25 ng/ml LPS stimulation. After 72 hours, the inserts were removed and the neurons were fixed in 4% paraformaldehyde and immunostained with an anti-MAP2 antibody. MAP2 positive cells were counted to assess viability. Experiments were performed with 8 replicates per condition. Graphs are the average (± SD) of three independent experiments. *** p < 0.001 compared to neurons only.

Mentions: Since we as well as others have demonstrated that TNF-α can alter neuronal activity and potentiate toxicity [32,61-66] it was reasonable to expect that α-synuclein over-expressing cells would demonstrate increased secretion of neurotoxic factors. To determine whether transfected BV2 cells had increased neurotoxic capacity, transfected cells were co-cultured with and without LPS in primary murine cortical neurons at 7 days in vitro for three days to assess effects on neuron survival. In spite of the elevated levels of cytokines and nitric oxide secreted from α-synuclein over-expressing cells, they demonstrated no increase in neurotoxic secretion above that induced by mock transfected cells with or without LPS stimulation (Figure 5). This demonstrated that although the reactive phenotype induced by α-synuclein over-expression included elevated proinflammatory secretion, this was not sufficient to induce an increased neurotoxic response in vitro in these particular culture conditions in which toxicity was already maximal in control BV2/neuron co-cultures.


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

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

Over-expression of α-synuclein did not increase neurotoxic secretion from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein then co-cultured onto a membrane insert with 7 days in vitro mouse cortical neurons for 72 hours in the A) absence or B) presence of 25 ng/ml LPS stimulation. After 72 hours, the inserts were removed and the neurons were fixed in 4% paraformaldehyde and immunostained with an anti-MAP2 antibody. MAP2 positive cells were counted to assess viability. Experiments were performed with 8 replicates per condition. Graphs are the average (± SD) of three independent experiments. *** p < 0.001 compared to neurons only.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Over-expression of α-synuclein did not increase neurotoxic secretion from BV2 cells. BV2 cells were transiently transfected to express WT, A30P, or A53T α-synuclein then co-cultured onto a membrane insert with 7 days in vitro mouse cortical neurons for 72 hours in the A) absence or B) presence of 25 ng/ml LPS stimulation. After 72 hours, the inserts were removed and the neurons were fixed in 4% paraformaldehyde and immunostained with an anti-MAP2 antibody. MAP2 positive cells were counted to assess viability. Experiments were performed with 8 replicates per condition. Graphs are the average (± SD) of three independent experiments. *** p < 0.001 compared to neurons only.
Mentions: Since we as well as others have demonstrated that TNF-α can alter neuronal activity and potentiate toxicity [32,61-66] it was reasonable to expect that α-synuclein over-expressing cells would demonstrate increased secretion of neurotoxic factors. To determine whether transfected BV2 cells had increased neurotoxic capacity, transfected cells were co-cultured with and without LPS in primary murine cortical neurons at 7 days in vitro for three days to assess effects on neuron survival. In spite of the elevated levels of cytokines and nitric oxide secreted from α-synuclein over-expressing cells, they demonstrated no increase in neurotoxic secretion above that induced by mock transfected cells with or without LPS stimulation (Figure 5). This demonstrated that although the reactive phenotype induced by α-synuclein over-expression included elevated proinflammatory secretion, this was not sufficient to induce an increased neurotoxic response in vitro in these particular culture conditions in which toxicity was already maximal in control BV2/neuron co-cultures.

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