Expression of mutant alpha-synuclein modulates microglial phenotype in vitro

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.

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.

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.

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.

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.

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.