Alpha-synuclein expression modulates microglial activation phenotype

Abstract

Recent Parkinson's disease research has focused on understanding the function of the cytosolic protein, alpha-synuclein, and its contribution to disease mechanisms. Within neurons, alpha-synuclein is hypothesized to have a role in regulating synaptic plasticity, vesicle release, and trafficking. In contrast, glial-expressed alpha-synuclein remains poorly described. Here, we examine the consequence of a loss of alpha-synuclein expression on microglial activation. Using a postnatal brain-derived culture system, we defined the phenotype of microglia from wild-type and knock-out alpha-synuclein mice (Scna-/-). Scna-/- microglia displayed a basally increased reactive phenotype compared with the wild-type cells and an exacerbated reactive phenotype after stimulation. They also exhibited dramatic morphologic differences compared with wild-type, presenting as large, ramified cells filled with vacuole-like structures. This corresponded with increased protein levels of activation markers, CD68 and beta1 integrin, in the Scna-/- cells. More importantly, Scna-/- microglia, after stimulation, secreted elevated levels of proinflammatory cytokines, TNFalpha (tumor necrosis factor alpha) and IL-6 (interleukin-6), compared with wild type. However, despite the reactive phenotype, Scna-/- cells had impaired phagocytic ability. We demonstrate for the first time that alpha-synuclein plays a critical role in modulating microglial activation state. We suggest that altered microglial alpha-synuclein expression will affect their phenotype as has already been demonstrated in neurons. This has direct ramifications for the contribution of microglia to the pathophysiology of disease, particularly in familial cases linked to altered alpha-synuclein expression.

Figure 1.

Microglia from Scna^−/− mice display a ramified, heavily vacuole-laden phenotype in vitro compared with wild-type microglia. Microglia were isolated from 14 d in vitro mixed glia cultures and plated onto tissue culture plastic for 24 h. A, Cells were fixed in 4% paraformaldehyde and immunostained with an anti-CD68 antibody. Antibody binding was visualized using Vector VIP as the chromagen. B, Cells were double-labeled using anti-CD68 and anti-α-synuclein antibodies with FITC- and Texas Red-conjugated secondary antibodies, respectively. Nuclei were stained with DAPI before mounting for confocal imaging. Contrast of merged images was increased to better illustrate double label. The arrows indicate large vacuole-like structures present in Scna^−/− microglia.

Figure 2.

Microglia from Scna^−/− mice display increased levels of reactive marker proteins compared with wild-type microglia. Microglia from P1–P2 Scna^−/− and wild-type mice were cultured for 14 d in vitro as mixed glia cultures. At 14 d in vitro, microglia were isolated and plated for 24 h. A, Cells were lysed, and proteins were resolved by 10% SDS-PAGE. Lysates were Western blotted using anti-β1 integrin, α-tubulin, and CD68 antibodies. Equal protein loading was verified using an anti-ERK2 antibody (loading control). B, Optical density of β1 integrin, α-tubulin, and CD68 protein bands from five independent culture preparations derived from different litters were normalized against respective ERK2 levels and averaged (±SD) (**p < 0.001 and *p < 0.05 from respective control). C, Microglia isolated at 14 d in vitro were stimulated with or without 50 ng/ml LPS for 24 h, and then lysed, and proteins were resolved by 10% SDS-PAGE. Lysates from three independent culture preparations from different litters were immunoblotted using anti-β1 integrin and α-tubulin antibodies. Blots were visualized via enhanced chemiluminescence.

Figure 3.

Microglia from Scna^−/− mice secrete increased levels of proinflammatory cytokines after stimulation compared with wild-type microglia. Microglia were isolated from Scna^−/− and wild-type mixed glia cultures at 14 d in vitro and plated in the absence or presence of 25 ng/ml LPS for 24 h. Conditioned medium was collected from the cells and secreted TNFα (A) and IL-6 (B) concentrations were determined using commercial ELISA. Graphs are representative of four independent experiments. Each experiment was performed with eight replicates per condition, and values were averaged (±SD). *p < 0.001 from respective control.

Figure 4.

Microglia from Scna^−/− mice have decreased phagocytic ability compared with wild-type microglia. Microglia from Scna^−/− and wild-type control mice were cultured as mixed glia for 14 d in vitro. Microglia were isolated at 14 d and plated with or without FITC-labeled E. coli bioparticles (0.25 mg/ml) for 3 h. After the incubation, the medium was removed and the signal from unphagocytosed or extracellular membrane-associated FITC-labeled bioparticles was quenched by rinsing with 0.25 mg/ml trypan blue solution. Fluorescence intensity [relative fluorescence units (RFU)] of phagocytosed bioparticles was measured via a fluorescent plate reader (480 nm excitation and 520 nm emission) and averaged (±SD). Each condition was performed with eight replicates, and the graph is representative of three independent experiments. *p < 0.001 from respective control.