α-Synuclein Alters Toll-Like Receptor Expression

Abstract

Parkinson's disease, an age-related neurodegenerative disorder, is characterized by the loss of dopamine neurons in the substantia nigra, the accumulation of α-synuclein in Lewy bodies and neurites, and neuroinflammation. While the exact etiology of sporadic Parkinson's disease remains elusive, a growing body of evidence suggests that misfolded α-synuclein promotes inflammation and oxidative stress resulting in neurodegeneration. α-Synuclein has been directly linked to microglial activation in vitro and increased numbers of activated microglia have been reported in an α-synuclein overexpressing mouse model prior to neuronal loss. However, the mechanism by which α-synuclein incites microglial activation has not been fully described. Microglial activation is governed in part, by pattern recognition receptors that detect foreign material and additionally recognize changes in homeostatic cellular conditions. Upon proinflammatory pathway initiation, activated microglia contribute to oxidative stress through release of cytokines, nitric oxide, and other reactive oxygen species, which may adversely impact adjacent neurons. Here we show that microglia are directly activated by α-synuclein in a classical activation pathway that includes alterations in the expression of toll-like receptors. These data suggest that α-synuclein can act as a danger-associated molecular pattern.

Keywords: DAMP; Parkinson's disease; inflammation; microglial activation; pattern recognition receptors.

Figure 1

Manipulation of α-synuclein results in the formation of aggregates. (A) α-Synuclein prepared in TEN buffer or TEN buffer alone was incubated at 33–37°C with mechanical rotation (1000 rpm) for 5 days (SYN^TR; Buffer^TR). SYN^TR was then subjected to anti-synuclein Western blot analysis following separation on a 4–20% Tris–glycine polyacrylamide gel under denaturing conditions. Arrows indicate SDS-stable α-synuclein aggregates. (B) Thioflavin T fluorescence measurements were obtained using an excitation wavelength of 450 nm and an emission wavelength of 490 nm. SYN^TR contains amyloid fibrils (*P < 0.05; each data point represents mean ± SD, n = 3). (C) SYN^TR and Buffer^TR were subjected to AFM. This analysis demonstrated the formation of α-synuclein aggregates in the SYN^TR samples. AFM height images of SYN^TRand similarly manipulated buffer are shown with the height of aggregates displayed along the z-axis. Height images shown at 2 μm × 2 μm × 2 μm. (D) AFM height measurements of SYN^TR. The largest proportion of SYN^TR had a height of <5 nm, likely representing monomeric α-synuclein, but aggregates of >10 nm were also present.

Figure 2

SYN^TR treatment stimulates nitric oxide (NO) release from microglia. BV-2 cells were treated with 50 nM of SYN^TR or Buffer^TR for 2–36 h. Following treatment, a Greiss reagent assay was performed on the conditioned media to determine NO release and subsequent nitrite production. NO release was significantly higher in cells treated with SYN^TR than buffer control beginning at 6 h of treatment (*P < 0.05, n = 3). NO release reached a maximum by 24 h of treatment. Nitrite concentrations are represented as mean ± SD; ND indicates none detected.

Figure 3

SYN^TR treatment increases the expression and release of proinflammatory molecules. (A) BV-2 cells were treated with 50 nM of SYN^TR or Buffer^TR for 24 h. Following treatment, the conditioned media were evaluated for TNF-α protein secretion using an ELISA. Cells treated with SYN^TR released significantly more TNF-α than Buffer^TR treated microglia (ND indicates none detected; *P < 0.05, n = 3). (B) Quantitative RT-PCR for IL1β was performed on cDNA from SYN^TR and buffer treated cells. Microglia treated with SYN^TR had significantly higher expression of IL1β than buffer treated cells (*P < 0.05; n = 3). Expression values were normalized to 18S rRNA as an internal control. Statistics were performed on ΔCt values.

Figure 4

SYN^TR treatment increases the expression of antioxidant response genes. (A) Quantitative RT-PCR for (A) peroxiredoxin-1 (PRDX1) and (B) heme oxygenase-1 (HMOX1) was performed on cDNA from Buffer^TR- or SYN^TR-treated BV-2 cells. Cells treated with SYN^TR (50 nM) had a significantly higher expression of PRDX1 and HMOX1 compared to buffer treated cells (Buffer^TR; *P < 0.05). Expression values were normalized to GAPDH as an internal control. Statistics were performed on ΔCt values. (C) Representative HO-1 Western blot analysis of BV-2 cell lysates following treatment. BV-2 cells were treated with 50 nM SYN^TR or Buffer^TR for 24 h. Protein lysates were prepared and subjected to 10% SDS-polyacrylamide gel electrophoresis and immunoblotted for HO-1. Blots were reprobed for α-tubulin as a loading control. (D) Immunocomplexes were quantified by densitometric analysis and normalized to the loading control. BV-2 cells treated with SYN^TR had significantly higher levels of HO-1 protein than buffer treated cells (Buffer^TR; *P < 0.05; n = 3).