Toll-like receptor 4 is required for α-synuclein dependent activation of microglia and astroglia

Abstract

Alpha-synucleinopathies (ASP) are neurodegenerative disorders, characterized by accumulation of misfolded α-synuclein, selective neuronal loss, and extensive gliosis. It is accepted that microgliosis and astrogliosis contribute to the disease progression in ASP. Toll-like receptors (TLRs) are expressed on cells of the innate immune system, including glia, and TLR4 dysregulation may play a role in ASP pathogenesis. In this study we aimed to define the involvement of TLR4 in microglial and astroglial activation induced by different forms of α-synuclein (full length soluble, fibrillized, and C-terminally truncated). Purified primary wild type (TLR4(+/+)) and TLR4 deficient (TLR4(-/-)) murine microglial and astroglial cell cultures were treated with recombinant α-synuclein and phagocytic activity, NFκB nuclear translocation, cytokine release, and reactive oxygen species (ROS) production were measured. We show that TLR4 mediates α-synuclein-induced microglial phagocytic activity, pro-inflammatory cytokine release, and ROS production. TLR4(-/-) astroglia present a suppressed pro-inflammatory response and decreased ROS production triggered by α-synuclein treatment. However, the uptake of α-synuclein by primary astroglia is not dependent on TLR4 expression. Our results indicate the C-terminally truncated form as the most potent inductor of TLR4-dependent glial activation. The current findings suggest that TLR4 plays a modulatory role on glial pro-inflammatory responses and ROS production triggered by α-synuclein. In contrast to microglia, the uptake of alpha-synuclein by astroglia is not dependent on TLR4. Our data provide novel insights into the mechanisms of α-synuclein-induced microglial and astroglial activation which may have an impact on understanding the pathogenesis of ASP.

Figure 1

Microglial phagocytic activity is increased after AS-treatments, but diminished in TLR4-deficient microglia. Initially, the purity and oligomeric state of the recombinant AS preparations was determined. A: Immunoblot was performed of full length soluble, fibrillized (14-day old), and C-terminally truncated AS (sAS, fAS, and tAS). In addition to the monomer band fAS shows different oligomer species. B: The fibrillization of AS incubated at 37°C was controlled using the Thioflavin T assay. The amount of fibrillized AS was significantly augmented after 14 days at 37°C. No significant change in the degree of fibrillization was detected between AS probes after 14-day and 20-day incubation. Therefore, 14-day-old fibrillized AS (fAS) was used for all the experiments. Data were analyzed by one-way analysis of variance for multiple comparisons. ***P < 0.001, **P < 0.01 compared with 5-day incubation. Next, primary murine microglia were treated with 3 μM sAS (full length soluble AS), fAS (full length fibrillized AS) or tAS (C-terminally truncated AS), and fluorescent microspheres (red) were added. Cells were fixed with 4% paraformaldehyde and immunostained for CD11b (green). C: Morphometric analysis of CD11b-labeled microglia with incorporated microspheres revealed that AS treatment (all forms) led to a significant increase of phagocyting cells. TLR4 deficiency induced a decline of microglial phagocytic activity. Data were analyzed by two-way ANOVA with post-hoc Bonferroni test. Results are presented as mean ± S.D. +++P < 0.001 compared with untreated TLR4^+/+ microglia, ***P < 0.001, **P < 0.01 comparison of TLR4^+/+ to TLR4^−/− microglia, $$$P < 0.001 compared with untreated TLR4^−/− microglia. n = 5. D: Confocal microscopy of microglia revealed AS-dependent increase of phagocytosed microspheres by TLR4^+/+ microglia (scale bar, 10 μm), whereas TLR4 ablated microglia featured reduced phagocytosis of fluorescent microspheres. Representative for all three treatments we show microglia treated with sAS and fluorescent microspheres. E: A representative confocal image stack confirmed the uptake of the microspheres (red) into CD11b-positive microglia, and further demonstrated that the amount of incorporated microspheres is decreased in TLR4^−/− microglia compared with TLR4^+/+ microglia (scale bar, 5 μm).

Figure 2

NF-κB nuclear translocation upon AS treatment in TLR4^+/+ microglia. Murine primary microglia were treated with different AS-forms (full length soluble AS–sAS, fibrillized AS–fAS, and C-terminally truncated AS–tAS). Cells were fixed with 4% paraformaldehyde, immunostained for NF-κB (green) and counterstained with DAPI (blue). NF-κB translocation in TLR4^+/+ microglia treated with AS was identified by co-localization of DAPI and NF-κB (see arrows). No co-localization was observed in untreated and AS treated TLR4^−/− microglia (scale bar, 10 μm).

Figure 3

Inflammatory responses of TLR4^+/+ and TLR4^−/− microglia upon AS treatment. Cytokine measurements were performed using the fluorometric multiplex bead-based immunoassay (BenderSystems). Primary murine microglia (TLR4^+/+ and TLR4^−/−) were treated with different AS forms (full length soluble AS–sAS, fibrillized AS–fAS, and C-terminally truncated AS–tAS) or LPS as positive assay control for 2, 12, and 24 h. The supernatants were collected and used for quantifying the amount of secreted TNF-α (A, B), IL-6 (C, D) and CXCL1 (E, F). TLR4 deficiency lead to a significantly decreased TNF-α, IL-6, and CXCL1 production, especially after tAS and control LPS treatment, whereas the treatment with tAS induced a significantly elevated TNF-α, IL-6, and CXCL1 production in TLR4^+/+ microglia. Data were analyzed by two-way ANOVA with post-hoc Bonferroni test. Data are the average (±SD) of three independent experiments. +++ P < 0.001, + P < 0.05 compared with untreated TLR4^+/+ microglia, ***P < 0.001 comparison of TLR4^+/+ to TLR4^−/− microglia.

Figure 4

TLR4 ablation results in a reduction of ROS production by glial cells after exposure to AS. Primary murine microglial and astroglial cells were treated with different AS forms (full length soluble AS–sAS, fibrillized AS–fAS, and C-terminally truncated AS–tAS). Nitroblue tetrazolium chloride was added to visualize ROS production. Cells were fixed with 4% paraformaldehyde and ROS-positive cells were counted using an inverse microscope. A: Treatment with all AS-forms led to an enhanced ROS release by TLR4^+/+ microglia. TLR4 deficient microglia showed a reduced production of ROS in comparison to TLR4^+/+ microglia. Data were analyzed by two-way ANOVA with post-hoc Bonferroni test. Results are presented as mean ± S.D. +++ P < 0.001, ++ P < 0.01 compared with untreated TLR4^+/+ microglia, ***P < 0.001 comparison of TLR4^+/+ to TLR4^−/− microglia. n = 3 (excl. untreated TLR4^+/+ and TLR4^−/− microglia n = 5). B: Light microscopy of microglia visualizing the difference of ROS production by TLR4^+/+ and TLR4^−/− microglia (scale bar, 10 μm). C: Treatment with all AS forms initiated augmented ROS production by TLR4^+/+ astroglia, whereas, TLR4 ablated astroglia showed a decreased ROS production in comparison to TLR4^+/+ astroglia. Data were analyzed by two-way ANOVA with post-hoc Bonferroni test. Results are presented as mean ± S.D. +++ P < 0.001, ++ P < 0.01, + P < 0.05 compared with untreated TLR4^+/+ astroglia, ***P < 0.001, **P < 0.01, *P < 0.05 comparison of TLR4^+/+ to TLR4^−/− microglia n = 3.

Figure 5

Uptake of AS by aged primary murine astroglial cells. Astroglial cells were treated with different AS-forms (full length soluble AS–sAS, fibrillized AS–fAS, and C-terminally truncated AS–tAS). Cells were fixed with 4% paraformaldehyde and immunostained for GFAP (green) and AS (red). A: Both TLR4^+/+ and TLR4^−/− astroglial cells presented incorporated AS in the cytoplasm (scale bar 20 μm). B: Representative three-dimensionally reconstructed confocal images of GFAP-positive astroglia with incorporated sAS are shown (scale bar 10 μm).

Figure 6

Analysis of cytokine and chemokine release upon AS treatment of aged primary astroglia. FlowCytomix analyses of TNF-α (A, B), IL-6 (C, D), and CXCL1 (E, F) were performed using supernatants of AS- or LPS-treated primary murine astroglial cells after 2, 12, and 24 h. Especially tAS caused a significant cytokine/chemokine release in TLR4^+/+ astroglia. In comparison, TLR4 ablation reduced the released cytokine amount significantly. Data were analyzed by two-way ANOVA with post-hoc Bonferroni test. Results are presented as mean ± S.D. +++ P < 0.001 compared with untreated TLR4^+/+ astroglia, *** P < 0.001, ** P < 0.01 comparison of TLR4^+/+ to TLR4^−/− astroglia, and $$ P < 0.01 compared with untreated TLR4^−/− astroglia. n = 3.