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. 2011 Aug;179(2):954-63.
doi: 10.1016/j.ajpath.2011.04.013. Epub 2011 Jun 14.

Toll-like receptor 4 promotes α-synuclein clearance and survival of nigral dopaminergic neurons

Nadia Stefanova  1 , Lisa Fellner, Markus Reindl, Eliezer Masliah, Werner Poewe, Gregor K Wenning
Affiliations

Toll-like receptor 4 promotes α-synuclein clearance and survival of nigral dopaminergic neurons

Nadia Stefanova et al. Am J Pathol. 2011 Aug.

Abstract

Toll-like receptors (TLRs) mediate innate immunity, and their dysregulation may play a role in α-synucleinopathies, such as Parkinson's disease or multiple system atrophy (MSA). The aim of this study was to define the role of TLR4 in α-synuclein-linked neurodegeneration. Ablation of TLR4 in a transgenic mouse model of MSA with oligodendroglial α-synuclein overexpression augmented motor disability and enhanced loss of nigrostriatal dopaminergic neurons. These changes were associated with increased brain levels of α-synuclein linked to disturbed TLR4-mediated microglial phagocytosis of α-synuclein. Furthermore, tumor necrosis factor-α levels were increased in the midbrain and associated with a proinflammatory astroglial response. Our data suggest that TLR4 ablation impairs the phagocytic response of microglia to α-synuclein and enhances neurodegeneration in a transgenic MSA mouse model. The study supports TLR4 signaling as innate neuroprotective mechanism acting through clearance of α-synuclein.

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Figures

Figure 1
Figure 1
Locomotor deterioration and neurodegeneration in AS,TLR4−/− mice. A: Quantification of vertical (rearing) and horizontal open field activity in the Flex Field Activity System shows significant decrease of rearing behavior of AS,TLR4−/− mice versus AS,TLR4+/+ mice (for each n = 8), whereas at baseline (wild type indicates without AS overexpression) no significant difference was detected between TLR4+/+ mice and TLR4−/− mice. B: Pole test revealed significant increase of the time needed to turn down on a vertical pole without significant change in the total time needed to descend the pole in AS,TLR4−/− mice. Again at baseline (wild type indicates without AS overexpression) no significant difference was detected between TLR4+/+ mice and TLR4−/− mice. Data were analyzed by two-way analysis of variance (with factors AS overexpression and TLR4 expression) with the post hoc Bonferroni test. Results are presented as mean ± SEM. C: Quantification of dopaminergic neurons by optical fractionator showed significantly augmented neuronal loss in SNc of AS,TLR4−/− mice but no nigral degeneration due to TLR4 deficiency only. Data were analyzed by two-way analysis of variance (with factors AS overexpression and TLR4 expression) with a post hoc Bonferroni test. Results are presented as mean ± SEM (for each group n = 6). D: Quantification of nigral neurons performed in cresyl violet–stained series throughout SNc confirmed amplified neuronal loss in AS,TLR4−/− compared with AS,TLR4+/+ mice. E: Parallel to the enhanced nigral neuronal loss in AS,TLR4−/− mice, TH optical density (OD) measurement in striatum indicated significant loss of striatal dopaminergic fibers in AS,TLR4−/− mice. F: DARPP-32 IHC for striatal medium spiny neurons showed no significant difference between AS,TLR4+/+ and AS,TLR4−/− mice. Groups were compared by unpaired two-tailed t-test. Results are presented as mean ± SEM (for each group n = 6). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P = 0.0001.
Figure 2
Figure 2
Phagocytosis of AS is modulated by TLR4. A: Genetically modified U373 cells with AS inclusion pathology as previously described were killed by freeze-thaw cycle, and the resulting suspension of AS debris was used to treat BV2 immortalized microglia. After 15 minutes of incubation, large AS debris marked by red fluorescence was observed in the vicinity of microglia. After 2 hours of incubation, AS debris was almost completely incorporated by microglia and small bodies containing AS (red fluorescence) could be observed in the cytoplasm of microglial cells. Scale bar = 50 μm. B: Phagocytosis of recombinant AS (labeled with 15G7 antibody) by BV2 microglia was visualized by Alexa 488 green fluorescence (arrows point toward labeled vesicular bodies in the microglial cytoplasm). Preincubation of the BV2 cells with functional TLR4 antibody resulted in suppression of AS phagocytosis as evidenced by disappearance of the Alexa 488–labeled vesicular cytoplasmic bodies. Main scale bar = 50 μm, inset scale bar = 10 μm. C: Phagocytosis of recombinant AS after 1-hour incubation with primary mouse microglia (TLR4+/+ or TLR4−/−) was visualized with anti-hAS immunocytochemistry and Alexa 594 red fluorescence (arrows). Cells were counterstained with fluorescein isothiocyanate–conjugated wheat germ agglutinin (WGA), and the percentage of phagocyting cells was defined to be 40% in TLR4+/+ microglia (123 of 300 cells analyzed) and 2% (11 of 514 cells analyzed) in TLR4−/− microglia. Scale bar = 10 μm.
Figure 3
Figure 3
A: Immunofluorescence for CD11b (red) and hAS (green) in the brain of AS,TLR4+/+ mice revealed punctuate hAS-positive structures (arrow) in the cytoplasm of CD11b-positive microglia opposing a hAS-expressing oligodendrocyte (scale bar = 7 μm) as evidenced by double immunofluorescence for hAS and CNP (2′,3′-cyclic nucleotide 3′-phosphohydrolase, an oligodendroglial marker, scale bar = 5 μm.). B: In contrast in AS,TLR4−/− mice hAS strictures (arrow) appeared only outside CD11b-positive microglia (scale bar = 15 μm). C: The localization of hAS in microglia of AS,TLR4+/+ mice was further confirmed by a conventional confocal image stack showing hAS (green) dot (arrow) and CD11b-labeled microglia (red). Below (xz) and right (yz) views through the depth of the image stack at the position represented by the white line, demonstrating hAS-positive punctuate structure engulfed by microglia. D: Advanced interactive three-dimensional image analysis with isosurface reconstruction of a Z-stack of x-y sections demonstrated hAS (green, arrow) inside a CD11b-positive microglial cell (red). Section view in the x- and y-axes was generated by using the clipping function of Imaris x64 software. Scale bar = 2 μm.
Figure 4
Figure 4
Electron microscopic analysis of the brains of AS,TLR4+/+ and AS,TLR4−/− mice. Microglia and macrophage cells were identified close to the perivascular areas by their elongated nucleus with condensed chromatin and abundant lysosomes and phagocytic organelles in the cytoplasm. In AS,TLR4+/+ mice, microglia and macrophages showed abundant gold particles in association with phagosomes and lysosomes. In contrast, microglia and macrophages of AS,TLR4−/− mice had fewer gold particles in phagocytic cytoplasmic organelles as defined by the number of gold grains per microglia and macrophage cell (n = 125 for each). Original magnification, ×5000 and ×25,000, respectively. ***P = 0.001.
Figure 5
Figure 5
Effect of TLR4 ablation on AS brain levels. Four animals per group were used to dissect the forebrain and midbrain and prepare lysates for measurement of hAS protein concentration by ELISA. Two-way analysis of variance (with factors genotype and area) with a post hoc Bonferroni test showed a significant increase of hAS protein concentration in both the forebrain and midbrain of AS,TLR4−/− mice. ***P = 0.001, ****P < 0.0001.
Figure 6
Figure 6
Inflammatory mediation in ASPs in the absence of TLR4. A: Flow cytomix analysis of IL-10, IFN-γ, IL-6, and GM-CSF in midbrain and forebrain lysates demonstrated no significant differences between AS,TLR4+/+ and AS,TLR4−/− mice. However, strongly increased production of TNF-α was detected in the midbrain of AS,TLR4−/− mice. For all analyses, n = 6 (excluding IL-10 AS,TLR4−/− mice, n = 4). Data were analyzed by two-way analysis of variance (with factors genotype and area) with a post hoc Bonferroni test. Results are presented as mean ± SEM. B: Immunofluorescence in SNc of AS,TLR4−/− mice identified GFAP-positive astroglia (red) as the main source of TNF-α (green). Scale bar = 10 μm. ****P < 0.0001.
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