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Cited by 156Neuroinflammation and Neurodegeneration MechanismsParkinson's Disease Mechanisms and TreatmentsNuclear Receptors and Signaling

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The acute inflammatory response to intranigral α-synuclein differs significantly from intranigral lipopolysaccharide and is exacerbated by peripheral inflammation

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Couch, Yvonne, Alvarez-Erviti, Lydia, Sibson, Nicola R, Wood, Matthew JA, Anthony, Daniel C·2011·Journal of Neuroinflammation
DOI 10.1186/1742-2094-8-166PMC PMC3239418
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Abstract

Background: Activated microglia are a feature of the host response to neurodegeneration in Parkinson's disease (PD) and are thought to contribute to disease progression. Recent evidence suggests that extracellular α-synuclein (eSNCA) may play an important role in the pathogenesis of PD and that this may be mediated by a microglial response. Methods: We wished to discover whether the host response to eSNCA would be sufficient to induce significant cytokine production. In vitro cultured BV-2 microglia were used to determine the basic inflammatory response to eSNCA. In vivo , 8-week old Biozzi mice were subjected to a single intranigral injection of either 3 μg SNCA, lipopolysaccharide (LPS) or serum protein (BSA) and allowed to recover for 24 hours. A second cohort of animals were peripherally challenged with LPS (0.5 mg/kg) 6 hours prior to tissue collection. Inflammation was studied by quantitative real-time PCR for a number of pro-inflammatory genes and immunohistochemistry for microglial activation, endothelial activation and cell death. Results: In vitro data showed a robust microglial response to SNCA, including a positive NFĸB response and the production of pro-inflammatory cytokines. Direct injection of SNCA into the substantia nigra resulted in the upregulation of mRNA expression of proinflammatory cytokines, the expression of endothelial markers of inflammation and microglial activation. However, these results were significantly different to those obtained after direct injection of LPS. By contrast, when the animals were injected intracerebrally with SNCA and subsequently challenged with systemic LPS, the level of production of IL-1β in the substantia nigra became comparable to that induced by the direct injection of LPS into the brain. The injection of albumin into the nigra with a peripheral LPS challenge did not provoke the production of a significant inflammatory response. Direct injection of LPS into the substantia nigra also induces cell death in a more robust manner than direct injection of either SNCA or BSA. Conclusion: These results suggest that the presence of eSNCA protein 'primes' microglia, making them susceptible to environmental proinflammatory challenge. For this reason, we hypothesise that where 'inflammation' contributes to the disease progression in PD, it does so in a punctuate manner (on-off) as a result of systemic events.

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Figures

Figure 1 NFĸB p65 subunit translocation to the nucleus 24 h after treatment with α-synuclein (SNCA) or amyloid-β. Cells were treated with vehicle (A-C); 3 μg SNCA (D-F) or 3 μg amyloid-β (G-I) for 24 hours at which point cells were fixed and immunostained for the p65 subunit of NFĸB (green; localization indicated by white arrows) and mounted in medium containing the nuclear stain DAPI. Note that SNCA and amyloid-β caused the p65 subunit to translocate to the nucleus (blue; co-localization indicated by red arrows). Scale bar represents 50 μm.
Figure 1 NFĸB p65 subunit translocation to the nucleus 24 h after treatment with α-synuclein (SNCA) or amyloid-β. Cells were treated with vehicle (A-C); 3 μg SNCA (D-F) or 3 μg amyloid-β (G-I) for 24 hours at which point cells were fixed and immunostained for the p65 subunit of NFĸB (green; localization indicated by white arrows) and mounted in medium containing the nuclear stain DAPI. Note that SNCA and amyloid-β caused the p65 subunit to translocate to the nucleus (blue; co-localization indicated by red arrows). Scale bar represents 50 μm.CC BY · PubMed Central
Figure 2 TNFα release from BV2 cells increases after treatment with LPS and SNCA but not with amyloid-β. Histogram shows TNFα release (pg/ml) from BV-2 cells as assessed by ELISA at time-points after treatment with vehicle (PBS), 3 μg SNCA, 3 μg amyloid-β or 10 ng LPS. Error bars represent mean ± S.E.M. * represents P < 0.05; ** represents P < 0.01 and *** represents P < 0.001 when compared to control values. # represents significance compared to amyloid-β.
Figure 2 TNFα release from BV2 cells increases after treatment with LPS and SNCA but not with amyloid-β. Histogram shows TNFα release (pg/ml) from BV-2 cells as assessed by ELISA at time-points after treatment with vehicle (PBS), 3 μg SNCA, 3 μg amyloid-β or 10 ng LPS. Error bars represent mean ± S.E.M. * represents P < 0.05; ** represents P < 0.01 and *** represents P < 0.001 when compared to control values. # represents significance compared to amyloid-β.CC BY · PubMed Central
Figure 3 Cytokine mRNA expression in the brain 24 hours after SNCA or BSA injection into the substantia nigra. mRNA levels of (A) NFĸB; (B) TNFα; (C) IL-1β; (D) IL-6; (E) TGFβ and (F) COX-2 measured as values relative to GAPDH and normalized to levels within the contralateral hemisphere of control animals. Error bars indicate mean ± SEM. Dotted line represents basal levels in naïve animals. * indicates a significance of P < 0.05 when compared to naïve animals; **indicates P < 0.01 and *** indicates P < 0.005.
Figure 3 Cytokine mRNA expression in the brain 24 hours after SNCA or BSA injection into the substantia nigra. mRNA levels of (A) NFĸB; (B) TNFα; (C) IL-1β; (D) IL-6; (E) TGFβ and (F) COX-2 measured as values relative to GAPDH and normalized to levels within the contralateral hemisphere of control animals. Error bars indicate mean ± SEM. Dotted line represents basal levels in naïve animals. * indicates a significance of P < 0.05 when compared to naïve animals; **indicates P < 0.01 and *** indicates P < 0.005.CC BY · PubMed Central
Figure 4 Cytokine mRNA expression in the brain 24 hours after LPS injection into the substantia nigra. mRNA levels of (A) NFkB; (B) TNF; (C) IL-1β; (D) IL-6; (E) TGFβ and (F) COX-2 measured as values relative to GAPDH and normalized to levels within the contralateral hemisphere of control animals. Error bars indicate mean ± SEM. Dotted line represents basal levels in naïve animals. * indicates a significance of P < 0.05 when compared to naïve animals and *** indicates P < 0.005.
Figure 4 Cytokine mRNA expression in the brain 24 hours after LPS injection into the substantia nigra. mRNA levels of (A) NFkB; (B) TNF; (C) IL-1β; (D) IL-6; (E) TGFβ and (F) COX-2 measured as values relative to GAPDH and normalized to levels within the contralateral hemisphere of control animals. Error bars indicate mean ± SEM. Dotted line represents basal levels in naïve animals. * indicates a significance of P < 0.05 when compared to naïve animals and *** indicates P < 0.005.CC BY · PubMed Central
Figure 5 Increased Iba-1 and ICAM-1 expression 24 hours after intranigral administration of SNCA. Representative microscopy showing typical Iba-1 (A & B) and ICAM-1 (C & D) staining seen 24 h after injection of SNCA (A & C) or BSA (B & D; both micrographs are counterstained with cresyl violet). Scale bar represents 10 μm. The number of Iba-1-positive microglia (E) or ICAM-1- positive vessels were quantified in both SNCA and BSA injected animals.
Figure 5 Increased Iba-1 and ICAM-1 expression 24 hours after intranigral administration of SNCA. Representative microscopy showing typical Iba-1 (A & B) and ICAM-1 (C & D) staining seen 24 h after injection of SNCA (A & C) or BSA (B & D; both micrographs are counterstained with cresyl violet). Scale bar represents 10 μm. The number of Iba-1-positive microglia (E) or ICAM-1- positive vessels were quantified in both SNCA and BSA injected animals.CC BY · PubMed Central
Figure 6 Cytokine mRNA expression in the brain 24 hours after SNCA, BSA or LPS injection into the substantia nigra with and without peripheral LPS challenge. mRNA levels of (A) NFkB; (B) TNFα; (C) IL-1β; (D) IL-6; (E) TGFβ and (F) COX-2 measured as values relative to GAPDH and normalized to levels within the contralateral hemisphere of control animals. Bars show mean ± SEM. * indicates a significance of P < 0.05 when compared to non-LPS-injected control animals; **indicates P < 0.01 and *** indicates P < 0.005.
Figure 6 Cytokine mRNA expression in the brain 24 hours after SNCA, BSA or LPS injection into the substantia nigra with and without peripheral LPS challenge. mRNA levels of (A) NFkB; (B) TNFα; (C) IL-1β; (D) IL-6; (E) TGFβ and (F) COX-2 measured as values relative to GAPDH and normalized to levels within the contralateral hemisphere of control animals. Bars show mean ± SEM. * indicates a significance of P < 0.05 when compared to non-LPS-injected control animals; **indicates P < 0.01 and *** indicates P < 0.005.CC BY · PubMed Central
Figure 7 p65 nuclear translocation in injected hemisphere 24 after BSA or SNCA injection into the substantia nigra with and without peripheral LPS challenge. (A) p65 protein was visible in both cytosolic and nuclear fractions of brain protein lysate of injected animals. p65 protein levels within the nuclear fraction (B) were measured as relative-fold changes compared to non-LPS injected control animals. * indicates a significance of P < 0.05 when compared to non-LPS injected controls; ** indicates a significance of P < 0.01 when compared to non-LPS SNCA controls.
Figure 7 p65 nuclear translocation in injected hemisphere 24 after BSA or SNCA injection into the substantia nigra with and without peripheral LPS challenge. (A) p65 protein was visible in both cytosolic and nuclear fractions of brain protein lysate of injected animals. p65 protein levels within the nuclear fraction (B) were measured as relative-fold changes compared to non-LPS injected control animals. * indicates a significance of P < 0.05 when compared to non-LPS injected controls; ** indicates a significance of P < 0.01 when compared to non-LPS SNCA controls.CC BY · PubMed Central
Figure 8 Cell death in injected hemisphere 24 h after SNCA, BSA or LPS injection into the substantia nigra with and without peripheral LPS challenge. TUNEL staining was clearly visible in LPS-injected (A) animals but TUNEL was rarely seen in SNCA injected animals (B). No TUNEL staining was observed in BSA-injected brain (C; representative image). Nuclease treated positive control section (D). Data are mean ± SEM, *** indicates a significance of P < 0.005 when compared to the contralateral hemisphere. Scale bar represents 10 μm.
Figure 8 Cell death in injected hemisphere 24 h after SNCA, BSA or LPS injection into the substantia nigra with and without peripheral LPS challenge. TUNEL staining was clearly visible in LPS-injected (A) animals but TUNEL was rarely seen in SNCA injected animals (B). No TUNEL staining was observed in BSA-injected brain (C; representative image). Nuclease treated positive control section (D). Data are mean ± SEM, *** indicates a significance of P < 0.005 when compared to the contralateral hemisphere. Scale bar represents 10 μm.CC BY · PubMed Central

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Metadata

Authors
Couch, Y., Alvarez-Erviti, L., Sibson, N. R., Wood, M. J., & Anthony, D. C.
Affiliations
  • University of Oxford (GB)
  • Churchill Hospital (GB)
Corresponding author
  • Couch, Yvonne
  • Sibson, Nicola R
  • Wood, Matthew JA
  • Anthony, Daniel C
ORCID
Venue
Journal of Neuroinflammation
Publisher
Springer Science and Business Media LLC
Published
2011-11-28
Volume
8
Issue
1
Pages
166-166
ISSN
1742-2094 (ISSN-L)
DOI
10.1186/1742-2094-8-166
PMC
PMC3239418
OpenAlex
W2064344027
Semantic Scholar
8d36da2a381f6e870ef72f2ae121c4a93eb5d6ac
Cited by
155
References
48
Citations by year
2026: 3 · 2025: 7 · 2024: 4 · 2023: 8 · 2022: 15 · 2021: 18 · 2020: 13 · 2019: 10 · 2018: 10 · 2017: 15 · 2016: 12 · 2015: 8 · 2014: 15 · 2013: 10 · 2012: 7
FWCI
5.40
Type
journal article
Language
en
Open access
Licence
cc-by
Locations
  • Journal of Neuroinflammation (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • UCL Discovery (University College London) (submittedVersion)
Funders
Biotechnology and Biological Sciences Research Council
Topics
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Health Sciences › Medicine › Clinical Neurology › Parkinson's Disease Mechanisms and Treatments
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Nuclear Receptors and Signaling
Keywords
extracellular alpha-synuclein, Parkinson's disease, microglial activation, neuroinflammation, lipopolysaccharide, substantia nigra, inflammatory cytokines, IL-1β, NF-κB, peripheral inflammation, microglial priming, neuronal cell death, endothelial activation, BV-2 cells, neurodegeneration
MeSH terms
Animals, Cell Line, Female, Inflammation, Lipopolysaccharides, Parkinson Disease, Serum Albumin, Substantia Nigra, Tumor Necrosis Factor-alpha, Cytokines, Amyloid beta-Peptides, Cell Death, Microglia, Mice, alpha-Synuclein, Transcription Factor RelA, Interleukin-1beta

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