Interferon-γ Potentiates α-Synuclein-induced Neurotoxicity Linked to Toll-like Receptors 2 and 3 and Tumor Necrosis Factor-α in Murine Astrocytes

  • Jintang Wang
  • Zheng Chen
  • Jeremy D. Walston
  • Peisong Gao
  • Maolong Gao
  • Sean X. LengEmail author


α-Synuclein (α-syn), a metabolite of neurons, induces glial activation and neuroinflammation and participates in pathogenesis of neurodegenerative diseases. This inflammatory response involves activation of toll-like receptors (TLRs) and its neurotoxic outcomes such as cytokine expression and release. However, regulatory role of cytokines on α-syn-induced neurotoxicity is still unclear. In this study, we used interferon (IFN)-γ to costimulate primary astrocytes with wild-type or A53T mutant α-syn, and evaluated inflammatory pathway activation. Four α-syn concentrations (0.5, 2, 8 and 20 μg/mL, 24 h) and four α-syn time-points (3, 12, 24 and 48 h, 2 μg/mL) were chosen to coincubate with one IFN-γ concentration (2 ng/mL). IFN-γ alone upregulated expressions of TLR3 and tumor necrosis factor (TNF)-α (mRNA level), and A53T mutant or wild-type α-syn alone activated the pathway components including TLR2, TLR3, nuclear factor-κB, TNF-α and interleukin (IL)-1β. Additive application of IFN-γ amplified this activation effect except for IL-1β at mRNA and protein levels or TNF-α release, displaying a synergistic effect of α-syn and IFN-γ. Blocking TLR2 other than TLR4 suppressed TLR3, TLR2 and TNF-α expressions induced by α-syn or plus IFN-γ, reflecting an interaction of TLR2 and TLR3 in TNF-α expression. These data collectively showed that IFN-γ potentiated α-syn stimulation and inflammatory outcomes via TLR2, TLR3 and TNF-α other than IL-1β in astrocytes, suggesting that involvement of IFN-γ in α-syn-induced innate immunity may be required for initiation and maintenance of glial activation, a novel neurotoxic mechanism underlying pathogenesis of neurodegenerative diseases.

Graphical Abstract

IFN-γ potentiates α-synuclein (A53T or wild-type)-induced innate immunity, involving expressions of TLR2, TLR3, NF-κB, and TNF-α, other than IL-1β. This effect is suppressed by blockage of TLR2 other than TLR4, reflecting an interaction of TLR2 and TLR3 in TNF-α expression. Thus, involvement of IFN-γ in α-syn-induced neurotoxicity may be required for initiation and maintenance of glial activation, a novel neurotoxic mechanism underlying pathogenesis of neurodegenerative diseases.


α-Synuclein Interferon-γ Toll-like receptors Cytokines Neuroinflammation Astrocytes 



Alzheimer’s disease


analysis of variance;






damage-associated molecular patterns




enzyme-linked immunoabsorbent assay


fetal bovine serum


glyceraldehyde phosphate dehydrogenase


glial fibrillary acidic protein








fisher’s least significant differences


nuclear factor-kappa B


phosphate-buffered saline


Parkinson’s disease

Poly I:C

polyinosinic-polycytidylic acid


quantitative real-time polymerase chain reaction


toll-like receptor


tumor necrosis factor-α


Authors’ Contributions

J.T.W designed, performed the research, and wrote the manuscript; Z.C. helped design and put the research into effect; J.W. and P.S.G. provided experiment facilities, techniques, and mice use; M.L.G. performed the statistical analysis and its interpretation; S.X.L. designed research, analyzed data, reviewed overall findings, and wrote the manuscript; and all authors approved the final draft of the manuscript.


This work was supported by the Irma and Paul Milstein Program for Senior Health fellowship from the Milstein Medical Asian American Partnership (MMAAP) Foundation ( (JT Wang: 1005352440), and in part by NIH grants R21-AG-043874, R01AI108907 (SX Leng).

Compliance with Ethical Standards

Competing Interests

The authors declare that they have no competing interests.

Ethics Approval

All animal procedures performed in this study were reviewed and approved by the Institution’s Animal Care Committee, and performed in accordance with the guidelines by Institution of Laboratory Resources, National Research Council (Department of Health and Human Service, National Institutes of Health, Bethesda, MD).


  1. 1.
    Norris EH, Giasson BI, Lee VM (2004) Alpha-synuclein: normal function and role in neurodegenerative diseases. Curr Top Dev Biol 60:17–54. CrossRefPubMedGoogle Scholar
  2. 2.
    Lawand NB, Saadé NE, El-Agnaf OM, Safieh-Garabedian B (2015) Targeting α-synuclein as a therapeutic strategy for Parkinson’s disease. Expert Opin Ther Targets 19(10):1351–1360. CrossRefPubMedGoogle Scholar
  3. 3.
    Woerman AL, Kazmi SA, Patel S, Aoyagi A, Oehler A, Widjaja K, Mordes DA, Olson SH et al (2018) Familial Parkinson’s point mutation abolishes multiple system atrophy prion replication. Proc Natl Acad Sci U S A 115(2):409–414. CrossRefPubMedGoogle Scholar
  4. 4.
    Lee HJ, Kim C, Lee SJ (2010) Alpha-synuclein stimulation of astrocytes: potential role for neuroinflammation and neuroprotection. Oxidative Med Cell Longev 3(4):283–287. CrossRefGoogle Scholar
  5. 5.
    Alvarez-Erviti L, Couch Y, Richardson J, Cooper JM, Wood MJ (2011) Alpha-synuclein release by neurons activates the inflammatory response in a microglial cell line. Neurosci Res 69(4):337–342. CrossRefPubMedGoogle Scholar
  6. 6.
    Ma D, Jin S, Li E, Doi Y, Parajuli B, Noda M, Sonobe Y, Mizuno T et al (2013) The neurotoxic effect of astrocytes activated with toll-like receptor ligands. J Neuroimmunol 254(1–2):10–18. CrossRefPubMedGoogle Scholar
  7. 7.
    Rivest S (2009) Regulation of innate immune responses in the brain. Nat Rev Immunol 9(6):429–439. CrossRefPubMedGoogle Scholar
  8. 8.
    Wang J, Chen Z, Walston JD, Gao P, Gao M, Leng SX (2018) α-Synuclein activates innate immunity but suppresses interferon-γ expression in murine astrocytes. Eur J Neurosci 48(1):1583–1599. CrossRefGoogle Scholar
  9. 9.
    Vidovic M, Sparacio SM, Elovitz M, Benveniste EN (1990) Induction and regulation of class II major histocompatibility complex mRNA expression in astrocytes by interferon-gamma and tumor necrosis factor-alpha. J Neuroimmunol 30(2–3):189–200CrossRefPubMedGoogle Scholar
  10. 10.
    Chung IY, Norris JG, Benveniste EN (1991) Differential tumor necrosis factor alpha expression by astrocytes from experimental allergic encephalomyelitis-susceptible and -resistant rat strains. J Exp Med 173(4):801–811CrossRefPubMedGoogle Scholar
  11. 11.
    Klegeris A, Pelech S, Giasson BI, Maguire J, Zhang H, McGeer EG, McGeer PL (2008) Alpha-synuclein activates stress signaling protein kinases in THP-1 cells and microglia. Neurobiol Aging 29(5):739–752. CrossRefPubMedGoogle Scholar
  12. 12.
    Kim S, Seo JH, Suh YH (2004) Alpha-synuclein, Parkinson’s disease, and Alzheimer’s disease. Parkinsonism Relat Disord 10(suppl 1):S9–S13. CrossRefPubMedGoogle Scholar
  13. 13.
    Mori F, Tanji K, Yoshimoto M, Takahashi H, Wakabayashi K (2002) Demonstration of alpha-synuclein immunoreactivity in neuronal and glial cytoplasm in normal human brain tissue using proteinase K and formic acid pretreatment. Exp Neurol 176(1):98–104CrossRefPubMedGoogle Scholar
  14. 14.
    Shin EC, Cho SE, Lee DK, Hur MW, Paik SR, Park JH, Kim J (2000) Expression patterns of alpha-synuclein in human hematopoietic cells and in Drosophila at different developmental stages. Mol Cell 10(1):65–70CrossRefGoogle Scholar
  15. 15.
    El-Agnaf OM, Salem SA, Paleologou KE, Cooper LJ, Fullwood NJ, Gibson MJ, Curran MD, Court JA et al (2003) Alpha-synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma. FASEB J 17(13):1945–1947. CrossRefPubMedGoogle Scholar
  16. 16.
    Lee HJ, Patel S, Lee SJ (2005) Intravesicular localization and exocytosis of alpha-synuclein and its aggregates. J Neurosci 25(25):6016–6024. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Sui YT, Bullock KM, Erickson MA, Zhang J, Banks WA (2014) Alpha-synuclein is transported into and out of the brain by the blood-brain barrier. Peptides 62:197–202. CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Mollenhauer B, Locascio JJ, Schulz-Schaeffer W, Sixel-Döring F, Trenkwalder C, Schlossmacher MG (2011) α-Synuclein and tau concentrations in cerebrospinal fluid of patients presenting with parkinsonism: a cohort study. Lancet Neurol 10(3):30–40. CrossRefGoogle Scholar
  19. 19.
    Lin CH, Yang SY, Horng HE, Yang CC, Chieh JJ, Chen HH, Liu BH, Chiu MJ (2017) Plasma α-synuclein predicts cognitive decline in Parkinson’s disease. J Neurol Neurosurg Psychiatry 88(10):818–824. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Béraud D, Twomey M, Bloom B, Mittereder A, Ton V, Neitzke K, Chasovskikh S, Mhyre TR et al (2011) α-Synuclein alters toll-like receptor expression. Front Neurosci 5:80. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Pasparakis M, Vandenabeele P (2015) Necroptosis and its role in inflammation. Nature 517(7534):311–320. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Farina C, Aloisi F, Meinl E (2007) Astrocytes are active players in cerebral innate immunity. Trends Immunol 28(3):138–145. CrossRefPubMedGoogle Scholar
  23. 23.
    Liddelow SA, Guttenplan KA, Clarke LE, Bennett FC, Bohlen CJ, Schirmer L, Bennett ML, Münch AE et al (2017) Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541(7638):481–487. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    La Vitola P, Balducci C, Cerovic M, Santamaria G, Brandi E, Grandi F, Caldinelli L, Colombo L et al (2018) Alpha-synuclein oligomers impair memory through glial cell activation and via toll-like receptor 2. Brain Behav Immun 69:591–602. CrossRefPubMedGoogle Scholar
  25. 25.
    Barcia C, Ros CM, Annese V, Gómez A, Ros-Bernal F, Aguado-Llera D, Martínez-Pagán ME, de Pablos V et al (2012) IFN-γ signaling, with the synergistic contribution of TNF-α, mediates cell specific microglial and astroglial activation in experimental models of Parkinson’s disease. Cell Death Dis 3:e379. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Papageorgiou IE, Lewen A, Galow LV, Cesetti T, Scheffel J, Regen T, Hanisch UK, Kann O (2016) TLR4-activated microglia require IFN-γ to induce severe neuronal dysfunction and death in situ. Proc Natl Acad Sci U S A 113(1):212–217. CrossRefPubMedGoogle Scholar
  27. 27.
    Mount MP, Lira A, Grimes D, Smith PD, Faucher S, Slack R, Anisman H, Hayley S et al (2007) Involvement of interferon-gamma in microglial-mediated loss of dopaminergic neurons. J Neurosci 27(12):3328–3337. CrossRefPubMedGoogle Scholar
  28. 28.
    Chakrabarty P, Ceballos-Diaz C, Lin WL, Beccard A, Jansen-West K, McFarland NR, Janus C, Dickson D et al (2011) Interferon-γ induces progressive nigrostriatal degeneration and basal ganglia calcification. Nat Neurosci 14(6):694–696. CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Cimoli G, Parodi S, Russo P (1996) Interferon-gamma enhances TNF sensitivity in A172 human glioblastoma cell line. Oncol Rep 3(2):369–370PubMedGoogle Scholar
  30. 30.
    Liscovitch N, French L (2014) Differential co-expression between α-synuclein and IFN-γ signaling genes across development and in Parkinson’s disease. PLoS One 9(12):e115029. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Roodveldt C, Labrador-Garrido A, Gonzalez-Rey E, Lachaud CC, Guilliams T, Fernandez-Montesinos R, Benitez-Rondan A, Robledo G et al (2013) Preconditioning of microglia by α-synuclein strongly affects the response induced by toll-like receptor (TLR) stimulation. PLoS One 8(11):e79160. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Springall R, Amezcua-Guerra LM, Gonzalez-Pacheco H, Furuzawa-Carballeda J, Gomez-Garcia L, Marquez-Velasco R, Mejía-Domínguez AM, Cossío-Aranda J et al (2013) Interferon-gamma increases the ratio of matrix metalloproteinase-9/tissue inhibitor of metalloproteinase-1 in peripheral monocytes from patients with coronary artery disease. PLoS One 8(8):e72291. CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25(4):402–408. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Smith GM, Rutishauser U, Silver J, Miller RH (1990) Maturation of astrocytes in vitro alters the extent and molecular basis of neurite outgrowth. Dev Biol 138(2):377–390CrossRefPubMedGoogle Scholar
  35. 35.
    Wang J, Song Y, Chen Z, Leng SX (2018a) Connection between systemic inflammation and neuroinflammation underlies neuroprotective mechanism of several phytochemicals in neurodegenerative diseases. Oxidative Med Cell Longev 2018:1–16. CrossRefGoogle Scholar
  36. 36.
    Kim C, Ho DH, Suk JE, You S, Michael S, Kang J, Joong Lee S, Masliah E et al (2013) Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia. Nat Commun 4:1562. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    González-Reyes RE, Nava-Mesa MO, Vargas-Sánchez K, Ariza-Salamanca D, Mora-Muñoz L (2017) Involvement of astrocytes in Alzheimer’s disease from a neuroinflammatory and oxidative stress perspective. Front Mol Neurosci 10:427. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Dzamko N, Gysbers A, Perera G, Bahar A, Shankar A, Gao J, Fu Y, Halliday GM (2017) Toll-like receptor 2 is increased in neurons in Parkinson’s disease brain and may contribute to alpha-synuclein pathology. Acta Neuropathol 133(2):303–319. CrossRefPubMedGoogle Scholar
  39. 39.
    Liu J, Zhou Y, Wang Y, Fong H, Murray TM, Zhang J (2007) Identification of proteins involved in microglial endocytosis of alpha-synuclein. J Proteome Res 6(9):3614–3627. CrossRefPubMedGoogle Scholar
  40. 40.
    Kettenmann H, Hanisch UK, Noda M, Verkhratsky A (2011) Physiology of microglia. Physiol Rev 91(2):461–553. CrossRefGoogle Scholar
  41. 41.
    Su X, Federoff HJ, Maguire-Zeiss KA (2009) Mutant alpha-synuclein overexpression mediates early proinflammatory activity. Neurotox Res 16(3):238–254. CrossRefPubMedPubMedCentralGoogle Scholar
  42. 42.
    Su X, Maguire-Zeiss KA, Giuliano R, Prifti L, Venkatesh K, Federoff HJ (2008) Synuclein activates microglia in a model of Parkinson’s disease. Neurobiol Aging 29(11):1690–1701. CrossRefPubMedGoogle Scholar
  43. 43.
    Couch Y, Alvarez-Erviti L, Sibson NR, Wood MJ, Anthony DC (2011) The acute inflammatory response to intranigral α-synuclein differs significantly from intranigral lipopolysaccharide and is exacerbated by peripheral inflammation. J Neuroinflammation 8:166. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Khandelwal PJ, Herman AM, Moussa CE (2011) Inflammation in the early stages of neurodegenerative pathology. J Neuroimmunol 238(1–2):1–11. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Hashioka S, Klegeris A, Schwab C, McGeer PL (2009) Interferon-gamma-dependent cytotoxic activation of human astrocytes and astrocytoma cells. Neurobiol Aging 30(12):1924–1935. CrossRefPubMedGoogle Scholar
  46. 46.
    Tichauer J, Saud K, von Bernhardi R (2007) Modulation by astrocytes of microglial cell-mediated neuroinflammation: effect on the activation of microglial signaling pathways. Neuroimmunomodulation 14:168–174. CrossRefPubMedGoogle Scholar
  47. 47.
    Gruden MA, Yanamandra K, Kucheryanu VG, Bocharova OR, Sherstnev VV, Morozova-Roche LA, Sewell RD (2012) Correlation between protective immunity to α-synuclein aggregates, oxidative stress and inflammation. Neuroimmunomodulation 19(6):334–342. CrossRefPubMedGoogle Scholar
  48. 48.
    Bick RJ, Poindexter BJ, Kott MM, Liang YA, Dinh K, Kaur B, Bick DL, Doursout MF et al (2008) Cytokines disrupt intracellular patterns of Parkinson’s disease-associated proteins alpha-synuclein, tau and ubiquitin in cultured glial cells. Brain Res 1217:203–212. CrossRefPubMedGoogle Scholar
  49. 49.
    Savarin C, Hinton DR, Valentin-Torres A, Chen Z, Trapp BD, Bergmann CC, Stohlman SA (2015) Astrocyte response to IFN-γ limits IL-6-mediated microglia activation and progressive autoimmune encephalomyelitis. J Neuroinflammation 12:79. CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Chung CY, Koprich JB, Siddiqi H, Isacson O (2009) Dynamic changes in presynaptic and axonal transport proteins combined with striatal neuroinflammation precede dopaminergic neuronal loss in a rat model of AAV alpha-synucleinopathy. J Neurosci 29(11):3365–3373. CrossRefPubMedPubMedCentralGoogle Scholar
  51. 51.
    Hu W, Jain A, Gao Y, Dozmorov IM, Mandraju R, Wakeland EK, Pasare C (2015) Differential outcome of TRIF-mediated signaling in TLR4 and TLR3 induced DC maturation. Proc Natl Acad Sci U S A 112(45):13994–13999. CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Jintang Wang
    • 1
  • Zheng Chen
    • 1
  • Jeremy D. Walston
    • 2
  • Peisong Gao
    • 3
  • Maolong Gao
    • 1
  • Sean X. Leng
    • 2
    Email author
  1. 1.Institute for Geriatrics and RehabilitationBeijing Geriatric HospitalBeijingPeople’s Republic of China
  2. 2.Division of Geriatric Medicine and Gerontology, Department of MedicineJohns Hopkins University School of MedicineBaltimoreUSA
  3. 3.Johns Hopkins Asthma and Allergy CenterJohns Hopkins University School of MedicineBaltimoreUSA

Personalised recommendations