Neurotoxicity Research

, Volume 33, Issue 4, pp 856–867 | Cite as

Lipopolysaccharide-Induced Microglia Activation Promotes the Survival of Midbrain Dopaminergic Neurons In Vitro

  • Xiaolai Zhou
  • Björn SpittauEmail author


Microglia are the resident immune cells of the central nervous system (CNS) and respond to a variety of endogenous and exogenous stimuli in order to restore cell and tissue homeostasis. Lipopolysaccharide (LPS) is one of these exogenous stimuli, constitutes a major component of the outer membrane of Gram-negative bacteria, and binds to the microglial pattern recognition receptor Toll-like receptor 4 (TLR4). LPS-induced microglia activation is believed to promote neurodegeneration by release of neurotoxic factors such as interleukin-1β, tumor necrosis factor α, or nitric oxide. In the present study, we investigated whether the physical presence of microglia is required to promote neurotoxicity and whether microglia-derived factors are essential. Interestingly, we observed that dopaminergic (mDA) neuron survival was only affected in mixed neuron-glia cultures containing microglia but not in neuron-enriched cultures. Moreover, we clearly demonstrate that microglia-conditioned medium (MCM) after LPS treatment increased mDA neuron survival, process numbers as well as process length. The observed protective effects of MCM was rather caused by microglia-derived factors and only partially dependent on the increase in reactive astrocytes. These results indicate that LPS-induced microglia activation does not necessarily have detrimental effects on mDA neurons and further support the hypothesis that activated microglia support neuron survival by release of neurotrophic and neuroprotective factors.


mDA neuron LPS Microglia Astrocytes TGFβ 



The authors would like to thank Susanna Glaser for excellent technical support.


  1. ’Episcopo FL, Tirolo C, Testa N, Caniglia S, Morale MC, Marchetti B (2013) Reactive astrocytes are key players in nigrostriatal dopaminergic neurorepair in the MPTP mouse model of Parkinson’s disease: focus on endogenous neurorestoration. Curr Aging Sci 6(1):45–55. CrossRefPubMedGoogle Scholar
  2. Abe M, Harpel JG, Metz CN, Nunes I, Loskuthoff DJ, Rifkin DB (1994) An assay for transforming growth factor-beta using cells transfected with a plasminogen activator inhibitor-1 promoter-luciferase construct. Anal Biochem 216(2):276–284. CrossRefPubMedGoogle Scholar
  3. Adams JC (1981) Heavy metal intensification of DAB-based HRP reaction product. J Histochem Cytochem 29(6):775. CrossRefPubMedGoogle Scholar
  4. Barcia C, Ros CM, Annese V, Carillo-de Sauvage MA, Ros-Bernal F, Gómez A, Yuste JE, Campuzano CM, de Pablos V, Fernandez-Villalba E, Herrero MT (2012) ROCK/Cdc42-mediated microglial motility and gliapse formation lead to phagocytosis of degenerating dopaminergic neurons in vivo. Sci Rep 2(1):809. CrossRefPubMedPubMedCentralGoogle Scholar
  5. Block ML, Zecca L, Hong JS (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8(1):57–69. CrossRefPubMedGoogle Scholar
  6. Bolin LM, Strycharska-Orczyk I, Murray R, Langston JW, Di Monte D (2002) Increased vulnerability of dopaminergic neurons in MPTP-lesioned interleukin-6 deficient mice. J Neurochem 83(1):167–175. CrossRefPubMedGoogle Scholar
  7. Chen H, Lin W, Zhang Y, Lin L, Chen J, Zeng Y, Zheng M, Zhuang Z, Du H, Chen R, Liu N (2016) IL-10 promotes neurite outgrowth and synapse formation in cultured cortical neurons after the oxygen-glucose deprivation via JAK1/STAT3 pathway. Sci Rep 6(1):30459. CrossRefPubMedPubMedCentralGoogle Scholar
  8. Chen X, Liu Z, Cao B-B, Qiu Y-H, Peng Y-P (2017) TGF-β1 neuroprotection via inhibition of microglial activation in a rat model of Parkinson’s disease. J NeuroImmune Pharmacol 12(3):433–446. CrossRefPubMedGoogle Scholar
  9. Funk GD, Rajani V, Alvares TS, Al R, Zhang Y, Chu NY, Biancardi V, Linhares-Taxini C, Katzell A, Reklov R (2015) Neuroglia and their roles in central respiratory control; an overview. Comp Biochem Physiol A Mol Integr Physiol 186:83–95. CrossRefPubMedGoogle Scholar
  10. Gayle DA, Ling Z, Tong C, Landers T, Lipton JW, Carvey PM (2002) Lipopolysaccharide (LPS)-induced dopamine cell loss in culture: roles of tumor necrosis factor-alpha, interleukin-1beta, and nitric oxide. Brain Res Dev Brain Res 133(1):27–35. CrossRefPubMedGoogle Scholar
  11. Gibbons HM, Dragunow M (2006) Microglia induce neural cell death via a proximity-dependent mechanism involving nitric oxide. Brain Res 1084(1):1–15. CrossRefPubMedGoogle Scholar
  12. Green LC, Wagner DA, Glogowski J, Skipper PL, Wishnok JS, Tannenbaum SR (1982) Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal Biochem 126(1):131–138. CrossRefPubMedGoogle Scholar
  13. Guha M, Mackman N (2001) LPS induction of gene expression in human monocytes. Cell Signal 13(2):85–94. CrossRefPubMedGoogle Scholar
  14. Herrera AJ, Castaño A, Venero JL, Cano J, Machado A (2000) The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system. Neurobiol Dis 7(4):429–447. CrossRefPubMedGoogle Scholar
  15. Hirsch EC, Hunot S (2009) Neuroinflammation in Parkinson’s disease: a target for neuroprotection? Lancet Neurol 8(4):382–397. CrossRefPubMedGoogle Scholar
  16. Hühner L, Rilka J, Gilsbach R, Zhou X, Machado V, Spittau B (2017) Interleukin-4 protects dopaminergic neurons in vitro but is dispensable for MPTP-induced neurodegeneration in vivo. Front Mol Neurosci 10:62. CrossRefPubMedPubMedCentralGoogle Scholar
  17. Iravani MM, Sadeghian M, Leung CCM, Jenner P, Rose S (2012) Lipopolysaccharide-induced nigral inflammation leads to increased IL-1β tissue content and expression of astrocytic glial cell line-derived neurotrophic factor. Neurosci Lett 510(2):138–142. CrossRefPubMedGoogle Scholar
  18. Jellinger KA (2001) The pathology of Parkinson’s disease. Adv Neurol 86:55–72PubMedGoogle Scholar
  19. Johnston LC, Su X, Maguire-Zeiss K, Horovitz K, Ankoudinova I, Guschin D, Hadaczek P, Federoff HJ, Bankiewicz K, Forsayeth J (2008) Human interleukin-10 gene transfer is protective in a rat model of Parkinson’s disease. Mol Ther 16(8):1392–1399. CrossRefPubMedPubMedCentralGoogle Scholar
  20. Kim WG, Mohney RP, Wilson B, Jeohn GH, Liu B, Hong JS (2000) Regional difference in susceptibility to lipopolysaccharide-induced neurotoxicity in the rat brain: role of microglia. J Neurosci 20(16):6309–6316CrossRefPubMedGoogle Scholar
  21. Kosloski LM, Kosmacek EA, Olson KE, Mosley RL, Gendelman HE (2013) GM-CSF induces neuroprotective and anti-inflammatory responses in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine intoxicated mice. J Neuroimmunol 265(1-2):1–10. CrossRefPubMedGoogle Scholar
  22. Krieglstein K, Suter-Crazzolara C, Fischer WH, Unsicker K (1995) TGF-beta superfamily members promote survival of midbrain dopaminergic neurons and protect them against MPP+ toxicity. EMBO J 14(4):736–742PubMedPubMedCentralGoogle Scholar
  23. Krieglstein K, Strelau J, Schober A, Sullivan A, Unsicker K (2002) TGF-beta and the regulation of neuron survival and death. J Physiol Paris 96(1-2):25–30. CrossRefPubMedGoogle Scholar
  24. Lawson LJ, Perry VH, Dri P, Gordon S (1990) Heterogeneity in the distribution and morphology of microglia in the normal adult mouse brain. Neuroscience 39(1):151–170. CrossRefPubMedGoogle Scholar
  25. Le W, Wu J, Tang Y (2016) Protective microglia and their regulation in Parkinson’s disease. Front Mol Neurosci.
  26. Li L, Lu J, Tay SSW, Moochhala SM, He BP (2007) The function of microglia, either neuroprotection or neurotoxicity, is determined by the equilibrium among factors released from activated microglia in vitro. Brain Res 1159:8–17. CrossRefPubMedGoogle Scholar
  27. Liu Y, Qin L, Wilson B, Wu X, Qian L, Granholm AC, Crews FT, Hong JS (2008) Endotoxin induces a delayed loss of TH-IR neurons in substantia nigra and motor behavioral deficits. Neurotoxicology 29(5):864–870. CrossRefPubMedPubMedCentralGoogle Scholar
  28. Liu W, Tang Y, Feng J (2011) Cross talk between activation of microglia and astrocytes in pathological conditions in the central nervous system. Life Sci 89(5-6):141–146. CrossRefPubMedGoogle Scholar
  29. Lu Y-C, Yeh W-C, Ohashi PS (2008) LPS/TLR4 signal transduction pathway. Cytokine 42(2):145–151. CrossRefPubMedGoogle Scholar
  30. Machado V, Haas SJ-P, von Bohlen Und Halbach O, Wree A, Krieglstein K, Unsicker K, Spittau B (2016a) Growth/differentiation factor-15 deficiency compromises dopaminergic neuron survival and microglial response in the 6-hydroxydopamine mouse model of Parkinson’s disease. Neurobiol Dis 88:1–15. CrossRefPubMedGoogle Scholar
  31. Machado V, Zöller T, Attaai A, Spittau B (2016b) Microglia-mediated neuroinflammation and neurotrophic factor-induced protection in the MPTP mouse model of Parkinson’s disease—lessons from transgenic mice. Int J Mol Sci.
  32. McCoy MK, Martinez TN, Ruhn KA, Szymkowski DE, Smith CG, Botterman BR, Tansey KE, Tansey MG (2006) Blocking soluble tumor necrosis factor signaling with dominant-negative tumor necrosis factor inhibitor attenuates loss of dopaminergic neurons in models of Parkinson’s disease. J Neurosci 26(37):9365–9375. CrossRefPubMedPubMedCentralGoogle Scholar
  33. Polazzi E, Altamira LEP, Eleuteri S, Barbaro R, Casadio C, Contestabile A, Monti B (2009) Neuroprotection of microglial conditioned medium on 6-hydroxydopamine-induced neuronal death: role of transforming growth factor beta-2. J Neurochem 110(2):545–556. CrossRefPubMedGoogle Scholar
  34. Prinz M, Priller J (2014) Microglia and brain macrophages in the molecular age: from origin to neuropsychiatric disease. Nat Rev Neurosci 15(5):300–312. CrossRefPubMedGoogle Scholar
  35. Qian L, Wei S-J, Zhang D, Hu X, Xu Z, Wilson B, El-Benna J, Hong JS, Flood PM (2008) Potent anti-inflammatory and neuroprotective effects of TGF-beta1 are mediated through the inhibition of ERK and p47phox-Ser345 phosphorylation and translocation in microglia. J Immunol 181(1):660–668. CrossRefPubMedPubMedCentralGoogle Scholar
  36. Qin L, Liu Y, Wang T, Wei SJ, Block ML, Wilson B, Liu B, Hong JS (2004) NADPH oxidase mediates lipopolysaccharide-induced neurotoxicity and proinflammatory gene expression in activated microglia. J Biol Chem 279(2):1415–1421. CrossRefPubMedGoogle Scholar
  37. Qin L, Wu X, Block ML, Liu Y, Breese GR, Hong JS, Knapp DJ, Crews FT (2007) Systemic LPS causes chronic neuroinflammation and progressive neurodegeneration. Glia 55(5):453–462. CrossRefPubMedPubMedCentralGoogle Scholar
  38. Rickert U, Grampp S, Wilms H, Spreu J, Knerlich-Lukoschus F, Held-Feindt J, Lucius R (2014) Glial cell line-derived neurotrophic factor family members reduce microglial activation via inhibiting p38MAPKs-mediated inflammatory responses. J Neurodegener Dis 2014:369468. PubMedPubMedCentralGoogle Scholar
  39. Roussa E, von Bohlen und Halbach O, Krieglstein K (2009) TGF-beta in dopamine neuron development, maintenance and neuroprotection. Adv Exp Med Biol 651:81–90. CrossRefPubMedGoogle Scholar
  40. Sharaf A, Krieglstein K, Spittau B (2013) Distribution of microglia in the postnatal murine nigrostriatal system. Cell Tissue Res 351(3):373–382. CrossRefPubMedGoogle Scholar
  41. Song S, Kong X, Acosta S, Sava V, Borlongan C, Sanchez-Ramos J (2016) Granulocyte-colony stimulating factor promotes brain repair following traumatic brain injury by recruitment of microglia and increasing neurotrophic factor expression. Restor Neurol Neurosci 34(3):415–431. PubMedGoogle Scholar
  42. Spittau B, Zhou X, Ming M, Krieglstein K (2012) IL6 protects MN9D cells and midbrain dopaminergic neurons from MPP+-induced neurodegeneration. NeuroMolecular Med 14(4):317–327. CrossRefPubMedGoogle Scholar
  43. Spittau B, Wullkopf L, Zhou X, Rilka J, Pfeifer D, Krieglstein K (2013) Endogenous transforming growth factor-beta promotes quiescence of primary microglia in vitro. Glia 61(2):287–300. CrossRefPubMedGoogle Scholar
  44. Spittau B, Rilka J, Steinfath E, Zöller T, Krieglstein K (2015) TGFβ1 increases microglia-mediated engulfment of apoptotic cells via upregulation of the milk fat globule-EGF factor 8. Glia 63(1):142–153. CrossRefPubMedGoogle Scholar
  45. Tanaka T, Oh-Hashi K, Shitara H, Hirata Y, Kiuchi K (2008) NF-kappaB independent signaling pathway is responsible for LPS-induced GDNF gene expression in primary rat glial cultures. Neurosci Lett 431(3):262–267. CrossRefPubMedGoogle Scholar
  46. Tang S-C, Arumugam TV, Xu X, Cheng A, Mughal MR, Jo DG, Lathia JD, Siler DA, Chigurupati S, Ouyang X, Magnus T, Camandola S, Mattson MP (2007) Pivotal role for neuronal toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci U S A 104(34):13798–13803. CrossRefPubMedPubMedCentralGoogle Scholar
  47. Tang S-C, Lathia JD, Selvaraj PK, Jo DG, Mughal MR, Cheng A, Siler DA, Markesbery WR, Arumugam TV, Mattson MP (2008) Toll-like receptor-4 mediates neuronal apoptosis induced by amyloid beta-peptide and the membrane lipid peroxidation product 4-hydroxynonenal. Exp Neurol 213(1):114–121. CrossRefPubMedPubMedCentralGoogle Scholar
  48. Unsicker K, Krieglstein K (2002) TGF-betas and their roles in the regulation of neuron survival. Adv Exp Med Biol 513:353–374CrossRefPubMedGoogle Scholar
  49. Watanabe H, Abe H, Takeuchi S, Tanaka R (2000) Protective effect of microglial conditioning medium on neuronal damage induced by glutamate. Neurosci Lett 289(1):53–56. CrossRefPubMedGoogle Scholar
  50. Wolf SA, Boddeke HWGM, Kettenmann H (2017) Microglia in physiology and disease. Annu Rev Physiol 79(1):619–643. CrossRefPubMedGoogle Scholar
  51. Zhou X, Zöller T, Krieglstein K, Spittau B (2015) TGFβ1 inhibits IFNγ-mediated microglia activation and protects mDA neurons from IFNγ-driven neurotoxicity. J Neurochem 134(1):125–134. CrossRefPubMedGoogle Scholar
  52. Zhu Y, Chen X, Liu Z, Peng YP, Qiu YH (2015) Interleukin-10 protection against lipopolysaccharide-induced neuro-inflammation and neurotoxicity in ventral mesencephalic cultures. Int J Mol Sci.
  53. Zhu C, Herrmann US, Falsig J, Abakumova I, Nuvolone M, Schwarz P, Frauenknecht K, Rushing EJ, Aguzzi A (2016) A neuroprotective role for microglia in prion diseases. J Exp Med 213(6):1047–1059. CrossRefPubMedPubMedCentralGoogle Scholar
  54. Zlotnik A, Spittau B (2014) GDNF fails to inhibit LPS-mediated activation of mouse microglia. J Neuroimmunol 270(1-2):22–28. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of NeuroscienceMayo Clinic JacksonvilleJacksonvilleUSA
  2. 2.Institute of AnatomyUniversity of RostockRostockGermany
  3. 3.Institute of Anatomy and Cell Biology, Department of Molecular Embryology, Faculty of MedicineUniversity of FreiburgFreiburgGermany

Personalised recommendations