Nrf2 Deficiency Exacerbates Cognitive Impairment and Reactive Microgliosis in a Lipopolysaccharide-Induced Neuroinflammatory Mouse Model

Abstract

The transcription factor Nrf2 is a central regulator of anti-inflammatory and antioxidant mechanisms that contribute to the development and progression of various neurological disorders. Although the direct and indirect Nrf2 regulatory roles on inflammation have been reviewed in recent years, the in vivo evidence of Nrf2 function on lipopolysaccharide (LPS)-induced cognitive decline and characteristic alterations of reactive microglia and astrocytes remains incomplete. During the 3–5 days after LPS or saline injection, 5–6-month-old wildtype (WT) and Nrf2−/− C57BL/6 mice were subjected to the novel object recognition task. Immunohistochemistry staining was employed for analyses of brain cells. The Nrf2−/− mice displayed exacerbated LPS-induced cognition impairment (28.1 ± 9.6% in the discrimination index of the novel object recognition task), enhanced hippocampal reactive microgliosis and astrogliosis, and an increased expression level of the water channel transmembrane protein aquaporin 4 when compared with WT controls. In addition, similar overt effects of Nrf2 deficiency on LPS-induced characteristic alterations of brain cells were observed in the cortex and striatum regions of mice. In summary, this transgenic loss-of-function study provides direct in vivo evidence that highlights the functional importance of Nrf2 activation in regulating LPS-induced cognitive alteration, glial responses, and aquaporin 4 expression. This finding provides a better understanding of the complex nature of Nrf2 signaling and neuroprotection.

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References

  1. Ammal Kaidery N, Ahuja M, Thomas B (2019) Crosstalk between Nrf2 signaling and mitochondrial function in Parkinson’s disease. Mol Cell Neurosci 101:103413. https://doi.org/10.1016/j.mcn.2019.103413

    CAS  Article  PubMed  Google Scholar 

  2. Arifin WN, Zahiruddin WM (2017) Sample size calculation in animal studies using resource equation approach. Malays J Med Sci 24:101–105. https://doi.org/10.21315/mjms2017.24.5.11

    Article  PubMed  PubMed Central  Google Scholar 

  3. Assentoft M, Larsen BR, MacAulay N (2015) Regulation and function of AQP4 in the central nervous system. Neurochem Res 40:2615–2627. https://doi.org/10.1007/s11064-015-1519-z

    CAS  Article  PubMed  Google Scholar 

  4. Bahn G, Jo D-G (2019) Therapeutic approaches to alzheimer’s disease through modulation of NRF2. Neuromolecul Med 21:1–11. https://doi.org/10.1007/s12017-018-08523-5

    CAS  Article  Google Scholar 

  5. Baxter PS, Hardingham GE (2016) Adaptive regulation of the brain’s antioxidant defences by neurons and astrocytes. Free Radic Biol Med 100:147–152. https://doi.org/10.1016/j.freeradbiomed.2016.06.027

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  6. Beaver SK, Mesa-Torres N, Pey AL, Timson DJ (2019) NQO1: a target for the treatment of cancer and neurological diseases, and a model to understand loss of function disease mechanisms. Biochim Biophys Acta Proteins Proteom 1867:663–676. https://doi.org/10.1016/j.bbapap.2019.05.002

    CAS  Article  PubMed  Google Scholar 

  7. Bevins RA, Besheer J (2006) Object recognition in rats and mice: a one-trial non-matching-to-sample learning task to study “recognition memory”. Nat Protoc 1:1306–1311. https://doi.org/10.1038/nprot.2006.205

    Article  PubMed  Google Scholar 

  8. Block ML, Zecca L, Hong J-S (2007) Microglia-mediated neurotoxicity: uncovering the molecular mechanisms. Nat Rev Neurosci 8:57–69. https://doi.org/10.1038/nrn2038

    CAS  Article  PubMed  Google Scholar 

  9. Buendia I, Michalska P, Navarro E et al (2016) Nrf2-ARE pathway: an emerging target against oxidative stress and neuroinflammation in neurodegenerative diseases. Pharmacol Ther 157:84–104. https://doi.org/10.1016/j.pharmthera.2015.11.003

    CAS  Article  PubMed  Google Scholar 

  10. Burda JE, Sofroniew MV (2017) Seducing astrocytes to the dark side. Cell Res 27:726–727. https://doi.org/10.1038/cr.2017.37

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  11. Catorce MN, Gevorkian G (2016) LPS-induced murine neuroinflammation model: main features and suitability for pre-clinical assessment of nutraceuticals. Curr Neuropharmacol 14:155–164. https://doi.org/10.2174/1570159x14666151204122017

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  12. Cekanaviciute E, Buckwalter MS (2016) Astrocytes: integrative regulators of neuroinflammation in stroke and other neurological diseases. Neurotherapeutics 13:685–701. https://doi.org/10.1007/s13311-016-0477-8

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  13. Chen J, Buchanan JB, Sparkman NL et al (2008) Neuroinflammation and disruption in working memory in aged mice after acute stimulation of the peripheral innate immune system. Brain Behav Immun 22:301–311. https://doi.org/10.1016/j.bbi.2007.08.014

    CAS  Article  PubMed  Google Scholar 

  14. Colombo E, Farina C (2016) Astrocytes: key regulators of neuroinflammation. Trends Immunol 37:608–620. https://doi.org/10.1016/j.it.2016.06.006

    CAS  Article  PubMed  Google Scholar 

  15. Cuadrado A, Manda G, Hassan A et al (2018) Transcription factor NRF2 as a therapeutic target for chronic diseases: a systems medicine approach. Pharmacol Rev 70:348–383. https://doi.org/10.1124/pr.117.014753

    CAS  Article  PubMed  Google Scholar 

  16. Cuadrado A, Rojo AI, Wells G et al (2019) Therapeutic targeting of the NRF2 and KEAP1 partnership in chronic diseases. Nat Rev Drug Discov 18:295–317. https://doi.org/10.1038/s41573-018-0008-x

    CAS  Article  PubMed  Google Scholar 

  17. Dai W, Yan J, Chen G et al (2018) AQP4-knockout alleviates the lipopolysaccharide-induced inflammatory response in astrocytes via SPHK1/MAPK/AKT signaling. Int J Mol Med 42:1716–1722. https://doi.org/10.3892/ijmm.2018.3749

    CAS  Article  PubMed  Google Scholar 

  18. Dinkova-Kostova AT, Talalay P (2010) NAD(P)H:quinone acceptor oxidoreductase 1 (NQO1), a multifunctional antioxidant enzyme and exceptionally versatile cytoprotector. Arch Biochem Biophys 501:116–123. https://doi.org/10.1016/j.abb.2010.03.019

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  19. Dodson M, de la Vega MR, Cholanians AB et al (2019) Modulating NRF2 in disease: timing is everything. Annu Rev Pharmacol Toxicol 59:555–575. https://doi.org/10.1146/annurev-pharmtox-010818-021856

    CAS  Article  PubMed  Google Scholar 

  20. Doré S (2002) Decreased activity of the antioxidant heme oxygenase enzyme: implications in ischemia and in Alzheimer’s disease. Free Radic Biol Med 32:1276–1282. https://doi.org/10.1016/s0891-5849(02)00805-5

    Article  PubMed  Google Scholar 

  21. Frühauf PKS, Ineu RP, Tomazi L et al (2015) Spermine reverses lipopolysaccharide-induced memory deficit in mice. J Neuroinflammation 12:3. https://doi.org/10.1186/s12974-014-0220-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  22. Gupta N, Shyamasundar S, Patnala R et al (2018) Recent progress in therapeutic strategies for microglia-mediated neuroinflammation in neuropathologies. Expert Opin Ther Targets 22:765–781. https://doi.org/10.1080/14728222.2018.1515917

    CAS  Article  PubMed  Google Scholar 

  23. Heneka MT, Golenbock DT, Latz E (2015) Innate immunity in Alzheimer’s disease. Nat Immunol 16:229–236. https://doi.org/10.1038/ni.3102

    CAS  Article  PubMed  Google Scholar 

  24. Hoogland ICM, Houbolt C, van Westerloo DJ et al (2015) Systemic inflammation and microglial activation: systematic review of animal experiments. J Neuroinflammation 12:114. https://doi.org/10.1186/s12974-015-0332-6

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  25. Huber CM, Yee C, May T et al (2018) Cognitive decline in preclinical Alzheimer’s disease: amyloid-beta versus tauopathy. J Alzheimers Dis 61:265–281. https://doi.org/10.3233/JAD-170490

    CAS  Article  PubMed  Google Scholar 

  26. Innamorato NG, Rojo AI, García-Yagüe AJ et al (2008) The transcription factor Nrf2 is a therapeutic target against brain inflammation. J Immunol 181:680–689. https://doi.org/10.4049/jimmunol.181.1.680

    CAS  Article  PubMed  Google Scholar 

  27. Leger M, Quiedeville A, Bouet V et al (2013) Object recognition test in mice. Nat Protoc 8:2531–2537. https://doi.org/10.1038/nprot.2013.155

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  28. Li L, Zhang H, Varrin-Doyer M et al (2011) Proinflammatory role of aquaporin-4 in autoimmune neuroinflammation. FASEB J 25:1556–1566. https://doi.org/10.1096/fj.10-177279

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  29. Liddelow SA, Guttenplan KA, Clarke LE et al (2017) Neurotoxic reactive astrocytes are induced by activated microglia. Nature 541:481–487. https://doi.org/10.1038/nature21029

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  30. Liu L, Vollmer MK, Fernandez VM et al (2018) Korean red ginseng pretreatment protects against long-term sensorimotor deficits after ischemic stroke likely through Nrf2. Front Cell Neurosci 12:74. https://doi.org/10.3389/fncel.2018.00074

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  31. Liu L, Locascio LM, Doré S (2019a) Critical role of nrf2 in experimental ischemic stroke. Front Pharmacol 10:153. https://doi.org/10.3389/fphar.2019.00153

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  32. Liu L, Vollmer MK, Ahmad AS et al (2019b) Pretreatment with Korean red ginseng or dimethyl fumarate attenuates reactive gliosis and confers sustained neuroprotection against cerebral hypoxic-ischemic damage by an Nrf2-dependent mechanism. Free Radic Biol Med 131:98–114. https://doi.org/10.1016/j.freeradbiomed.2018.11.017

    CAS  Article  PubMed  Google Scholar 

  33. Mattson MP, Guthrie PB, Kater SB (1989) Intrinsic factors in the selective vulnerability of hippocampal pyramidal neurons. Prog Clin Biol Res 317:333–351

    CAS  PubMed  Google Scholar 

  34. Medvedeva YV, Ji SG, Yin HZ, Weiss JH (2017) Differential vulnerability of CA1 versus CA3 pyramidal neurons after ischemia: possible relationship to sources of Zn2+ accumulation and its entry into and prolonged effects on mitochondria. J Neurosci 37:726–737. https://doi.org/10.1523/JNEUROSCI.3270-16.2016

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  35. Miwa M, Tsuboi M, Noguchi Y et al (2011) Effects of betaine on lipopolysaccharide-induced memory impairment in mice and the involvement of GABA transporter 2. J Neuroinflammation 8:153. https://doi.org/10.1186/1742-2094-8-153

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  36. Nagelhus EA, Ottersen OP (2013) Physiological roles of aquaporin-4 in brain. Physiol Rev 93:1543–1562. https://doi.org/10.1152/physrev.00011.2013

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  37. Nomoto M, Ohkawa N, Nishizono H et al (2016) Cellular tagging as a neural network mechanism for behavioural tagging. Nat Commun 7:12319. https://doi.org/10.1038/ncomms12319

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  38. Oliveira JF, Sardinha VM, Guerra-Gomes S et al (2015) Do stars govern our actions? Astrocyte involvement in rodent behavior. Trends Neurosci 38:535–549. https://doi.org/10.1016/j.tins.2015.07.006

    CAS  Article  PubMed  Google Scholar 

  39. Pannasch U, Rouach N (2013) Emerging role for astroglial networks in information processing: from synapse to behavior. Trends Neurosci 36:405–417. https://doi.org/10.1016/j.tins.2013.04.004

    CAS  Article  PubMed  Google Scholar 

  40. Papadopoulos MC, Verkman AS (2013) Aquaporin water channels in the nervous system. Nat Rev Neurosci 14:265–277. https://doi.org/10.1038/nrn3468

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  41. Paraiso HC, Kuo P-C, Curfman ET et al (2018) Dimethyl fumarate attenuates reactive microglia and long-term memory deficits following systemic immune challenge. J Neuroinflammation 15:100. https://doi.org/10.1186/s12974-018-1125-5

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  42. Ransohoff RM, Schafer D, Vincent A et al (2015) Neuroinflammation: ways in which the immune system affects the brain. Neurotherapeutics 12:896–909. https://doi.org/10.1007/s13311-015-0385-3

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  43. Rojo AI, Pajares M, Rada P et al (2017) NRF2 deficiency replicates transcriptomic changes in Alzheimer’s patients and worsens APP and TAU pathology. Redox Biol 13:444–451. https://doi.org/10.1016/j.redox.2017.07.006

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  44. Ryter SW, Choi AMK (2016) Targeting heme oxygenase-1 and carbon monoxide for therapeutic modulation of inflammation. Transl Res 167:7–34. https://doi.org/10.1016/j.trsl.2015.06.011

    CAS  Article  PubMed  Google Scholar 

  45. Sofroniew MV (2015) Astrocyte barriers to neurotoxic inflammation. Nat Rev Neurosci 16:249–263. https://doi.org/10.1038/nrn3898

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  46. Sparkman NL, Martin LA, Calvert WS, Boehm GW (2005) Effects of intraperitoneal lipopolysaccharide on Morris maze performance in year-old and 2-month-old female C57BL/6J mice. Behav Brain Res 159:145–151. https://doi.org/10.1016/j.bbr.2004.10.011

    CAS  Article  PubMed  Google Scholar 

  47. Suzuki T, Motohashi H, Yamamoto M (2013) Toward clinical application of the Keap1-Nrf2 pathway. Trends Pharmacol Sci 34:340–346. https://doi.org/10.1016/j.tips.2013.04.005

    CAS  Article  PubMed  Google Scholar 

  48. Vargas MR, Johnson JA (2009) The Nrf2-ARE cytoprotective pathway in astrocytes. Expert Rev Mol Med 11:e17. https://doi.org/10.1017/S1462399409001094

    Article  PubMed  PubMed Central  Google Scholar 

  49. Verkman AS, Smith AJ, Phuan P-W et al (2017) The aquaporin-4 water channel as a potential drug target in neurological disorders. Expert Opin Ther Targets 21:1161–1170. https://doi.org/10.1080/14728222.2017.1398236

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  50. Wang B, Cao W, Biswal S, Doré S (2011) Carbon monoxide-activated Nrf2 pathway leads to protection against permanent focal cerebral ischemia. Stroke 42:2605–2610. https://doi.org/10.1161/STROKEAHA.110.607101

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  51. Webster SJ, Bachstetter AD, Nelson PT et al (2014) Using mice to model Alzheimer’s dementia: an overview of the clinical disease and the preclinical behavioral changes in 10 mouse models. Front Genet 5:88. https://doi.org/10.3389/fgene.2014.00088

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  52. Yamamoto M, Kensler TW, Motohashi H (2018) The KEAP1-NRF2 system: a thiol-based sensor-effector apparatus for maintaining redox homeostasis. Physiol Rev 98:1169–1203. https://doi.org/10.1152/physrev.00023.2017

    CAS  Article  PubMed  Google Scholar 

  53. Yang Z, Wang KKW (2015) Glial fibrillary acidic protein: from intermediate filament assembly and gliosis to neurobiomarker. Trends Neurosci 38:364–374. https://doi.org/10.1016/j.tins.2015.04.003

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  54. Yang Q-Q, Zhou J-W (2019) Neuroinflammation in the central nervous system: symphony of glial cells. Glia 67:1017–1035. https://doi.org/10.1002/glia.23571

    Article  PubMed  Google Scholar 

  55. Yang B, Zador Z, Verkman AS (2008) Glial cell aquaporin-4 overexpression in transgenic mice accelerates cytotoxic brain swelling. J Biol Chem 283:15280–15286. https://doi.org/10.1074/jbc.M801425200

    CAS  Article  PubMed  PubMed Central  Google Scholar 

  56. Zhao X, Sun G, Zhang J et al (2015) Dimethyl fumarate protects brain from damage produced by intracerebral hemorrhage by mechanism involving nrf2. Stroke 46:1923–1928. https://doi.org/10.1161/STROKEAHA.115.009398

    CAS  Article  PubMed  PubMed Central  Google Scholar 

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Funding

This study was supported in part by National Institutes of Health grants R01AT007429, R01NS046400, and R21NS110008 (SD) and the American Heart Association Postdoctoral Fellowship 16POST31220032 (LL).

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LL and SD designed the study and experiments. LL, XRY, MGK, TGF, ELW, and AS performed the experiments, data collection, and analyses. LL and SD wrote the manuscript. All authors discussed, commented, and approved the final manuscript.

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Correspondence to Sylvain Doré.

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All procedures performed in studies involving animals were in accordance with the ethical standards of the National Institutes of Health Guide for the care and use of laboratory animals and approved by the Institutional Animal Care and Use Committee of the University of Florida (#201608068).

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10571_2020_807_MOESM1_ESM.jpg

Supplementary Fig. 1 Under basal conditions, the Nrf2−/− mice do not display an observable difference in the novel object recognition task. Both saline-treated wildtype (WT) and Nrf2−/− mice displayed a preference of exploration to the novel object compared with the familiar one, which were similar to the mixed group (combined with all saline-treated WT and Nrf2−/− mice; left panel; familiar vs. novel object; **P < 0.01, ***P < 0.001). Both post-saline groups displayed a very similar tendency in the discrimination index (middle panel). No significant difference was detected in the total exploration time of both objects among groups (right panel). There is no significant difference in the total exploration time between genotypes.

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Liu, L., Kelly, M.G., Yang, X.R. et al. Nrf2 Deficiency Exacerbates Cognitive Impairment and Reactive Microgliosis in a Lipopolysaccharide-Induced Neuroinflammatory Mouse Model. Cell Mol Neurobiol 40, 1185–1197 (2020). https://doi.org/10.1007/s10571-020-00807-4

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Keywords

  • Aquaporin 4
  • Astrocyte
  • Microglia
  • Neurodegeneration
  • Neuroinflammation
  • Neuroprotection
  • Novel object recognition
  • Oxidative stress