Skip to main content
SpringerLink
Log in

Toll-Like Receptor 4 Signaling in Neurons Mediates Cerebral Ischemia/Reperfusion Injury

  • Published:
Molecular Neurobiology Aims and scope Submit manuscript

Abstract

In microglia, Toll-like receptor 4 (TLR4) is well known to contribute to neuroinflammatory responses following brain ischemia. TLR4 is also expressed in neurons and can mediate the conduction of calcium (Ca2+) influx, but the mechanistic link between neuronal TLR4 signaling and brain ischemic injury is still poorly understood. Here, primary neuronal cell cultures from TLR4 knockout mice and mice with conditional TLR4 knockout in glutamatergic neurons (TLR4cKO) were used to establish ischemic models in vitro and in vivo, respectively. We found that deleting TLR4 would reduce the neuronal death and intracellular Ca2+ increasement induced by oxygen and glucose deprivation (OGD) or lipopolysaccharide treatment. Infarct volume and functional deficits were also alleviated in TLR4cKO mice following cerebral ischemia/reperfusion (I/R). Furthermore, TLR4 and N-methyl-d-aspartate receptor subunit 2B (NMDAR2B) were colocalized in neurons. Deletion of TLR4 in neurons rescued the upregulation of phosphorylated NMDAR2B induced by ischemia via Src kinase in vitro and in vivo. Downstream of NMDAR2B signaling, the interaction of neuronal nitric oxide synthase (nNOS) with postsynaptic density protein-95 (PSD-95) was also disrupted in TLR4cKO mice following cerebral I/R. Taken together, our results demonstrate a novel molecular neuronal pathway in which TLR4 signaling in neurons plays a crucial role in neuronal death and provide a new target for neuroprotection after ischemic stroke.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

Data Availability

The datasets used and/or analyzed during the present study are available from the corresponding author on reasonable request.

Abbreviations

TLR4:

Toll-like receptor 4

Ca2+ :

Calcium

TLR4cKO :

Conditional knockout mice of TLR4 in glutamatergic neurons

OGD:

Oxygen and glucose deprivation

I/R:

Ischemia/reperfusion

NMDAR2B:

N-methyl-d-aspartate receptor subunit 2B

nNOS:

Neuronal nitric oxide synthase

PSD-95:

Postsynaptic density protein-95

LPS:

Lipopolysaccharide

VGLUT2:

Vesicular glutamate transporters 2

MCAO:

Middle cerebral artery occlusion

WT:

Wild type

TTC:

Triphenyl tetrazolium chloride

FJB:

Fluoro-Jade B

References

  1. Leitner GR et al (2019) Targeting toll-like receptor 4 to modulate neuroinflammation in central nervous system disorders. Expert Opin Ther Targets 23(10):865–882

    Article  Google Scholar 

  2. Buchanan MM, Hutchinson M, Watkins LR, Yin H (2010) Toll-like receptor 4 in CNS pathologies. J Neurochem 114(1):13–27

    CAS  Google Scholar 

  3. Luo L et al (2022) Intermittent theta-burst stimulation improves motor function by inhibiting neuronal pyroptosis and regulating microglial polarization via TLR4/NFkappaB/NLRP3 signaling pathway in cerebral ischemic mice. J Neuroinflammation 19(1):141

    Article  CAS  Google Scholar 

  4. Tang SC et al (2007) Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits. Proc Natl Acad Sci USA 104(34):13798–13803

    Article  CAS  Google Scholar 

  5. Lipton SA (2006) Paradigm shift in neuroprotection by NMDA receptor blockade: memantine and beyond. Nat Rev Drug Discov 5(2):160–170

    Article  CAS  Google Scholar 

  6. Granzotto A et al (2022) Long-term dynamic changes of NMDA receptors following an excitotoxic challenge. Cells 11(5):911

    Article  CAS  Google Scholar 

  7. Yu XM, Askalan R, Keil GJ 2nd, Salter MW (1997) NMDA channel regulation by channel-associated protein tyrosine kinase Src. Science 275(5300):674–678

    Article  CAS  Google Scholar 

  8. Calvo-Rodriguez M et al (2017) Aging and amyloid beta oligomers enhance TLR4 expression, LPS-induced Ca(2+) responses, and neuron cell death in cultured rat hippocampal neurons. J Neuroinflammation 14(1):24

    Article  Google Scholar 

  9. Balosso S, Liu J, Bianchi ME, Vezzani A (2014) Disulfide-containing high mobility group box-1 promotes N-methyl-d-aspartate receptor function and excitotoxicity by activating Toll-like receptor 4-dependent signaling in hippocampal neurons. Antioxid Redox Signal 21(12):1726–1740

    Article  CAS  Google Scholar 

  10. Wang PF et al (2014) Polyinosinic-polycytidylic acid has therapeutic effects against cerebral ischemia/reperfusion injury through the downregulation of TLR4 signaling via TLR3. J Immunol 192(10):4783–4794

    Article  CAS  Google Scholar 

  11. Thatipamula S, Hossain MA (2014) Critical role of extracellularly secreted neuronal pentraxin 1 in ischemic neuronal death. BMC Neurosci 15:133

    Article  Google Scholar 

  12. Tu W et al (2010) DAPK1 interaction with NMDA receptor NR2B subunits mediates brain damage in stroke. Cell 140(2):222–234

    Article  CAS  Google Scholar 

  13. Cao CX et al (2007) Reduced cerebral ischemia-reperfusion injury in Toll-like receptor 4 deficient mice. Biochem Biophys Res Commun 353(2):509–514

    Article  CAS  Google Scholar 

  14. Tsien RY, Pozzan T, Rink TJ (1982) T-cell mitogens cause early changes in cytoplasmic free Ca2+ and membrane potential in lymphocytes. Nature 295(5844):68–71

    Article  CAS  Google Scholar 

  15. Rahmati M, Taherabadi SJ (2021) The effects of exercise training on Kinesin and GAP-43 expression in skeletal muscle fibers of STZ-induced diabetic rats. Sci Rep 11(1):9535

    Article  CAS  Google Scholar 

  16. Rahmati M, Rashno A (2021) Automated image segmentation method to analyse skeletal muscle cross section in exercise-induced regenerating myofibers. Sci Rep 11(1):21327

    Article  CAS  Google Scholar 

  17. Duan CM et al (2020) SRT2104 attenuates chronic unpredictable mild stress-induced depressive-like behaviors and imbalance between microglial M1 and M2 phenotypes in the mice. Behav Brain Res 378:112296

    Article  CAS  Google Scholar 

  18. Ma Y et al (2006) Toll-like receptor 8 functions as a negative regulator of neurite outgrowth and inducer of neuronal apoptosis. J Cell Biol 175(2):209–215

    Article  CAS  Google Scholar 

  19. Ludhiadch A, Sharma R, Muriki A, Munshi A (2022) Role of calcium homeostasis in ischemic stroke: a review. CNS Neurol Disord Drug Targets 21(1):52–61

    Article  CAS  Google Scholar 

  20. Wang J et al (2003) Cdk5 activation induces hippocampal CA1 cell death by directly phosphorylating NMDA receptors. Nat Neurosci 6(10):1039–1047

    Article  CAS  Google Scholar 

  21. Wu QJ, Tymianski M (2018) Targeting NMDA receptors in stroke: new hope in neuroprotection. Mol Brain 11(1):15

    Article  Google Scholar 

  22. Salter MW, Kalia LV (2004) Src kinases: a hub for NMDA receptor regulation. Nat Rev Neurosci 5(4):317–328

    Article  CAS  Google Scholar 

  23. Dawson VL et al (1996) Resistance to neurotoxicity in cortical cultures from neuronal nitric oxide synthase-deficient mice. J Neurosci: Off J Soc Neurosci 16(8):2479–2487

    Article  CAS  Google Scholar 

  24. Viviani B et al (2003) Interleukin-1beta enhances NMDA receptor-mediated intracellular calcium increase through activation of the Src family of kinases. J Neurosci: Off J Soc Neurosci 23(25):8692–8700

    Article  CAS  Google Scholar 

  25. O’Neill LA, Bowie AG (2007) The family of five: TIR-domain-containing adaptors in Toll-like receptor signalling. Nat Rev Immunol 7(5):353–364

    Article  CAS  Google Scholar 

  26. Hanke JH et al (1996) Discovery of a novel, potent, and Src family-selective tyrosine kinase inhibitor Study of Lck- and FynT-dependent T cell activation. J Biol Chem 271(2):695–701

    Article  CAS  Google Scholar 

  27. Sattler R et al (1999) Specific coupling of NMDA receptor activation to nitric oxide neurotoxicity by PSD-95 protein. Science 284(5421):1845–1848

    Article  CAS  Google Scholar 

  28. Zhou L et al (2010) Treatment of cerebral ischemia by disrupting ischemia-induced interaction of nNOS with PSD-95. Nat Med 16(12):1439–1443

    Article  CAS  Google Scholar 

  29. Sadat-Hatamnezhad L, Tanomand A, Mahmoudi J, SandoghchianShotorbani S (2016) Activation of Toll-like receptors 2 by high-mobility group box 1 in monocytes from patients with ischemic stroke. Iran Biomed J 20(4):223–8

    Google Scholar 

  30. Li Y et al (2021) Food reward depends on TLR4 activation in dopaminergic neurons. Pharmacol Res 169:105659

    Article  CAS  Google Scholar 

  31. Famakin BM, Vemuganti R (2020) Toll-like receptor 4 signaling in focal cerebral ischemia: a focus on the neurovascular unit. Mol Neurobiol 57(6):2690–2701

    Article  CAS  Google Scholar 

  32. Wang Y, Ge P, Zhu Y (2013) TLR2 and TLR4 in the brain injury caused by cerebral ischemia and reperfusion. Mediators Inflamm 2013:124614

    Article  Google Scholar 

  33. Zhang P et al (2022) Neuroprotective effects of TRPM7 deletion in parvalbumin GABAergic vs. glutamatergic neurons following ischemia. Cells 11(7):1178

  34. Wang F, Xie X, Xing X, Sun X (2022) Excitatory synaptic transmission in ischemic stroke: a new outlet for classical neuroprotective strategies. Int J Mol Sci 23(16):381

    Article  Google Scholar 

  35. Mari Y, Katnik C, Cuevas J (2010) ASIC1a channels are activated by endogenous protons during ischemia and contribute to synergistic potentiation of intracellular Ca(2+) overload during ischemia and acidosis. Cell Calcium 48(1):70–82

    Article  CAS  Google Scholar 

  36. Lipton SA, Rosenberg PA (1994) Excitatory amino acids as a final common pathway for neurologic disorders. N Engl J Med 330(9):613–622

    Article  CAS  Google Scholar 

  37. Weilinger NL et al (2016) Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity. Nat Neurosci 19(3):432–442

    Article  CAS  Google Scholar 

Download references

Funding

The work was supported by grants from the National Natural Science Foundation of China (No. 81901217) and the Doctoral Research Start-up Foundation of Liaoning (2019-BS-269).

Author information

Authors and Affiliations

  1. Department of Neurology, The General Hospital of Northern Theater Command, No. 83 Wenhua Street, Shenhe District, ShenyangLiaoning, 110016, China

    Liang Liu, Tian-Ce Xu, Zi-Ai Zhao, Nan-Nan Zhang, Jing Li & Hui-Sheng Chen

Authors
  1. Liang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tian-Ce Xu
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Zi-Ai Zhao
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Nan-Nan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jing Li
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hui-Sheng Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

L. L., H. S., and T. C. X. conceived the study. Z. A. Z. wrote the main manuscript text, and N. N. Z. and J. L. prepared the figures. H. S. revised the manuscript and language. All authors reviewed the manuscript.

Corresponding author

Correspondence to Hui-Sheng Chen.

Ethics declarations

Ethics Approval and Consent to Participate

All experimental procedures were performed in accordance with approved principles of the Animal Ethics Committee of the General Hospital of Northern Theater Command.

Consent for Publication

Not applicable.

Competing Interests

The authors declare no competing interests.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 5347 kb)

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Liu, L., Xu, TC., Zhao, ZA. et al. Toll-Like Receptor 4 Signaling in Neurons Mediates Cerebral Ischemia/Reperfusion Injury. Mol Neurobiol 60, 864–874 (2023). https://doi.org/10.1007/s12035-022-03122-9

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12035-022-03122-9

Keywords

Search

Navigation