Pivotal role for neuronal Toll-like receptors in ischemic brain injury and functional deficits

Abstract

The innate immune system senses the invasion of pathogenic microorganisms and tissue injury through Toll-like receptors (TLR), a mechanism thought to be limited to immune cells. We now report that neurons express several TLRs, and that the levels of TLR2 and -4 are increased in neurons in response to IFN-gamma stimulation and energy deprivation. Neurons from both TLR2 knockout and -4 mutant mice were protected against energy deprivation-induced cell death, which was associated with decreased activation of a proapoptotic signaling cascade involving jun N-terminal kinase and the transcription factor AP-1. TLR2 and -4 expression was increased in cerebral cortical neurons in response to ischemia/reperfusion injury, and the amount of brain damage and neurological deficits caused by a stroke were significantly less in mice deficient in TLR2 or -4 compared with WT control mice. Our findings establish a proapoptotic signaling pathway for TLR2 and -4 in neurons that may render them vulnerable to ischemic death.

Fig. 1.

Neurons express TLRs and respond to IFNγ stimulation. (a) TLR2 and -4 mRNA are present in cultured cortical neurons determined by single-cell RT-PCR analysis. The numbers indicate the number of neurons from which RNA was amplified; 3, 6, and 10 cortical neurons consistently yielded a positive PCR signal for the TLR2 and -4 with exactly predicted size. (b) Level of TLR mRNAs in neurons treated with IFNγ (500 μg/ml). Neurons expressed TLR2, -3, -4, and MYD88 mRNAs, and their expression levels increased in response to IFNγ. (c) Immunoblot showing relative levels of TLR2 (95 kDa), TLR3 (95 kDa), TLR4 (90 kDa), and MYD88 (33 kDa) in lysates of cultured neurons treated with vehicle (Con) or IFNγ. (d) TLR2, -3, and -4 immunoreactivities (green) in cultured neurons; cells were counterstained with DAPI (blue) to label all nuclei and with the neuron-specific marker NeuN (red). (e) Results of microarray analysis for the indicated TLR mRNAs in RNA samples isolated from cultured cortical neurons (six separate RNA samples run in triplicate; c1–c18). Relative expression levels are indicated by the color scale shown.

Fig. 2.

Expression of TLR2 and -4 increases after GD, and neurons deficient in TLR2 or -4 receptors are resistant to GD-induced death. (a) Results of a microarray analysis comparing levels of the indicated TLR mRNAs in neurons in control cultures with the level in cultures subjected to GD for 0.5, 6, or 18 h. Note that GD induced increased expression of TLR2 and -4 but not the other TLRs. (b) Immunoblot analysis of proteins in cell lysates of neurons in control cultures and cultures subjected to GD for 3 or 6 h. GD resulted in increased levels of TLR2, -4, HSP70, and p-JNK. (c) Immunoblot analysis of proteins in cell lysates of neurons or astrocytes in control cultures and cultures subjected to GD for the indicated time periods. (d) Cultured cortical neurons from WT mice, TLR2 knockout mice, and TLR4 mutant mice were subjected to GD for the indicated time periods, and cell survival was quantified. Values are the mean and SEM (n = 24–36 cultures). *, P < 0.05 compared with the WT value.

Fig. 3.

TLR2 and -4 mediate energy deprivation-induced activation of the JNK-AP-1 pathway and caspase-3 in cultured cortical neurons. (a and b) Immunoblot analysis of proteins in cell lysates of neurons from WT, TLR2 knockout, and TLR4 mutant mice that had been subjected to GD for the indicated time periods. GD resulted in increased levels of p-JNK and the cleaved form of caspase-3 (19-kDa) in WT neurons but little or no increase in p-JNK or activated caspase-3 in neurons deficient in TLR2 or -4. (c and d) Gel-shift analysis showing NF-κB and AP-1 DNA-binding activities in lysates of neurons from WT, TLR2 knockout mice, or TLR4 mutant mice that had been subjected to GD for 1, 3, or 6 h. Levels of AP-1 DNA-binding activity were lower in samples from TLR2 and -4 mutant mice compared with WT mice. (e) Neuronal cultures were treated with 1 or 10 μM of the JNK inhibitor SP600125 and then subjected to GD (or not) for 20 h. Proteins in cell lysates were then subjected to immunoblot analysis by using the indicated antibodies. The JNK inhibitor attenuated GD-induced increases in levels of p-JNK and activated caspase-3.

Fig. 4.

TLR2 and -4 mediate ischemic neuronal death and functional deficits in a mouse stroke model. Mice of the indicated genotypes were subjected to sham surgery or ischemia/reperfusion (I/R), neurological function was evaluated daily for 3 d, and brain damage was evaluated at 3 d. (a and b) Neurological scores (a) and infarct volumes (b) for WT mice subjected to sham surgery (n = 5), and WT (n = 12) and TLR2 knockout (n = 11) mice subjected to I/R. *, P < 0.001 compared with the sham value. +, P < 0.01 compared with the WT I/R value. (c and d) Neurological scores (c) and infarct volumes (d) for WT mice subjected to sham surgery (n = 5), and WT (n = 8) and TLR4 mutant (n = 7) mice subjected to I/R. *, P < 0.001 compared with the sham value. +, P < 0.01 compared with the WT I/R value.

Fig. 5.

Cerebral ischemia induces a rapid increase in TLR2 and -4 immunoreactivities in neurons and a delayed appearance of TLR2-positive microglia. (a) Immunoblot analysis of protein samples from the cerebral cortex of sham-operated control mice and mice killed either 1 or 3 h after I/R. (b–d) Images of brain sections showing TLR2 and -4 immunoreactivities (green), DAPI staining (blue), and NeuN (neuronal marker) or IBA1 (microglial marker) immunoreactivities (red) in WT mice subjected to sham surgery of I/R for the indicated time periods. Ischemia resulted in rapid increases in the levels of TLR2 and -4 immunoreactivities in neurons in the cerebral cortex and a delayed appearance of TLR2 immunoreactive IBA1-positive microglial cells.