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. 2008 Sep;213(1):114-21.
doi: 10.1016/j.expneurol.2008.05.014. Epub 2008 May 29.

Toll-like receptor-4 mediates neuronal apoptosis induced by amyloid beta-peptide and the membrane lipid peroxidation product 4-hydroxynonenal

Sung-Chun Tang  1 , Justin D Lathia, Pradeep K Selvaraj, Dong-Gyu Jo, Mohamed R Mughal, Aiwu Cheng, Dominic A Siler, William R Markesbery, Thiruma V Arumugam, Mark P Mattson
Affiliations

Toll-like receptor-4 mediates neuronal apoptosis induced by amyloid beta-peptide and the membrane lipid peroxidation product 4-hydroxynonenal

Sung-Chun Tang et al. Exp Neurol. 2008 Sep.

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 recently found that neurons express several TLRs, and that the levels of TLR2 and TLR4 are increased in neurons in response to energy deprivation. Here we report that TLR4 expression increases in neurons when exposed to amyloid beta-peptide (Abeta1-42) or the lipid peroxidation product 4-hydroxynonenal (HNE). Neuronal apoptosis triggered by Abeta and HNE was mediated by jun N-terminal kinase (JNK); neurons from TLR4 mutant mice exhibited reduced JNK and caspase-3 activation and were protected against apoptosis induced by Abeta and HNE. Levels of TLR4 were decreased in inferior parietal cortex tissue specimens from end-stage AD patients compared to aged-matched control subjects, possibly as the result of loss of neurons expressing TLR4. Our findings suggest that TLR4 signaling increases the vulnerability of neurons to Abeta and oxidative stress in AD, and identify TLR4 as a potential therapeutic target for AD.

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Figures

Figure 1
Figure 1
Cultured cortical neuronal cells up-regulate expression of TLR4 when exposed to Aβ1-42. A. Levels of TLR 2, 3 and 4 mRNAs in neurons treated with Aβ (10 μM) for the indicated time periods. TLR4 mRNA levels increased in response to Aβ treatment, whereas TLR2 and TLR3 mRNA levels did not change. B. Immunoblot showing relative levels of TLR2 (95 KDa) and TLR4 (90 KDa) in lysates of cultured neurons that had been treated for 20 hours with vehicle (Control) or Aβ (1, 5 or 10 μM). Levels of TLR4, but not TLR2 or TLR3, were increased in response to Aβ treatment. Analyses were performed on pooled samples from 4-8 cultures.
Figure 2
Figure 2
Expression of TLR2 and TLR4 increase in cortical neuronal cultures in response to exposure to HNE. A. The levels of TLR 2, 3 and 4 mRNAs in neurons treated with HNE (10 μM). B and C. Immunoblots showing protein levels of TLR2 (95 KDa) and TLR4 (90 KDa) in cultured cortical neuronal cells that had been exposed to the indicated concentrations of HNE for the indicated time periods. Levels of both TLR2 and TLR4 were increased within 3 hours and remained elevated through 20 hours of exposure to HNE. Analyses were performed on pooled samples from 4-8 cultures.
Figure 3
Figure 3
Aβ and HNE induce increases in TLR4 immunoreactivity in cultured cortical neurons. Images of TLR4 immunofluorescence (green) in cultured neurons in a control culture, and cultures that had been exposed for 12 hours to either 10 μM Aβ1-42 or 5 μM HNE. Scale bar = 100μM.
Figure 4
Figure 4
Evidence that TLR4 mediates activation of JNK and caspase-3 in response to Aβ and HNE in cortical neurons. Immunoblot analysis of phospho-JNK (46 kDa), total JNK (46 kDa) and cleaved caspase-3 (19 kDa) in cell lysates of neurons in cortical cultures established from either wild-type (WT) or TLR4 deficient (TLR4mu) mice. The neurons were left untreated or were exposed to 5 μM Aβ1-42 (panel A) or 5 μM HNE (panel C) for the indicated time points. Results of densitometric analysis of phospho-JNK and cleaved caspase-3 protein levels in WT compared with TLR4 mutant cells from Aβ treated (panel B) and HNE treated (panel D) samples at 24 hours. Analyses were performed on pooled samples from at least 3 separate cultures that had been established from 10-14 embryos in each group.
Figure 4
Figure 4
Evidence that TLR4 mediates activation of JNK and caspase-3 in response to Aβ and HNE in cortical neurons. Immunoblot analysis of phospho-JNK (46 kDa), total JNK (46 kDa) and cleaved caspase-3 (19 kDa) in cell lysates of neurons in cortical cultures established from either wild-type (WT) or TLR4 deficient (TLR4mu) mice. The neurons were left untreated or were exposed to 5 μM Aβ1-42 (panel A) or 5 μM HNE (panel C) for the indicated time points. Results of densitometric analysis of phospho-JNK and cleaved caspase-3 protein levels in WT compared with TLR4 mutant cells from Aβ treated (panel B) and HNE treated (panel D) samples at 24 hours. Analyses were performed on pooled samples from at least 3 separate cultures that had been established from 10-14 embryos in each group.
Figure 5
Figure 5
Evidence that JNK mediates activation of the apoptotic caspase-3 protein in response to Aβ and HNE in cortical neurons. Immunoblot analysis of phospho-JNK (46 kDa), total JNK (46 kDa) and cleaved caspase-3 (19 kDa) in cell lysates of neurons in cortical cultures that were pretreated for 10 minutes with the JNK inhibitor SP600125 (10 μM) or vehicle (DMSO). The cultures were then exposed for the indicated time periods to either 5 μM Aβ1-42 (panel A) or 5 μM HNE (panel C). Results of densitometric analysis of phospho-JNK and cleaved caspase-3 protein levels in DMSO treated cells compared with SP600125 (10 μM) cells from Aβ treated (panel B) and HNE treated (panel D) samples at 24 hours.
Figure 5
Figure 5
Evidence that JNK mediates activation of the apoptotic caspase-3 protein in response to Aβ and HNE in cortical neurons. Immunoblot analysis of phospho-JNK (46 kDa), total JNK (46 kDa) and cleaved caspase-3 (19 kDa) in cell lysates of neurons in cortical cultures that were pretreated for 10 minutes with the JNK inhibitor SP600125 (10 μM) or vehicle (DMSO). The cultures were then exposed for the indicated time periods to either 5 μM Aβ1-42 (panel A) or 5 μM HNE (panel C). Results of densitometric analysis of phospho-JNK and cleaved caspase-3 protein levels in DMSO treated cells compared with SP600125 (10 μM) cells from Aβ treated (panel B) and HNE treated (panel D) samples at 24 hours.
Figure 6
Figure 6
Evidence that Aβ1-42 and HNE can activate multiple kinases including p44/42, p38 and Akt kinases. Immunoblot analysis using antibodies that selectively recognize activated forms of the indicated kinases (p-p44/42, p-p38 and p-Akt) in lysates of cortical cells established from wild-type mice. The neurons were treated with 5 μM Aβ1-42 or 5 μM HNE for the indicated time periods.
Figure 7
Figure 7
Levels of TLR4 protein are decreased in association with neuronal degeneration in postmortem brain tissue samples from AD patients compared to control subjects. A. Immunoblots showing protein levels of TLR4 (90 kDa), synaptophysin (38 kDa) and IBA-1 (17 kDa) in samples of inferior parietal cortex of AD and control human subjects. B. Results of densitometric analysis of TLR4 protein levels in samples from AD and control subjects. Values are the mean and SEM. *p = 0.038 (unpaired t test).
Figure 7
Figure 7
Levels of TLR4 protein are decreased in association with neuronal degeneration in postmortem brain tissue samples from AD patients compared to control subjects. A. Immunoblots showing protein levels of TLR4 (90 kDa), synaptophysin (38 kDa) and IBA-1 (17 kDa) in samples of inferior parietal cortex of AD and control human subjects. B. Results of densitometric analysis of TLR4 protein levels in samples from AD and control subjects. Values are the mean and SEM. *p = 0.038 (unpaired t test).
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