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Comparative Study
. 2017 Aug 11;292(32):13415-13427.
doi: 10.1074/jbc.M117.784983. Epub 2017 Jun 27.

Intramembrane attenuation of the TLR4-TLR6 dimer impairs receptor assembly and reduces microglia-mediated neurodegeneration

Liraz Shmuel-Galia  1 , Yoel Klug  1 , Ziv Porat  2 , Meital Charni  1 , Batya Zarmi  1 , Yechiel Shai  3
Affiliations
Comparative Study

Intramembrane attenuation of the TLR4-TLR6 dimer impairs receptor assembly and reduces microglia-mediated neurodegeneration

Liraz Shmuel-Galia et al. J Biol Chem. .

Abstract

Recently, a single study revealed a new complex composed of Toll-like receptor 4 (TLR4), TLR6, and CD36 induced by fibrillary Aβ peptides, the hallmark of Alzheimer's disease. Unlike TLRs located on the plasma membrane that dimerize on the membrane after ligand binding to their extracellular domain, the TLR4-TLR6-CD36 complex assembly has been suggested to be induced by intracellular signals from CD36, similar to integrin inside-out signaling. However, the assembly site of TLR4-TLR6-CD36 and the domains participating in Aβ-induced signaling is still unknown. By interfering with TLR4-TLR6 dimerization using a TLR4-derived peptide, we show that receptor assembly is abrogated within the plasma membrane. Furthermore, we reveal that the transmembrane domains of TLR4 and TLR6 have an essential role in receptor dimerization and activation. Inhibition of TLR4-TLR6 assembly was associated with reduced secretion of proinflammatory mediators from microglia cells, ultimately rescuing neurons from death. Our findings support TLR4-TLR6 dimerization induced by Aβ. Moreover, we shed new light on TLR4-TLR6 assembly and localization and show the potential of inhibiting TLR4-TLR6 dimerization as a treatment of Alzheimer's disease.

Keywords: Alzheimer disease; CD36; TLR4; TLR6; amyloid-β; inflammation; microglia; neurodegenerative disease; neuroinflammation; toll-like receptor.

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Conflict of interest statement

The authors declare that they have no conflicts of interest with the contents of this article

Figures

Figure 1.
Figure 1.
TLR4 and TLR6 TMDs form a heterodimer within the membrane that can be inhibited by TMD-derived peptides. A and B, TLR4 and TLR6 TMDs heterodimerize within the E. coli membrane. A, schematic illustration of the GALLEX heterodimerization system. The TMD anchors the chimera in the cytoplasmic membrane of E. coli with the MBP domain located in the periplasm and the LexA DNA-binding domain in the cytoplasm. Interaction of the TMDs leads to the formation of LexA heterodimers, which can bind to the operator region. The binding of the LexA dimer results in repression of the reporter gene (lacZ) and to reduced expression levels of β-gal. B, β-gal activity was measured after expression of the chimera proteins with either the N or C termini of TLR4 or TLR6 TMD. As a positive control we expressed the GPA TMD, which is well characterized for its ability to homodimerize in the membrane and as a negative control the homodimerization-deficient mutant sequence of GPA, G83I. β-Gal activity was normalized to the expression levels of the chimera proteins. Results are the mean ± S.E. of three independent experiments (6–8 repeats for each experiment). C, TLR-derived peptides inhibit the secretion of the proinflammatory cytokine, IL-1β, in BV2 microglia cells. Cells were incubated for 2 h with 20 μm concentrations of the indicated peptides, then washed and stimulated with 10 μm Aβ for 24 h. Secreted levels of IL-1β in the supernatant were assessed by ELISA. The results are normalized to Aβ only (absolute number: 106 ± 14pg/ml). Results are the mean ± S.E. of two independent experiments (*, p < 0.05; **, p < 0.01, 3 repeats for each experiment).
Figure 2.
Figure 2.
A peptide derived from the TLR4 TMD inhibited heterodimerization by interacting with its target receptor within the membrane. A and B, the TLR4 and TLR6 TMDs form a heterodimer within a membrane mimicking environment. 0.2 μm TLR6C2 peptide (A) scrTLR4C peptide (B) were labeled with NBD (donor) and added to 100 μm PC:Chol LUVs in PBS buffer. Changes in the intensity of the emission signal were monitored between 500 and 600 nm upon the addition of successive amounts of rhodamine labeled TLR4C peptide (acceptor). Acceptor peptide was added at ratios of 1:32 to 1:4. For each experiment the spectra was normalized to the value of the donor alone. Curves were calculated based on the average of three independent experiments. C and D, the TLR6C2 peptide interacts with the LUVs. C, 0.2 μm TLR6C2 peptide was labeled with NBD, and PC:cholesterol LUVs were added at different peptide lipid ratios: 1:2000, 1:1500, 1:1000, 1;750, 1:500, 1;250, 1:100. Changes in the intensity of the emission signal were monitored between 500 and 600 nm. D, the Kd of the TLR6C2 peptide was calculated. Curves were calculated based on the average of three independent experiments. E, the TLR4C peptide physically interacted with its corresponding receptor, TLR6. BV2 microglia cells were incubated with 20 μm concentrations of rhodamine-labeled peptide for 2 h at 37 °C. Then the cells were lysed using radio RIPA, and the soluble fraction was used for immunoprecipitation with antibodies against TLR6 or TLR2. As a negative control we used the scrTLR2C peptide. Protein samples were run on SDS-PAGE, and the presence of the peptide was detected with a fluorescence scanner (excitation at 532 nm and emission at 585 nm). Nonspecific binding of the peptides to G protein beads was subtracted. Subsequently, the gel was transferred to a membrane and subjected to Western blotting (W.B.) for TLR6 and TLR2 in the appropriate samples. Equal loading was measured by detecting of anti-tubulin in the cell lysate. Results are presented as three independent experiments. A.U., absorbance units.
Figure 3.
Figure 3.
TLR4C peptide specifically blocked the TLR4-TLR6 heterodimer in BV2 microglia cells after Aβ activation. A, A scheme showing the FRET reaction. B, representative images of cellular interaction between TLR4 and TLR6 in BV2 microglia cells with the indicated treatments observed by FRET using ImageStreamX. Scale bars, 10 μm. Cells were incubated with 20 μm concentrations of the TLR4C or scrTLR6C peptide for 0.5 h and then washed and incubated with 10 μm Aβ for another 0.5 h at 37 °C. Cells were probed with an anti-TLR6-PE-conjugated antibody (donor) and an anti-TLR4 antibody followed by staining with an APC-labeled secondary antibody (acceptor). PE intensity (middle panel) and FRET intensity (right panel) were measured. C, a graphic summary of the FRET percentage normalized to cells only. Results are the mean ± S.E. of two independent experiments (***, p < 0.005, n ≥ 17,000 for each experiment). A.U., absorbance units.
Figure 4.
Figure 4.
The TLR4C peptide attenuated TLR4-TLR6 heterodimer downstream signaling of BV2 microglia cells. A and B, the TLR4C peptide inhibits NF-κB translocation to the nucleus upon activation with Aβ. A, representative images of cellular NF-κB staining after 20min in BV2 microglia cells. B, a graphic summary of cellular NF-κB translocation to the nucleus at different time points in BV2 microglia cells with the indicated treatments as observed by fluorescence resonance energy transfer using ImageStreamX. Cells were treatment with 10 μm Aβ (●) for the indicated times after incubation with 20 μm concentrations of either the TLR4C (□) or the scrTLR6C (♢) peptide for 1 h. For labeling of NF-κB, rabbit anti-NF-κB was added (1:50) overnight at 4 °C (Santa Cruz Biotechnology) followed by staining with a secondary anti-rabbit-APC (1:100) for 2 h at room temperature (Biolegend). For nuclear staining, Hoechst was added to the cells (1:1000) for 5 min. Results are the mean ± S.E. of six independent experiments (***, p < 0.005, n ≥ 1300 cells for each experiment). C, the TLR4C peptide inhibits ERK1/2 phosphorylation after activation with Aβ. Shown is a representative image from two independent experiments of ERK1/2 phosphorylation levels in BV2 microglia cells after Aβ exposure. Cells were pretreated for 0.5 h with 20 μm concentrations of the TLR4C peptide, scrTLR6C peptide, or untreated and then washed and incubated with 10 μm Aβ for the indicated times. ERK1/2 phosphorylation levels and total ERK1/2 levels were detected by Western blotting. Equal loading was detected by measuring tubulin.
Figure 5.
Figure 5.
TLR4C specifically inhibited the secretion of proinflammatory mediators in a BV2 microglia cell line. Cells were incubated for 2 h with 20 μm concentrations of the indicated peptides, then washed and stimulated with 10 μm Aβ for 24h. Secreted levels of IL-6 (A), TNF-α (B), MCP-1 (C), and NO− (D) in the supernatant were assessed by ELISA and Greiss kit. The results are the means ± S.E. of 2–4 independent experiments and are normalized to Aβ only for each cytokine as follows: IL-6, 559.47 ± 35 pg/ml; TNF-α, 671.12 ± 70 pg/ml; MCP-1, 4707.762 ± 802 pg/ml; NO, 95.6 ± 6.62 μm (***, p < 0.005, 3 repeats for each experiment). E, The specificity of the TLR4C peptide toward the TLR4-TLR6 heterodimer was evaluated by testing its inhibitory activity (white bars, μg/ml); 0.5 μg/ml and 0.25 μg/ml LTA for TLR2-TLR6 heterodimer, PAM3CSK for TLR2-TLR1 heterodimer, and LPS for TLR4-TLR4 homodimer (black bars). Results are the mean ± S.E. of two independent experiments (three repeats for each experiment). ns, not significant.
Figure 6.
Figure 6.
TLR4C inhibited the secretion of IL-6, TNF-α and MCP-1 in primary murine microglia cells. Cells were incubated for 2 h with 20 μm concentrations of the indicated peptides, then washed and stimulated with 10 μm Aβ for 24 h. Secreted levels of IL-6 (A), TNF-α (B), and MCP-1 (C) in the supernatant were assessed by ELISA. The results are the mean ± S.E. of 3 independent experiments and are normalized to Aβ for each cytokine as follows: IL-6, 304.98 ± 13.42 pg/ml; TNF-α, 732.137 ± 250pg/ml; MCP-1, 620.49 ± 20.57 pg/ml. ***, p < 0.005, 3 repeats for each experiment.
Figure 7.
Figure 7.
The TLR4C peptide rescued neurons from death mediated by microglia-induced inflammation. A, graphic summary of neurons dead/live ratio caused by microglia-induced inflammation. CAD neuronal cells were differentiated for 4 days by serum withdrawal. Then microglia cells pretreated with 20 μm concentrations of either TLR4C or scrTLR6C for 2 h were seeded into a transwell insert on top of the neuronal cells. After 72 h of stimulation with 10 μm Aβ, neurons were collected and labeled with propidium iodide for detection of dead cells. Live and dead neurons were analyzed by FACS. The results are the ratio between the dead and the live neurons and are the mean ± S.E. of four independent experiments (***, p < 0.005). B, representative images from two independent experiments of neuron survival were observed using confocal microscopy. For labeling of neurons, rabbit anti-tubulin antibody was added (1:50) overnight at 4 °C followed by incubation with a secondary anti-rabbit-APC (1:100) for 2 h at room temperature (Biolegend). The nucleus was stained by Hoechst. Scale bar, 20 μm.
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