Journal: Scientific Reports

Article Title: 7,8-Dihydroxyflavone improves neuropathological changes in the brain of Tg26 mice, a model for HIV-associated neurocognitive disorder

doi: 10.1038/s41598-021-97220-8

Figure Lengend Snippet: List of primary antibodies.

Article Snippet: Anti TLR4 , Toll-like receptor 4 , Novus Biotechne , 1:200 (IHC).

Techniques: Concentration Assay, Marker

Journal: Scientific Reports

Article Title: 7,8-Dihydroxyflavone improves neuropathological changes in the brain of Tg26 mice, a model for HIV-associated neurocognitive disorder

doi: 10.1038/s41598-021-97220-8

Figure Lengend Snippet: DHF treatment downregulates activation of the TLR4 and NFkB. Legend: TLR4 and NFkB expression in the hippocampus and cortex of Wild Type (WT), Tg26, and DHF treated Tg26 mice ( A - P ). Immunohistochemical stained sections show TLR4 and NFkB expressing cells in the hippocampal and cortex regions of the mice brains. 3 and 3 WT mice, respectively, 3 Tg26 mice, and 4 TG + DHF mice were used. A, B, C, E, F, G, I, J, K, M, N, and O are 400 × magnification pictures of the hippocampus and cortex regions of the mice. ( D ) Quantification of TLR4 expressing cells in the hippocampus: p < 0.001, WT versus Tg; p < 0.001 Tg versus Tg + DHF. ( H ) Quantification of TLR4 expressing cells in cortex: p < 0.05, WT versus Tg; p < 0.001, Tg versus Tg + DHF. ( L ) Quantification of NFkB expressing cells in the hippocampus: p < 0.001, WT versus Tg; p < 0.001 Tg versus Tg + DHF. ( P ) Quantification of NFkB expressing cells in cortex: p < 0.001, WT versus Tg; p > 0.05, Tg versus Tg + DHF. The total numbers of hippocampal fields analyzed (WT, Tg26, TG + DHF) for each antibody were TLR4 (55, 87, 104), NFkB (154, 72, 101). The total numbers of cortex fields analyzed (WT, Tg26, TG + DHF) for each antibody were TLR4 (15, 15, 20), NFkB (15, 15, 20). One Way ANOVA with Bonferroni's Multiple Comparison post- test. scale bar, 100 μm.

Article Snippet: Anti TLR4 , Toll-like receptor 4 , Novus Biotechne , 1:200 (IHC).

Techniques: Activation Assay, Expressing, Immunohistochemical staining, Staining

Journal: eBioMedicine

Article Title: The S1P receptor 1 antagonist Ponesimod reduces TLR4-induced neuroinflammation and increases Aβ clearance in 5XFAD mice

doi: 10.1016/j.ebiom.2023.104713

Figure Lengend Snippet: Ponesimod prevents Aβ-induced increase of TLR4 and S1PR1 levels . A. Levels of TLR4 and S1PR1 in 5XFAD (N = 3 controls and 3 5XFAD mice, T-test). P < 0.05, ∗∗P < 0.01. B. Levels of TLR4 and S1PR1 in AD patient brain grey matter (N = 5 healthy and 5 AD brains, T-test). Bar graphs show means ± SEM. ∗P < 0.05, ∗∗P < 0.01. C, D. Primary cultures of mixed glia were treated with 1 μM Aβ 42 in the presence or absence of 10 nM (in C) or 100 nM Ponesimod (Pone, in C and D) for 24 h. Levels of TLR4 and S1PR1 were determined using immunoblotting. N = 3. One-way ANOVA followed by Turkey's multiple comparisons test.

Article Snippet: Lysates were pre-cleared with 20 μL of Pierce protein A/G magnetic beads (Pierce Biotechnology, Cat#88802, Rockford, USA) for 1 h at 4 °C under rotational movement and then incubated overnight with mouse TLR4 antibody (Novus, Cat#NB100-56566) at 4 °C under rotational movement.

Techniques: Western Blot

Journal: eBioMedicine

Article Title: The S1P receptor 1 antagonist Ponesimod reduces TLR4-induced neuroinflammation and increases Aβ clearance in 5XFAD mice

doi: 10.1016/j.ebiom.2023.104713

Figure Lengend Snippet: Aβ 42 induces a complex between TLR4 and S1PR1. A, B. Primary cultures of mixed glia cells were treated with 1 μM Aβ 42 in the presence or absence of 100 nM Ponesimod (Pone) for 24 h. Proximity ligation assays (PLAs) were performed using anti-TLR4 and S1PR1 antibodies (PLA, red) and co-labeled for Aβ 42 (purple in A or green in B) and phagosomes (CD68+, green) visualized with the respective antibodies. The quantitation of PLA signals is shown in C . Data shows means ± SEM. N = 3, One-way ANOVA followed by Turkey's multiple comparisons test, ∗∗P < 0.01, ∗∗∗∗P < 0.0001. D. Hela cells were transfected with TLR4-YFP and Flag-S1PR1 and then treated with 1 μM Aβ 42 in the presence or absence of 100 nM Ponesimod (Pone) for 4 h. Cell lysates were incubated with 1 μg of TLR4 mouse antibody to co-immunoprecipitate S1PR1. Captured protein was analyzed by SDS-immunoblotting with anti-S1PR1 rabbit antibody. N = 3.

Article Snippet: Lysates were pre-cleared with 20 μL of Pierce protein A/G magnetic beads (Pierce Biotechnology, Cat#88802, Rockford, USA) for 1 h at 4 °C under rotational movement and then incubated overnight with mouse TLR4 antibody (Novus, Cat#NB100-56566) at 4 °C under rotational movement.

Techniques: Ligation, Labeling, Quantitation Assay, Transfection, Incubation, Western Blot

Journal: eBioMedicine

Article Title: The S1P receptor 1 antagonist Ponesimod reduces TLR4-induced neuroinflammation and increases Aβ clearance in 5XFAD mice

doi: 10.1016/j.ebiom.2023.104713

Figure Lengend Snippet: Working model for the effect of Ponesimod on microglial activation and phagocytosis . Aβ 42 binds to TLR4 and induces a complex with S1PR1. S1P co-activates the downstream pro-inflammatory cell signaling pathways ERK, p38 MAPK and JNK, which leads to phosphorylation activation of the transcription factors Stat1/3 and induction of neuroinflammation, particularly via gene expression of TNF-α. Ponesimod blocks S1P mediated co-activation of TLR4 and instead, leads to phagocytosis and proteolytic degradation of the receptor complex and Aβ 42 . In addition to inhibition of the canonical cell signaling for neuroinflammation downstream of TLR4, Ponesimod upregulates microglial phagocytosis via IL-33/Stat6 in a non-canonical cell signaling pathway, thereby contributing to persistent clearance of Aβ and alleviation of AD pathology. Generated using Biorender.

Article Snippet: Lysates were pre-cleared with 20 μL of Pierce protein A/G magnetic beads (Pierce Biotechnology, Cat#88802, Rockford, USA) for 1 h at 4 °C under rotational movement and then incubated overnight with mouse TLR4 antibody (Novus, Cat#NB100-56566) at 4 °C under rotational movement.

Techniques: Activation Assay, Protein-Protein interactions, Phospho-proteomics, Gene Expression, Inhibition, Generated