Figure 4.

TLR4 mediates Spike-induced memory impairment in mice and is associated with post-COVID-19 cognitive impairment in a human cohort

(A and B) Mice received an i.c.v. infusion of 6.5 μg of SARS-CoV-2 Spike or Veh, and TLR4 mRNA levels in the hippocampi of Veh- or Spike-infused mice were evaluated at early (A; 3 days, t = 0.8892, p = 0.4034, Student’s t test) or late (B; 45 days, ^∗p = 0.0303, Mann-Whitney U test) time points (n = 4–6 mice per group).

(C) Swiss mice received an i.c.v. infusion of 6.5 μg of Spike, were treated with Veh or the TLR4 antagonist TAK-242 (2 mg/kg, intraperitoneal [i.p.], once daily for 7 days), and were tested in the late stage of the model in the NOR test (D; t = 2.713, ^∗p = 0.0301 for Spike/TAK-242). One-sample Student’s t test compared with the chance level of 50%; n = 8–9 mice per group.

(E) Plasma NFL levels evaluated in the late stage of the Spike infusion model (F = 6.329, ^∗p = 0.0133). One-way ANOVA test followed by Tukey’s test, n = 4–6 mice per group.

(F) Wild-type (WT) and TLR4 knockout (TLR4^−/−) mice received an i.c.v. infusion of 6.5 μg of SARS-CoV-2 Spike and were tested in the NOR test in the late stage of the model (F; t = 2.033, p = 0.0883 for WT/Spike and t = 2.744, ^∗p = 0.0336 for TLR4^−/−/Spike). One-sample Student’s t test compared with the chance level of 50%, n = 7 mice per group.

(G and H) Representative images of the DG hippocampal region of WT/Spike (G) and TLR4^−/−/Spike (H) mice immunolabeled for Homer1 (red) and SYP (green). Scale bar, 20 μm.

(I–K) Number of puncta for Homer-1 (I; t = 1.272, p = 0.2506) and SYP (J; t = 1.592, p = 0.1624) and colocalized Homer-1/SYP puncta (K; t = 2.945, ^∗p = 0.0258). Student’s t test; n = 4 mice per group.

(L and M) Representative images of Iba-1 immunolabeling in the DG hippocampal subregion of WT (L) and TLR4^−/− (M) mice infused with Spike. Scale bar, 25 μm; inset scale bar, 10 μm.

(N) Iba-1⁺ cells in the DG (t = 5.088, ^∗p = 0.0014) hippocampal subregion of WT or TLR4^−/− mice infused with Spike.

(O) Quantification of the different morphological types of Iba-1⁺ cells in the hippocampus of Spike-infused WT and TLR4^−/− mice (O; t = 2.229, #p = 0.0611 for type I; t = 3.340, ^∗p = 0.0124 for type II; t = 3.277, ^∗p = 0.0135 for type IV; t = 3.316, ^∗p = 0.0128 for type V). Student’s t test, n = 4–5 mice per group. Type I and type II cells have smaller somata and fewer than 5 thin branches, surveillant microglia. Type III, IV, and V cells have more than 4 branches, thicker branches, and bigger somata, reactive microglia.

(P and Q) Representative images of microglia (Iba-1⁺, green) engulfing pre-synaptic terminals immunolabeled for SYP (red) in the DG hippocampal subregion of WT (P) and TLR4^−/− (Q) mice infused with Spike. Scale bar, 50 μm; inset scale bar, 10 μm.

(R and S) Quantification of microglia-SYP colocalization in the CA3 (R; t = 2.200, #p = 0.0637) and DG (S; t = 4.012, ^∗p = 0.0051) hippocampal subregions. Student’s t test; n = 4–5 mice per group. Symbols represent individual mice, and bars represent mean ± SEM.

(T) Pipeline to analyze the impact of TLR4 variants on the cognitive status of patients with post-COVID-19 syndrome.

(U and V) Forest plots showing the OR and 95% confidence interval for risk of cognitive impairment post COVID-19 by genotype for SNPs TLR4 - 2604G>A (U, rs10759931) and TLR4-2272A>G (V, rs2737190). Each square represents the OR for each genotype, and each horizontal line shows the 95% confidence interval.

(W) The expression levels of TLR4 for genotypes of SNP TLR4-2604G>A (rs10759931) was determined from peripheral blood mononuclear cells (PBMCs) treated with 1 μg of Spike protein for 24 h (t = 5.612, ^∗p < 0.0001). Student’s t test; n = 7–8 patients per group. Data represents the mean ± SD.