Fig 2: Cav-1 deficiency reduces brain endothelial cell VCAM-1 expression in SARS-CoV-2 infection. A) Flow cytometry of VCAM-1 and CD31 in brain endothelial cells. B) Quantification of VCAM-1high cells as a percent of total CD31+ viable cells. n = 3–4 mice/group. Two-way ANOVA demonstrated significant effect of genotype [F(1, 9) = 42.64, p = 0.0001], infection [F(1, 9) = 5.188, p = 0.0487], and genotype*infection interaction [F(1, 9) = 7.231, p = 0.0248]. Tukey’s multiple comparison tests revealed significantly increased BEC VCAM-1 in Cav-1+/+ (p < 0.01) but not Cav-1−/− mice with SARS-CoV-2. VCAM-1high BEC were significantly fewer in Cav-1−/− mice with SARS-CoV-2 than in Cav-1+/+ mice with SARS-CoV-2 (p < 0.0001). C-D) Immunofluorescence detection of VCAM-1 (red) and podocalyxin (white) in Cav-1+/+ and Cav-1−/− mice at 5DPI SARS-CoV-2. n = 5 mice/group, with three sections analyzed per mouse. E) Quantification of VCAM-1+ vascular area. Podocalyxin immunoreactivity was used to define vascular area. There was significantly less VCAM-1+ immunoreactive vascular area in Cav-1−/− mice with SARS-CoV-2 than in Cav-1+/+ mice with SARS-CoV-2 (p < 0.01, unpaired t-test with Welch’s correction for unequal variance).

Fig 3: Cav-1 is increased in brain endothelial cells in SARS-CoV-2 infection. A-B) Immunofluorescence detection of Cav-1 (green) in hippocampal sections from wild-type mice euthanized 4 days post inoculation (DPI) with SARS-CoV-2. Monochromatic images (A, B) and overlays between Caveolin-1 (green), brain endothelial cell protein Glut-1 (red), and DAPI (blue) (A’, B′). C) Quantification of % area immunoreactive for Cav-1 in hippocampus sections. Three brain sections analyzed per mouse. **p < 0.01, unpaired student t-test. D) Flow cytometric plot of CD31 and viable exclusion dye demonstrates gating strategy for endothelial cells (box) in single cell suspension prepared from isolated brain microvessels of WT mice euthanized 4 days after respiratory inoculation with SARS-CoV-2. E-F) Histograms of Cav-1 fluorescence intensity in viable CD31+ endothelial cells isolated as in D. G) Quantification of Cav-1 fluorescence intensity in viable CD31+ endothelial cells from mice euthanized at 4 DPI with SARS-CoV-2, expressed as ratio to healthy WT. n = 6 mice/group from two independent experiments. p < 0.05, unpaired student t-test.

Fig 4: Cav-1 deficiency reduces short term learning and memory impairment in SARS-CoV-2 infection. A) Novel object discrimination index during novel object recognition testing at 5 DPI with SARS-CoV-2. n = 5–8 mice/group. Two-way ANOVA demonstrated significant effect of infection [F(1, 23) = 11.15, p = 0.0028], and genotype*infection interaction [F(1, 23) = 5.103, p = 0.0337]. Tukey’s multiple comparison test revealed significant decrease in novel object discrimination in Cav-1+/+ (p < 0.001) but not Cav-1−/− mice with SARS-CoV-2. Cav-1−/− mice with SARS-CoV-2 performed significantly better than did Cav-1+/+ mice with SARS-CoV-2. B) Object discrimination index measured during the training task demonstrates unbiased exploration during the training phase in all groups.

Fig 5: Cav-1 deficiency reduces hippocampal T cell density in SARS-CoV-2 infection. A-D) Immunofluorescence detection of CD3+ T cells (red) and Glut-1+ cerebrovasculature (green) in hippocampal sections of mice euthanized at 5 DPI with SARS-CoV-2. E) Quantification of CD3+ T cells in hippocampal sections. n = 6–8 mice/group, with two sections analyzed per mouse. Two-way ANOVA demonstrated significant effect of genotype [F(1,23) = 8.409, p = 0.0081], infection [F(1, 23) = 41.25, p < 0.0001], and genotype*infection interaction [F(1, 23) = 13.99, p = 0.0011]. Tukey’s multiple comparison tests revealed SARS-CoV-2 significantly increased T cells in the hippocampus of Cav-1+/+ mice (p < 0.001) but not Cav-1−/− mice (p = 0.09). There were significantly fewer T cells in hippocampus of Cav-1−/− mice with SARS-CoV-2 as compared to Cav-1+/+ mice with SARS-CoV-2 (p < 0.01).