Expression of SARS-CoV-2-related receptors in cells of the neurovascular unit: implications for HIV-1 infection

Abstract

Background: Neurological complications are common in patients affected by COVID-19 due to the ability of SARS-CoV-2 to infect brains. While the mechanisms of this process are not fully understood, it has been proposed that SARS-CoV-2 can infect the cells of the neurovascular unit (NVU), which form the blood-brain barrier (BBB). The aim of the current study was to analyze the expression pattern of the main SARS-CoV-2 receptors in naïve and HIV-1-infected cells of the NVU in order to elucidate a possible pathway of the virus entry into the brain and a potential modulatory impact of HIV-1 in this process.

Methods: The gene and protein expression profile of ACE2, TMPRSS2, ADAM17, BSG, DPP4, AGTR2, ANPEP, cathepsin B, and cathepsin L was assessed by qPCR, immunoblotting, and immunostaining, respectively. In addition, we investigated if brain endothelial cells can be affected by the exposure to the S1 subunit of the S protein, the domain responsible for the direct binding of SARS-CoV-2 to the ACE2 receptors.

Results: The receptors involved in SARS-CoV-2 infection are co-expressed in the cells of the NVU, especially in astrocytes and microglial cells. These receptors are functionally active as exposure of endothelial cells to the SARS CoV-2 S1 protein subunit altered the expression pattern of tight junction proteins, such as claudin-5 and ZO-1. Additionally, HIV-1 infection upregulated ACE2 and TMPRSS2 expression in brain astrocytes and microglia cells.

Conclusions: These findings provide key insight into SARS-CoV-2 recognition by cells of the NVU and may help to develop possible treatment of CNS complications of COVID-19.

Keywords: ACE2; Blood-brain barrier; HIV-1; SARS-CoV-2; TMPRSS2.

Fig. 1

Expression of ACE2 in cells of the NVU. Expression levels of ACE2 were measured by q-PCR (A), immunoblotting (B), and immunostaining (C). GAPDH was used as a housekeeping gene and loading control. Graphs indicate the mean ± SD from three independent experiments. ****p < 0.0001, ***p = 0.0002, **p = 0.003, *p < 0.0449, n = 3–6 per group; scale bars, 30 μm

Fig. 2

Expression of TMPRSS2 in cells of the NVU. Expression levels of TMPRSS2 were measured by q-PCR (A), immunoblotting (B), and immunostaining (C). GAPDH was used as a housekeeping gene and loading control. Graphs indicate the mean ± SD from three independent experiments. ****p < 0.0001, ***p = 0.0002, **p = 0.003, *p < 0.0449, n = 3–6 per group; scale bars, 30 μm

Fig 3

Expression of SARS-CoV-2 entry molecules in cells of the NVU. Expression levels of ADAM17 (A), BSG (B), DPP4 (C), AGTR2 (D), ANPEP (E), cathepsin B (F), and cathepsin L (G) were measured by q-PCR. GAPDH was used as a housekeeping. Graphs indicate the mean ± SD from three independent experiments. ****p < 0.0001, ***p = 0.0002, **p = 0.003, *p < 0.0449, n = 3–6 per group

Fig. 4

Impact of the SARS-CoV-2 S1 protein subunit on claudin-5 and ZO-1 expression in EC. Human primary endothelial cells were exposed to 15 nM of the SARS-CoV-2 S1 protein subunit for 3, 12, 48, or 72 h and the expression levels of claudin-5 (A) and ZO-1 (B) were measured by immunoblotting. GAPDH was used as a loading control. Graphs indicate the mean ± SD. *p < 0.0449, **p = 0.003; n = 3 per group, three independent experiments

Fig. 5

Impact of HIV-1 infection on ACE2 and TMPRSS2 expression in astrocytes. Human primary astrocytes were either mock-infected or infected with HIV-1 with 60 ng/mL HIV-1 p24 for 24 h or 48 h and the expression levels of ACE2 and TMPRSS2 were measured by q-PCR (A and D, respectively), immunoblotting (B and E, respectively), and immunostaining (C and F, respectively). GAPDH was used as a housekeeping gene and α-tubulin as a loading control. Graphs indicate the mean ± SD from three independent experiments. **p = 0.003, *p < 0.0449, n = 4–5 per group; scale bars, 30 μm

Fig. 6

Impact of HIV-1 infection on ACE2 and TMPRSS2 expression in pericytes. Human primary pericytes were either mock-infected or infected with HIV-1 as in Fig. 5 and the expression levels of ACE2 and TMPRSS2 were measured by q-PCR (A and D, respectively), immunoblotting (B and E, respectively), and immunostaining (C and F, respectively). GAPDH was used as a housekeeping gene and α-tubulin as a loading control. Graphs indicate the mean ± SD from three independent experiments; n = 4 per group; scale bars, 30 μm

Fig. 7

Impact of HIV-1 infection on ACE2 and TMPRSS2 expression in microglial cells. Microglia were either mock-infected or infected with HIV-1 as in Fig. 5 and the expression levels of ACE2 and TMPRSS2 were measured by q-PCR (A and D, respectively), immunoblotting (B and E, respectively), and immunostaining (C and F, respectively). GAPDH was used as a housekeeping gene and α-tubulin as a loading control. Graphs indicate the mean ± SD from three independent experiments. ***p = 0.0002, *p < 0.0449, n = 4 per group; scale bars, 30 μm