SARS-CoV-2 Spike Protein Disrupts Blood-Brain Barrier Integrity via RhoA Activation

Abstract

The SARS-CoV-2 spike protein has been shown to disrupt blood-brain barrier (BBB) function, but its pathogenic mechanism of action is unknown. Whether angiotensin converting enzyme 2 (ACE2), the viral binding site for SARS-CoV-2, contributes to the spike protein-induced barrier disruption also remains unclear. Here, a 3D-BBB microfluidic model was used to interrogate mechanisms by which the spike protein may facilitate barrier dysfunction. The spike protein upregulated the expression of ACE2 in response to laminar shear stress. Moreover, interrogating the role of ACE2 showed that knock-down affected endothelial barrier properties. These results identify a possible role of ACE2 in barrier homeostasis. Analysis of RhoA, a key molecule in regulating endothelial cytoskeleton and tight junction complex dynamics, reveals that the spike protein triggers RhoA activation. Inhibition of RhoA with C3 transferase rescues its effect on tight junction disassembly. Overall, these results indicate a possible means by which the engagement of SARS-CoV-2 with ACE2 facilitates disruption of the BBB via RhoA activation. Understanding how SARS-CoV-2 dysregulates the BBB may lead to strategies to prevent the neurological deficits seen in COVID-19 patients.

Keywords: Blood–brain barrier; Fluid shear stress; Mechanotransduction; RhoA; SARS-CoV-2.

Fig. 1

A−C Fluorescent images of 3D vessels stained with DAPI (blue), Phalloidin (green), ZO-1( red) (isolated in ii) and ACE2 (magenta)(isolated in iii) for 3 conditions: Static (A), Flow (B), (C) Flow + S1 subunit exposure for 3 h. D Schematic of rheometer setup used to apply fluid shear stress to 2D cultures for protein expression assays. E Western blot of cells exposure to static, flow, or flow + S1 protein (3 h) (i), and ratio of ACE2 to beta-actin normalized to a flow sample (ii). * Indicates p < 0.05 compared to flow. n = 3 for all experiments

Fig. 2

A Western blot of cells treated with DsiRNA targeting ACE2 (i), and relative intensity of ACE2 in DsiRNA and control cells normalized to beta-actin (ii). B Permeability coefficients measured in channels seeded with ACE2-Knockdown (KD) HCMEC/D3 cells. *indicates p < 0.05 compared to control. n = 3 for all experiments. C−D Fluorescent images of vessels stained with DAPI (blue), Phalloidin (green), ZO-1( magenta) (isolated in ii)for 3 conditions: siRNA control (C), and siRNA ACE2-KD (D)

Fig. 3

A Relative intensity (RQ) of RhoA activation in channels exposed to flow measured with ELISA. B Permeability measurement of vessels exposed to experimental conditions at 0-h and 3-h time points. *Indicates p < 0.05 compared to all conditions. C TEER values of vessels exposed to experimental conditions at 0-h,1.5 h and 3-h time points. * Indicates p < 0.05 compared to all 0-h and 1.5-h time points and ** indicates p < 0.05 compared all other conditions. n = 3 for all experiments. D Fluorescent images of vessels treated with C3 Transferase and S1 spike protein stained with DAPI (blue), Phalloidin (green), ACE2 (magenta), and ZO-1 ( red) (isolated in ii)