SARS-CoV-2 S1 Protein Induces Endolysosome Dysfunction and Neuritic Dystrophy

Abstract

SARS-CoV-2 is the viral cause of the COVID-19 pandemic. Increasingly, significant neurological disorders have been associated with COVID-19. However, the pathogenesis of these neurological disorders remains unclear especially because only low or undetectable levels of SARS-CoV-2 have been reported in human brain specimens. Because SARS-CoV-2 S1 protein can be released from viral membranes, can cross the blood-brain barrier, and is present in brain cells including neurons, we tested the hypothesis that SARS-CoV-2 S1 protein can directly induce neuronal injury. Incubation of primary human cortical neurons with SARS-CoV-2 S1 protein resulted in accumulation of the S1 protein in endolysosomes as well as endolysosome de-acidification. Further, SARS-CoV-2 S1 protein induced aberrant endolysosome morphology and neuritic varicosities. Our findings suggest that SARS-CoV-2 S1 protein directly induces neuritic dystrophy, which could contribute to the high incidence of neurological disorders associated with COVID-19.

Keywords: SARS-CoV-2 S1; endocytosis; endolysosomal acidification; lysosome; neuritic dystrophy; neuron.

FIGURE 1

SARS-CoV2 S1 induced endolysosome dysfunction in mouse neuronal cells. (A) Immunoblotting showed the expression of ACE2 receptors (90 kDa) in mouse primary hippocampal neurons (Ms-pHN, Lane 1) and in mouse CLU199 hippocampal neuronal cells (Lane 2). (B) Representative confocal images show the presence of SARS-CoV2 S1 Receptor Binding domain (RBD) with C-terminal GFP (S1-RBD-GFP, 250 ng/mL for 30 min) in endolysosomes (LysoTracker Red) and lysosomes (LAMP1-RFP) lysosomes (red) in CLU199 cells. (C) Representative confocal images show the presence of S1-RBD-GFP (250 ng/mL for 30 min) in endolysosomes (LysoTracker Red) in primary cultured mouse hippocampal neurons. (D) Line graph shows that SARS-CoV2 S1 de-acidified endolysosomes in a concentration- and time-dependent manner. Whereas heat inactivated SARS-CoV2 S1 (control, 50 ng/ml), full length SARS-CoV2 S (50 ng/ml), or SARS-CoV1 S1 (50 ng/ml) did not induced endolysosome de-acidification. Bar graph shows that SARS-CoV2 S1 (compared with control at 10 min) de-acidified endolysosomes in a concentration-dependent manner (n = 3; *p < 0.05, ***p < 0.001, ****p < 0.0001). (E) Bar graph and representative confocal images show that SARS-CoV2-S1 (50 ng/ml for 30 min), but not SARS-CoV1 S1 (50 ng/ml), significantly decreased the percentage of active Cathepsin D positive lysosomes (BODIPY FL-Pepstatin A- green) vs. total endolysosomes (Dextran-Texas Red) (n = 3 repeats).

FIGURE 2

SARS-CoV2 S1 protein entered endolysosomes and induced lysosome dysfunction in human neurons. (A) Representative confocal images show the accumulation of SARS-CoV2 S1 Receptor Binding domain (RBD) with C-terminal GFP (S1-RBD-GFP, 250 ng/mL for 48h) in endolysosomes of primary human cortical neurons. Endolysosomes were identified with LysoTracker Red DND-99 or transduced with Rab7-RFP for late endosomes and Lysosome-RFP for lysosomes. 3D Imaris reconstruction of images show the presence of S1-RBD-GFP inside late endosome (Rab7-RFP) and lysosomes (LAMP1-RFP). (B) Graph shows time-dependent lysosome de-acidification effects of SARS-CoV2 S1 (50 and 100 ng/ml) in primary human neurons and lack of de-acidification effects of heat-inactivated S1 (controls). Lysosome pH was measured ratio-metrically by transfecting with RpH-LAMP1-3XFLAG. Representative confocal images of RpH-LAMP1-3XFLAG transfected human primary neurons at 0- and 10-min were shown on the right (n = 3 repeats). (C) Representative confocal images and bar graph show that SARS-CoV2 S1 (100 ng/ml for 30 min) decreased the percentage of active cathepsin D positive lysosomes (BODIPY FL-Pepstatin A-green) vs. total endolysosomes (LysoTracker Red) in primary human cortical neurons (n = 3).

FIGURE 3

SARS-CoV2 S1 induced lysosome enlargement and neurite dystrophy. (A) SARS-CoV2 S1 (50 ng/ml for 72 h) induced morphological changes of lysosomes (LAMP1-GFP) in neuronal soma and along the neurites of mouse primary hippocampal neurons (DIV5-7) transduced with GFP; heat-inactivated S1 was used as a control. Bar graph shows that SARS-CoV2 S1 (50 ng/ml for 72 h) induced significant enlargement of lysosomes along the neurites but not in the soma (n = 3). (B) SARS-CoV2 S1 (50 ng/ml for 72 h) significantly reduced the length of neurites in primary mouse hippocampal neurons transduced with GFP; see representative confocal images and bar graph (n = 5). (C) Representative confocal images and 3D reconstruction (right panel) show the presence of late endosomes (Rab7-RFP) and lysosomes (LAMP1-RFP) in varicosities induced by S1 protein (50 ng/ml for 72 h) in primary mouse hippocampal neurons transduced with cytosolic GFP. (D) Varicosity-like structures along the neurites were observed in S1 treated neurons transfected with GFP and LAMP1-RFP. Bar graph shows that SARS-CoV2 S1 (50 ng/ml for 72 h) significantly increased the diameter and density of varicosities along the neurites of S1 treated neurons (n = 6).