Abstract
It is unclear whether severe acute respiratory syndrome coronavirus 2, which causes coronavirus disease 2019, can enter the brain. Severe acute respiratory syndrome coronavirus 2 binds to cells via the S1 subunit of its spike protein. We show that intravenously injected radioiodinated S1 (I-S1) readily crossed the blood–brain barrier in male mice, was taken up by brain regions and entered the parenchymal brain space. I-S1 was also taken up by the lung, spleen, kidney and liver. Intranasally administered I-S1 also entered the brain, although at levels roughly ten times lower than after intravenous administration.
APOE genotype and sex did not affect whole-brain I-S1 uptake but had variable effects on uptake by the olfactory bulb, liver, spleen and kidney. I-S1 uptake in the hippocampus and olfactory bulb was reduced by lipopolysaccharide-induced inflammation. Mechanistic studies indicated that I-S1 crosses the blood–brain barrier by adsorptive transcytosis and that murine angiotensin-converting enzyme 2 is involved in brain and lung uptake, but not in kidney, liver or spleen uptake.
Discussion
The results from this study show that I-S1 from two different commercial sources readily crosses the mouse BBB, at least when injected intravenously. I-S1 was taken up by all 11 brain regions examined. Such widespread entry into brain of I-S1 could explain the diverse effects of S1 and/or SARS-CoV-2 such as encephalitis, respiratory difficulties and anosmia1,3,4. S1 is the SARS-CoV-2 protein that initially binds to cell-surface receptors, setting the stage for viral internalization. For transport across the BBB, viral binding proteins often behave similarly to the virus itself. For example, interactions (including binding and transport) between the HIV-1 glycoprotein gp120 and the BBB are similar to those for the complete virus18,28. Additionally, many if not most viral proteins themselves can be biologically highly active; for example, gp120 is highly toxic11,12,13,14,15,16,17. Coronavirus spike proteins are often cleaved from the virus by host cell proteases. Once cleaved, coronavirus spike S1 and S2 subunits are not held covalently by disulfide bonds and so S1 could be shed from virions34. It is possible that during infection by SARS-CoV-2, shed S1 is available to cross the BBB, triggering responses in the brain itself, without necessarily involving crossing of intact virus particles. Thus, determining whether S1 crosses the BBB is important for understanding whether SARS-CoV-2 and S1 itself could induce responses in the brain.
Our method of studying S1 pharmacokinetics has many advantages over the more traditional approach of determining viral uptake and distribution that is based on virus recovery. Radioactive labeling allows S1 to be detected at very low levels and quantification of the rates of uptake for brain and other tissues. Factors that might affect viral protein uptake can be manipulated experimentally in healthy, rather than infected, animals. Recovery of I-S1 from a tissue reflects only factors related to permeability, whereas recovery from infected mice also reflects other factors, such as rate of virus replication in that tissue.
A crucial question which we partially answered here was: what receptor does I-S1 use to enter brain and other tissues? Based on experience with SARS, it is assumed that SARS-CoV-2 will bind to human ACE2, but not murine ACE2. SARS can infect wild-type (WT) mice, but it doesn’t produce severe symptoms and death, except in transgenic mice overexpressing human ACE2 (ref. 35), although this could also simply be because these transgenic mice express 8–12 times more ACE2 than WT mice. The mice used here only expressed murine receptors, so our findings suggest that the assumption that ACE2 must be the human protein is incorrect and demonstrate that WT mice can be used in kinetics studies of S1 and probably SARS-CoV-2.
We did find reasonably strong evidence that murine ACE2 is involved in I-S1 uptake in lung tissue, as co-injection of I-S1 with soluble ACE2 and ACE2 substrates increased the uptake of I-S1 (this perhaps surprising observation is discussed further below). The evidence for ACE2 involvement in brain I-S1 uptake is weaker than that for lung, as here, uptake was affected by co-injection with soluble ACE2, but not by ACE2 substrates. The finding that I-S1 uptake in the kidney, liver and spleen was unaffected by soluble ACE2 or by ACE2 substrates indicate that receptors other than, or in addition to, ACE2 are involved in uptake of I-S1 in some tissues.
That S1 and even SARS-CoV-2 would use more than one receptor is not surprising when one considers that many viruses use multiple receptors. For example, HIV-1 uses the CD4 and mannose-6-phosphate receptors, and the rabies virus uses the acetylcholine receptor, a nerve growth factor receptor and the neural cell adhesion molecule to enter cells25,36. Receptors (besides ACE2) that can bind or are predicted to bind SARS-CoV-2 based on modeling include basigin, cyclophilins, dipeptidyl peptidase-4 (refs. 37,38) and GRP78 (ref. 39).
One reason that a virus can use such a diversity of receptors is that viruses bind with less specificity than do endogenous receptor ligands. The binding sites for viral proteins are often highly charged regions on the cell-membrane glycoprotein owing to high concentrations of sialic acid, N-acetylglucosamine or heparan sulfate. Coronaviruses in general bind to glycoproteins high in sialic acid40. Pioneering work showed that WGA binding to BBB regions rich in sialic acid or N-acetylglucosamine resulted in the transportation of WGA across the BBB through the mechanism of adsorptive transcytosis24. WGA coadministered with a weaker inducer of adsorptive transcytosis will often increase rather than block the BBB penetration of the weaker inducer41. In the current study, the ability of WGA to increase the I-S1 uptake in brain tissue suggests that S1 crosses the BBB through adsorptive transcytosis.
Because the spike protein of SARS-CoV-2 is more highly charged than the spike protein of SARS, it has been suggested that it may bind to a larger number of receptors42. Some viruses bind receptors rich in heparan sulfate; uptake of those viruses is inhibited by heparin25. We showed that I-S1 uptake in liver was inhibited by heparin, but uptake in brain and other peripheral tissues was not. These results show that S1 uses heparan sulfate to bind to liver but not to other tissues. We conclude that a number of receptors are likely involved in S1 uptake; which receptor is most important varies from tissue to tissue. It will be important to identify the membrane-bound glycoproteins that serve as receptors for SARS-CoV-2.
It is unclear why, in our study, co-injection with ACE2 or ACE2 substrates enhanced rather than inhibited uptake of intravenously injected I-S1, but there are some possible explanations. Since S1 does not bind to the ACE2 catalytic site42,43, traditional ligand–receptor dose-dependent inhibition kinetics may not occur. The ACE2 we co-injected with I-S1 may have bound circulating Ang II that would normally have competed with I-S1 for binding to membrane-bound ACE2. In addition, S1 is attached to SARS-CoV-2 as a homotrimer38, but we studied monomeric S1; it is possible that co-injection of ACE2 ligands altered ACE2 conformation in such a way that it facilitated binding of the S1 monomer.
Risk factors for both contracting COVID-19 and having a poor outcome include male individuals who are positive for the ApoE4 allele in comparison to the ApoE3 allele31,32,33, whereas cytokine storm is a characteristic of severe disease44. We found that the influence of sex, ApoE genotype and inflammatory state on I-S1 uptake varied among the tissues. Sex and human ApoE status in mice did not affect uptake of intravenously injected I-S1 in whole brain or lung, but did affect its uptake in olfactory bulb, liver, spleen and kidney, with higher uptake in males. This suggests that some of the risk of poor outcome for males may be related to the degree to which their tissues have an enhanced uptake of S1 or SARS-CoV-2. However, ApoE3, not ApoE4, was associated with higher uptake rates of I-S1 by the olfactory bulb, liver, spleen and kidney, suggesting that the risk associated with ApoE status is unlikely to be due to increased S1 or SARS-CoV-2 tissue uptake.
Inflammation induced by LPS injection increased the amount of intravenously injected I-S1 entering the brain, but this increase was likely due to BBB disruption and not due to enhancement of adsorptive transcytosis; indeed, uptake of I-S1 after correction for BBB disruption was actually lower in one brain region, the olfactory bulb. LPS-exposed mice had higher I-S1 uptake in lung but lower uptake in spleen and liver; the latter likely explains why these mice had reduced I-S1 clearance from blood. Notably, this decrease in clearance from blood observed in mice in an inflammatory state suggests that all tissues will be exposed to higher S1 levels than in the noninflammatory state.
A lethal infection can occur after intranasal administration of SARS35. It has been postulated that nasal virus spreads to the lung and from there to blood and brain35, but others suggest that SARS-CoV-2 in the nares could spread to the brain through the olfactory nerve45, as do many other viruses30. Although our findings show that intranasally administered I-S1 can enter mouse brain tissue, they highlight the BBB as the major route for I-S1 entry into the brain. Moreover, a very small amount (0.66% bioavailability after intranasal administration) of I-S1 was found in blood, suggesting poor nasal-to-blood transfer. However, our studies were designed to assess the ability of I-S1 to enter the brain through the olfactory nerve and not to assess its ability to enter the blood via the nasal vasculature. Nevertheless, our results favor some sites other than the nares, such as the lungs, as being the entry point of S1 detected in blood.
It is important to note that although the study shows that I-S1 crosses the BBB in mice, this may not be the case in humans. For that reason, we used in vitro models of the human BBB, which can be useful in studying mechanisms of BBB permeability. The model used in this study is derived from human iPSCs and develops a brain endothelial cell-like phenotype that includes functional BBB influx and efflux transporters and strong barrier properties that permit the study of transport without confounding effects of high baseline leakage46,47. In this model, we did not observe significant differences in permeability for I-S1 compared to T-Alb. The apparent absence of I-S1 transport across the BBB in this in vitro model could be due to technical issues, such as blockers of I-S1 binding in the buffers. It could also mean that the iBECs did not express the cell-membrane glycoproteins necessary for I-S1 transport, or that I-S1 is not able to cross the human BBB. A note of caution regarding the validity of using monomeric S1 as a model for SARS-CoV-2 is that S1 is normally attached to SARS-CoV-2 as a trimer. However, the S1 protein may be shed from the virus in vivo, and therefore studying S1 monomers may have validity by itself—although there is currently no direct evidence that spike proteins are shed from SARS-CoV-2. Altogether, our results strongly suggest that the S1 protein can cross the murine BBB through a mechanism resembling adsorptive transcytosis and be taken up by peripheral tissues independently of human ACE2.
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