key: cord-0901273-aj885nl0
authors: Rhea, Elizabeth M.; Logsdon, Aric F.; Hansen, Kim M.; Williams, Lindsey M.; Reed, May J.; Baumann, Kristen K.; Holden, Sarah J.; Raber, Jacob; Banks, William A.; Erickson, Michelle A.
title: The S1 protein of SARS-CoV-2 crosses the blood–brain barrier in mice
date: 2020-12-16
journal: Nat Neurosci
DOI: 10.1038/s41593-020-00771-8
sha: cba48df18b770a9de412c34df8c3d4084094ebad
doc_id: 901273
cord_uid: aj885nl0

It is unclear whether SARS-CoV-2, which causes COVID-19, can enter the brain. SARS-CoV-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 (BBB) in male mice, was taken up by brain regions and entered the parenchymal brain space. I-S1 was also taken up by lung, spleen, kidney, and liver. Intranasally administered I-S1 also entered the brain, though at ~10 times lower levels 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 BBB 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.

brain influx constant (Ki) using multiple-time regression analysis (MTRA). MTRA plots the tissue/serum ratios for I-S1 against exposure time, which is a measure of time that has been corrected for the clearance of I-S1 from blood. The slope of the linear portion of this plot measures Ki, i.e. the unidirectional influx constant for I-S1.

We co-injected 99m Tc -labeled albumin (T-Alb) along with the I-S1. T-Alb crosses the intact BBB poorly and so can be used to measure the vascular space of the brain. Any brain/serum ratios for I-S1 that exceed the brain/serum ratios of T-Alb therefore represent extravascular I-S1; that is, material which has crossed the BBB. T-Alb can also be used to measure the leakiness of peripheral tissue beds and of a BBB that has been disrupted by disease or inflammation.

The blood-to-brain influx constants (Ki's) of the I-SI proteins from the two sources differed by about 3% (Figure 1 ). These results show that unlike T-Alb, I-S1 readily crosses the mouse BBB.

We determined the clearance of intravenously injected I-S1 from RayBiotech from blood and its uptake in brain and other tissues, again using MTRA ( Figure 2 ). Clearance of I-S1 from blood was linear for the first 10 min, with a half-time clearance of about 6.6 min (Figure 2A) , and a plateau after that. In Figure 2 (and in subsequent figures), tissue/serum ratios for T-Alb were subtracted from the tissue/serum ratios for I-S1. These "delta" tissue/ serum ratios were thus corrected for the I-S1 in the vascular space and for any I-S1 that would have entered tissue because of a leaky capillary bed. The resulting delta values thus represent specific uptake of I-S1 by the tissues.

All tissues showed uptake of I-S1 ( Figure 2B -F). Spleen and liver uptake was nonlinear, suggesting that their tissue beds were coming into equilibrium with blood. Most substances in blood are cleared by kidney or liver; the much higher I-S1 uptake in liver compared to kidney suggests that I-S1 is cleared from blood predominantly by the liver. To determine if there were regional differences in brain uptake, we collected the olfactory bulb and dissected the whole brain into 10 regions (Extended Data Figure 2 ). We found that I-S1 entered all brain regions, with no statistically significant differences among them.

We similarly determined the clearance of I-S1 (AMSBIO) from blood and its uptake by brain and other tissues ( Figure 3) . The results were similar to those obtained with I-S1 derived from RayBiotech ( Figure 2 ), but there were a few differences. The AMSBIO-derived I-S1 was cleared from blood faster (half-life of 3.6 min) and the rate of liver uptake was nearly 5 times faster than liver uptake of RayBiotech-derived I-S1 (p<0.0001). Together, these results show that I-S1 is cleared from blood primarily by the liver and that S1 has access to other tissues relevant for COVID-19, including kidney, lung, and spleen.

We measured the stability of intravenously injected I-S1 from RayBiotech in blood and brain through acid precipitation (Supplementary Table 1 ). I-S1 added to tissues (processing controls) ex vivo showed little degradation. Radioactivity recovered from brain 10 min after intravenous injection and serum 10 and 30 min after the intravenous injection of I-S1 was stable, with most of the radioactivity precipitated by acid. Radioactivity recovered from brain 30 min after the intravenous injection of I-S1 was mostly degraded. This indicates that I-S1 enters the BBB intact but is eventually degraded in the brain, although it is not clear whether this involves cleavage of the radioactive iodine from I-S1 and/or whether S1 protein itself is degraded.

Most of the I-S1 taken up by the capillary bed enters the brain parenchyma.

In rare cases, substances that seem to cross the BBB are actually sequestered by the capillary bed, which prevents them from entering the brain parenchymal/interstitial fluid space. Therefore, we next assessed whether intravenously injected I-S1 fully crosses the capillary wall to enter brain parenchyma. For this, we used a modified version of the 'capillary depletion' method that also enabled us to measure the amount of intravenously injected I-S1 that is reversibly adhering to the luminal side of the capillary bed. We found that binding of I-S1 to the luminal side remained static over time, whereas there was a small increase over time in the amount of I-S1 retained by brain capillaries (Extended Figure 3 ). The largest change over time was the amount of I-S1 entering the parenchyma. These results show that by thirty minutes, over 50% of I-S1 has crossed the capillary wall fully to enter into the brain parenchymal and interstitial fluid spaces.

WGA enhances I-S1 uptake in brain and some peripheral tissues Wheatgerm agglutinin (WGA) is a plant lectin that crosses the BBB through adsorptive transcytosis 24 -a process by which proteins or peptides bind to glycoproteins of the luminal surface of endothelial cells, are internalized into vesicles and then transported across the membrane. In the case of WGA, adsorptive transcytosis occurs when WGA binds to cellsurface glycoproteins containing sialic acid and N-acetylglucosamine. Many viral proteins also bind to sialic acid-and N-acetylglucosamine-containing glycoproteins, and therefore, when WGA is co-injected with such viral proteins, it enhances their transport across the BBB and uptake in peripheral tissues 18 . Here, we found that intravenously injected I-S1 (RayBiotech or AMSBIO) uptake into brain, lung, spleen, and kidney was increased compared when WGA was included in the injection ( Figure 4B -E). WGA co-injection also increased clearance of I-S1 (RayBiotech), but not I-S1 (AMSBIO), as shown by a decrease in I-S1 in blood ( Figure 4A ). WGA co-injection decreased uptake of I-S1 (AMSBIO) but not I-S1 (RayBiotech) in the liver ( Figure 4F ), although it did with the WGA arm of the heparin experiment ( Figure 4H ).

These results strongly suggest that I-S1 crosses the BBB and peripheral tissue beds through the mechanism of adsorptive transcytosis, binding to cell surface glycoproteins that contain sialic acid or N-acetylglucosamine.

Heparin blocks uptake of I-S1 in liver but not in brain.

Some viruses use heparan sulfate on the cell membrane as a receptor 25 . Heparin can block virus uptake in tissues, presumably by binding to viral proteins and thereby blocking these viral proteins from binding to the heparan sulfate on the cell membranes 19, 26 . However, we found that heparin co-injected with I-S1 did not affect uptake of I-S1 (RayBiotech) in brain, lung, spleen, or kidney and did not influence the effect of WGA co-injection on I-S1 uptake in these issues (data not shown). Heparin co-injected with I-S1 did block uptake of I-S1 in liver (Figure 4 H), which likely explained the observed decreased clearance of I-S1 from blood (as evidenced by the increase in I-S1 levels in serum; Figure 4G ), but did not block the effects of WGA in I-S1 uptake in liver. These results indicate that I-S1 uses heparin-sensitive sites to bind to the capillary bed of the liver, but not to bind to the capillary beds of brain, spleen, lung, or kidney.

I-S1 uptake in brain was not blocked by unlabeled S1

We next determined whether transport of intravenously injected I-S1 (RayBiotech) into the brain could be blocked by including in the injection an excess of unlabeled S1 protein (AMSBIO). Unlabeled S1 did not affect brain/serum ratios for delta I-S1 at any of the tested doses ( Figure 5A ), although it did enhance uptake of I-S1 in lung ( Figure 5C ). This indicates that the binding site for I-S1 in brain tissue is not easily saturated -a characteristic of adsorptive transcytosis. Unlabeled S1 also did not affect brain/serum ratios for T-Alb (data not shown), which indicates that even the high dose of 10ug S1 did not acutely disrupt the BBB.

ACE2 likely mediates I-S1 uptake in brain and lung, but not other tissues

The ability of SARS-CoV-2 to enter cells is thought to depend on S protein binding to the membrane-bound enzyme (and glycoprotein) ACE2. To assess whether ACE2 has a role in I-S1 uptake in brain and peripheral tissue, we intravenously co-injected ACE2 or the ACE2 substrates AngII and ghrelin with I-S1 (RayBiotech). This did not affect blood levels of intravenously injected I-S1 or T-Alb (data not shown), indicating that these proteins did not affect the volume of distribution or clearance of either S1 or albumin. Co-injection of ACE2 increased kidney levels of intravenously injected T-Alb: F(4,36) = 2.63, p = 0.0505; vehicle: 119 ± 4.4 μL/g (n = 8 mice); ACE2: 151 ± 15.6 μL/g (n = 8 mice), p = 0.02), possibly indicating that ACE2 affects renal clearance of albumin. Only ACE2 co-injection enhanced I-S1 uptake in brain ( Figure 5B ). Uptake in lung was increased by co-injection of S1, ghrelin or ACE2, but not angiotensin II ( Figure 5C ), whereas uptake in liver, kidney, or spleen was not altered by co-injection of any of these substances (data not shown). These results suggest that ACE2 is involved in S1 uptake in lung and probably brain, but not for I-S1 uptake in spleen, liver, or kidney.

Since SARS-CoV-2 infection induces an inflammatory state, we next determined whether an inflammatory state -in this case, induced by LPS injection -affects uptake of intravenously injected I-S1 (RayBiotech) in brain and peripheral tissues. Mice received a 3mg/kg injection of LPS derived from Salmonella typhimurium (Sigma, St. Louis, MO) at t = 0 and again 6 and 24 h later and received an intravenous I-S1 or T-Alb injection 28 h after the first LPS injection; this LPS regimen can disrupt the BBB, increase its permeability to viruses and viral proteins 27, 28 , and increase the blood level of many of the cytokines found to be elevated in the cytokine storm associated with COVID-19 5, 29 .

We found that LPS did not affect T-Alb blood levels, indicating that inflammation did not induce volume contraction of the vascular space nor leakiness of the peripheral capillary beds in these mice. Mice that had received LPS did have higher I-S1 serum levels ( Figure  6A ), indicating a decrease in clearance from blood (likely because of the decreased I-S1 uptake in liver observed in these mice ( Figure 6B )), as well as higher T-Alb brain/serum ratios, indicating BBB disruption [control: 8.52 ± 0.19 μL/g (n = 10); LPS: 12.2 ± 0.68 μL/g (n = 8), t = 5.74, p<0.0001]. These mice also had higher I-S1 brain/serum ratios (i.e. ratios not corrected for I-S1 in the vascular space and for any I-S1 brain entry due to leakage), indicating increased I-S1 passage across the BBB [Control: 12.06 ± 0.26 μL/g (n = 10); LPS: 14.9 ± 0.64 μL/g (n =8), t = 4.38, p = 0.0005], although their delta brain/serum ratios for I-S1 (i.e. corrected ratios) were not different from controls (except in the olfactory bulb) ( Figure 6C ). The only tissue showing an increased uptake of I-S1 after receiving LPS was the lung, which increased 101% ( Figure 6B ). These results show that inflammation could increase S1 toxicity for lung by increasing its uptake. The results also show that in mice, inflammation can increase intravenously injected I-S1 entry into brain, but this is likely due to BBB disruption rather than by enhancing absorptive transcytosis.

Some viruses can enter the brain via the olfactory nerve, a cranial nerve with projections through the cribriform plate 30 . Other viruses may cross the BBB after entering the blood stream from the nasal compartment. It is unclear whether SARS-CoV-2 in the nose can enter brain by either of these routes. We therefore assessed the ability of intranasally administered I-S1 to enter brain. Following a 1μL vehicle solution containing I-S1 to each naris at the level of the cribriform plate (where the olfactory nerve emerges from the cranial vault) I-S1 was detected in all brain regions ( Figure 7A -B). I-S1 levels in whole brain, frontal cortex, cerebellum, midbrain, and pons-medulla were higher at 30 min compared to 10 min post administration. I-S1 distribution in the brain was mostly homogenous, although levels in the olfactory bulb and hypothalamus were higher than in other brain regions at 10 min and, in the olfactory bulb also at 30 min post-administration. Whole-brain I-S1 levels expressed as the percent of the administered dose were about 10 times higher after intravenous injection than after intranasal injection ( Figure 7A ). Radioactivity appeared in blood after intranasal administration of I-S1, indicating that some I-S1 had entered the blood stream (data not shown). The AUC for I-S1 in blood after intranasal administration was 9.42 (%Inj/ml)-min, whereas the AUC for I-S1 in blood after iv injection was 1430 (%Inj/ml)-min, indicating that the bioavailability for the nasal route is 0.66%. Together, these results show that I-S1 can enter the brain and distribute in a time-dependent manner to all brain regions after intranasal administration. However, I-S1 uptake in the brain via this route is much less efficient than uptake after transport across the BBB.

Effects of sex and APOE genotype on I-S1 uptake.

Male sex and APOE4 genotype in comparison to the APOE3 genotype are risk factors for both contracting and having a poor outcome to COVID-19 [31] [32] [33] . Therefore, we determined uptake of intravenously injected I-S1 (RayBiotech) or T-Alb in male and female mice expressing human APOE3 or APOE4 under the expression of the mouse Apoe promoter. APOE genotype and sex did not affect T-Alb brain/serum ratios ( Figure 8A ). Two-way ANOVA showed an effect of sex on I-S1 uptake in olfactory bulb and spleen and an effect of APOE genotype on I-S1 uptake in liver, spleen, and kidney ( Figure 8B -H). Multiple comparisons tests showed that in comparison to the other three groups male mice expressing human APOE3 had the fastest I-S1 uptake of I-S1 in olfactory bulb liver, and kidney, whereas in comparison to the other three groups female mice expressing human APOE3 has the fastest I-S1 uptake in spleen. These results suggest that enhanced uptake of I-S1 by some tissues could contribute to the increased risk of COVID-19 associated with being male, but not to the risk associated with the APOE4 genotype.

To determine whether the I-S1 crosses endothelial-like cells in an in vitro model of the human BBB, we compared transport of I-S1 vs T-Alb across monolayers of human iPSC-derived brain endothelial-like cells (iBECs) seeded on transwells. Three independent experiments showed that rate of passage of I-S1 (RayBiotech) in the luminal-to-abluminal direction was not statistically significant when compared to that of T-Alb (Extended Data Figure 4A ). One experiment indicated that unlabeled S1 did not inhibit I-S1 (RayBiotech) passage (Extended Data Figure 4B ). WGA also had no effect on RayBiotech I-S1 transport (Extended Data Figure 4C ). I-S1 (RayBiotech) had a significantly higher permeability coefficient than I-S1 (AMSBIO I-S1) in vitro (Extended Data Figure 4D ); however this difference was not significant after correcting for T-Alb transport. In summary, these results suggest that iBECs may be more permeable to I-S1 than to T-Alb, but the difference is too small to support mechanistic studies using this model.

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 anosmia 1, 3, 4 . S1 is the SARS-CoV-2 protein that initially binds to cell-surface receptors, setting the stage for viral internalization. In terms of 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 virus 18, 28 . Additionally, many if not most viral proteins themselves can be biologically highly active; for example, gp120 is highly toxic [11] [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 virions 34 . It is possible that during infection by SARS-CoV-2, shed S1 is available to cross the BBB, triggering responses in the brain in 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 neurotoxicity.

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 has been assumed that SARS-CoV-2 will bind to human ACE2, but not murine ACE2. SARS can infect WT mice, but doesn't produce severe symptoms and death, except in transgenic mice overexpressing human ACE2 35 -although this could also simply be due to the fact that these transgenic mice express 8-12 times more ACE2 than WT mice. The mice we used here only expressed murine receptors and so the assumption that ACE2 must be the human protein is clearly wrong; this demonstrates 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, as co-injection of I-S1 with soluble ACE2 and ACE2 substrates increased the uptake of I-S1 (the perhaps surprising observed increase rather than decrease 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 kidney, liver, and spleen were 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 cells 25, 36 . Receptors (besides ACE2) that can bind or are predicted to bind SARS-CoV-2 based on modeling include basigin, cyclophilins, dipeptidyl peptidase-4 37, 38 and GRP78 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 acid 40 . Pioneering work showed that WGA binding to BBB regions rich in sialic acid or N-acetylglucosamine resulted in WGA being transported across the BBB through the mechanism of adsorptive transcytosis 24 . WGA co-administered with a weaker inducer of adsorptive transcytosis will often increase rather than block the BBB penetration of the weaker inducer 41 . In the current study, the ability of WGA to increase the I-S1 uptake in brain 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 receptors 42 . Some viruses bind receptors rich in heparan sulfate; uptake of those viruses is inhibited by heparin 25 . 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 it is likely that a number of receptors are 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 site 43, 42 , traditional ligandreceptor dose-dependent inhibition kinetics may not occur. The ACE2 we co-injected with I-S1 may have bound circulating AngII 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 homotrimer 38 , but we studied monomeric S1; it is possible that co-injection of ACE2 ligands altered ACE2 conformation in such a way that it facilitates binding of S1 monomer.

Risk factors for both contracting COVID-19 and having a poor outcome are being male and being ApoE4 positive in comparison to ApoE3 positive [31] [32] [33] , whereas cytokine storm is a characteristic of severe disease 44 . 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 a higher uptake rates of I-S1 by 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 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 non-inflammatory state.

A lethal infection can occur after intranasal administration of SARS 35 . It has been postulated that nasal virus spreads to the lung and from there to blood and brain 35 , but others suggest that SARS-CoV-2 in the nares could spread to brain by way of olfactory nerve 45 , as do many other viruses 30 . Although our findings show that intranasally administered I-S1 is able to enter mouse brain, they suggest the BBB is the major route for I-S1 entry into 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 brain by way of the olfactory nerve and not to assess its ability to enter the blood by way of nasal vasculature. Nevertheless, our results favor some site other than the nares, such as lung, 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 leakage 46, 47 . In this model, we did not observe significant differences in permeability for I-S1 vs 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 it could mean that I-S1 is not able to cross the human BBB. A note of caution with regard to 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 studying S1 monomers therefore may have validity by itself -although there is currently no direct evidence that spike proteins are shed from SARS-CoV-2. Altogether, our results do strongly suggest that the S1 protein is able to cross the murine BBB through a mechanism resembling adsorptive transcytosis and to be taken up by peripheral tissues independently of human ACE2.

All mouse studies were approved by the local IACUC at OHSU and the VA Puget Sound Healthcare System and conducted in AAALAC approved facilities. Male CD-1 mice (6-10 weeks old) were purchased from Charles River Laboratories (Hollister, CA). A total of 204 CD-1 males were used for all studies. Human E3-and E4-targeted replacement (TR) male and female mice, generated as described 48 , were bred at the Oregon Health & Sciences University (OHSU) prior to transfer to the Veterans Affairs Puget Sound Health Care System (VAPSHCS) for the experiments of this study. E3 and E4 TR mice were approximately 4 months of age on the day of the study. A total of 21 E3 and 20 E4 TR mice were used for all studies. Mice had ad libitum access to food and water and were kept on a 12/12 hour light/dark cycle. Temperature was maintained at 18-23°C and humidity was maintained at 40-60%. For all experiments, mice were anesthetized with an intraperitoneal (ip) injection of 0.15-0.2 mL of 40% urethane to minimize pain and distress. Anesthetized mice were placed on a heating pad until time of use. At the end of each study, mice were euthanized by decapitation while under anesthesia.

The S1 proteins were provided by the manufacturers (RayBiotech, Cat no 230-30161, Peachtree Corners, GA, Val16-Gln690; AMSBIO, AMS.S1N-C52H3, Abingdon, UK, Val16-Arg685). The proteins were dissolved in phosphate buffered saline (PBS, pH 7.4) at a concentration ranging from 0.45-0.61 mg/mL. The proteins were produced in HEK293 cell lines, had C-terminal His tags, were calculated to be about 76 kDa in protein, but migrated on gel at 100-120 kDa because of glycosylation. Upon receipt, the S1 proteins were thawed and aliquoted into 5μg portions, and either used immediately or stored at −80 °C until use. The 5 μg of thawed S1 protein was radioactively labeled with 1 mCi 125 I (Perkin Elmer, Waltman, MA) using the chloramine-T method, as described 49 . The radioiodinated S1 (I-S1) was purified on a column of G10 Sephadex (GE Healthcare, Uppsala, SE) and eluted with phosphate buffer (PB) into glass tubes containing 1% bovine serum albumin in lactated Ringer's solution (BSA-LR). We estimated based on the amount of protein iodinated that the specific activity of I-S1 to be approximately 11 Ci/g or about 12.5 ng/300,000 cpm. Bovine serum albumin (Sigma, St. Louis, MO) was labeled with 99m Tc (GE Healthcare, Seattle, WA) using the stannous tartrate method 50 . The 99m Tc -labeled albumin (T-Alb) was purified on a column of G-10 Sephadex. Both of the I-S1's and the T-Alb were more than 90% acid precipitable. The molecular weight of the labeled proteins was confirmed by running 200,000-600,000 CPM activity in 1x LDS buffer (Invitrogen) with or without reducing agent (Invitrogen) on a 4-12% bis-tris gel (Genescript) in 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (Invitrogen). The gel was then fixed for 30 min in 10% acetic acid/50% methanol, washed 3x with water, and then dried using a DryEase® Mini-Gel Drying System (Invitrogen). Dried gels were exposed on autoradiography film for 24 hours and then developed. Major bands of I-S1 from RayBiotech and AMSBIO migrated at their predicted molecular weight patterns, based on manufacturer data (Extended Data Figure 1 ).

Multiple-time regression analysis (MTRA) 51, 52 was used to measure the unidirectional blood-to-brain influx constant (Ki) for I-S1. In anesthetized mice, the left jugular vein was exposed for an intravenous (iv) injection of 0.1 mL BSA-LR containing 3×10 5 cpm of I-S1. T-Alb (6×10 5 cpm) was also included in the injection for measurement of the vascular space for the brain and the albumin space for peripheral tissues. At time points between 1 and 30 min, blood was collected from the carotid artery. Blood was centrifuged at 3200×g for 10min and 50uL serum was collected. The whole brain, kidney, and spleen and portions of the lung and liver were removed and weighed. Tissues and serum were placed into a Wizard 2 gamma counter (Perkin Elmer), and the levels of radioactivity were measured. Results for brain and other tissues were expressed as the tissue/serum ratio in units of μL/g for both I-S1

and T-Alb. For each individual tissue, its tissue/serum ratio for T-Alb was subtracted from its tissue/serum ratio for I-S1, yielding "delta" value. These delta values were thus corrected for vascular space and any nonspecific leakage into tissue and so represent I-S1 protein uptake that was not due to trapping in the vascular space or to leakage. The delta brain/serum ratios were plotted against exposure time, a calculation that corrects for clearance from blood: (1) where t is the time between the iv injection and sampling, Cp is the cpm/mL of arterial serum, Cpt is the cpm/mL of arterial serum at time t, and τ is the dummy variable for time. The slope of the linear portion of the relation of tissue/serum ratio vs exposure time measures the unidirectional influx rate (Ki in units of μL/g-min) and the Y-intercept measures Vi, the vascular space and initial luminal binding at t = 0 51, 52 :

Tissue/serum ratio = Ki(Expt) + Vi (2) 

The area under the curve for the level of radioactivity in blood from 0-30 min was calculated using Prism 8.0 software (GraphPad Inc, San Diego, CA). Results for serum were expressed as the percent of the injected dose per mL of blood (%Inj/mL). The percent of the injected dose per gram of brain (%Inj/g) was calculated by multiplying the delta brain/serum value by the %Inj/mL for I-S1.

Anesthetized mice received an injection into the jugular vein of 0.1 mL of BSA-LR containing 6×10 5 cpm of I-S1 plus 6×10 5 cpm of T-Alb. Ten minutes after the injection, arterial blood was collected from the abdominal aorta, the thorax opened and the descending thoracic artery clamped, both jugular veins severed, and 20 mL of lactated Ringer's solution perfused through the left ventricle of the heart to wash out the vascular space of the brain. The whole brain was then removed. Whole blood was centrifuged and 10 uL of serum was added to 500 uL BSA-LR and combined with an equal part of 30% tricholoroacetic acid, mixed, centrifuged, and the supernatant and pellet counted. Whole brain was homogenized in BSA-LR plus complete mini protease inhibitor (Roche, Mannheim, DE; one tab per 10 mL buffer) with a hand-held glass homogenizer, and centrifuged. The resulting supernatant was combined with equal parts of 30% trichloroacetic acid, mixed, centrifuged, and the supernatant and pellet counted. To determine the amount of degradation of I-S1 or T-Alb that occurred during processing, I-S1 and T-Alb was added to brains and arterial whole blood from animals that had not been injected with radioactivity and processed immediately as above. The percent of radioactivity that was precipitated by acid (%P) in all of these samples was calculated by the equation: %P = 100(S)/(S + P) (3) where S is the cpm in the supernatant and P is the cpm in the pellet.

The capillary depletion method as adapted to mice was used to separate cerebral capillaries and vascular components from brain parenchyma 53, 54 . We used the variant of the technique that also estimates reversible binding to the capillary lumen. The latter is done by assessing two groups of mice, one with a vascular washout step which removes material reversibly bound to the capillary lumen (washout group) and those without the washout step (nonwashout group). The difference between these groups represents material that was reversibly adhering to the capillary lumen (see equation 4 below). Mice were anesthetized and received an iv injection of 6×10 5 cpm Tc-Alb with 6×10 5 cpm I-S1 in 0.1 mL BSA-LR. 5, 10, and 30 min later, blood was obtained from the carotid artery, and the brain (non-washout group) was extracted. In a separate group of mice, blood was taken from the abdominal aorta at 5, 10, and 30 min, the thorax was opened and the descending thoracic artery clamped, both jugulars severed, and 20 mL of lactated Ringer's solution infused via the left ventricle of the heart in order to washout the vascular contents of the brain and to remove any material reversibly associated with the capillary lumen (washout group). Each whole brain was homogenized in glass with physiological buffer (10mM HEPES buffer, 141 mM NaCl, 4mM KCl, 2.8 mM CaCl 2 , 1 mM MgSO 4 , 1 mM NaH 2 PO 4 -H 2 O, 10 mM D-glucose, pH 7.4) and mixed thoroughly with 26% dextran. The homogenate was centrifuged at 4255×g for 15 min at 4°C. The pellet, containing the capillaries, and the supernatant, representing the brain parenchymal space, were carefully separated. Radioactivity levels in the capillary pellet, the brain supernatant, and the arterial serum were determined for both T-Alb and I-S1 and expressed as the capillary/serum and brain parenchyma/serum ratios. The I-S1 parenchymal ratios were corrected for vascular contamination by subtracting the corresponding ratios for T-Alb; these results are reported as the delta brain parenchyma/serum ratios. The amount of S1 in the brain parenchymal space was taken as the delta brain parenchymal space from the washout group (PW), the amount in the capillary as the capillary from the washout group (CW), and the amount of material loosely binding to the luminal surface (Luminal) as: Luminal = (P + C) − (PW + CW) (4) Where P are the delta brain parenchymal space and the capillary space, both from the nonwashout groups.

We co-injected WGA with I-S1 to determine the role of sialic acid-and Nacetylglucosamine-containing glycoproteins in the uptake of S1 by the BBB and other tissues. Mice were anesthetized, after which the jugular vein and right carotid artery were exposed. Mice then received a jugular vein injection of 0.1 mL BSA-LR containing 3×10 5 cpm of I-S1 and 6×10 5 cpm of T-Alb and, in a subset of mice, 10 μg of the plant lectin wheat germ agglutinin (WGA, Sigma, St. Louis, MO). Brain, tissues, and serum samples were collected 5 min later and tissue/serum ratios calculated as above in units of μL/g. Heparan sulfate is used as a receptor by some viruses 25 and heparin by binding to viral proteins can block viral entry into brain 19, 26 . Therefore, separate groups of mice had heparin (12 U/mouse) included in the injection.

In anesthetized mice, the left jugular vein was exposed for an iv injection of 0.1 mL BSA-LR containing 3×10 5 cpm of I-S1 (RayBiotech) and 6×10 5 cpm of T-Alb. For some mice, the injection contained 1 μg/mouse of unlabeled S1 (AMSBIO), mouse acyl ghrelin (CBio, Menlo Park, CA), angiotensin II (Tocris, Bristol, UK), or human ACE2 (R&D, Minneapolis, MN). Ten min after iv injection, whole blood was obtained from the carotid artery and centrifuged after clotting. The whole brain, olfactory bulb, kidney, spleen, and portions of the liver and lung removed. The levels of radioactivity in the arterial serum and the tissues were determined and the results expressed as the percent of the injected I-S1 per mL for serum and delta tissue/serum ratios (μL/g) for the tissues.

Male CD-1 mice aged 6-10 weeks were given an ip injection of 3 mg/kg LPS from Salmonella typhimurium (Sigma, St. Louis, MO) dissolved in sterile normal saline at 0, 6, and 24 h. At 28 h, mice were anesthetized and the left jugular vein and right carotid artery exposed. The mice were given an iv injection of 6×10 5 cpm of I-S1 and 6×10 5 cpm T-Alb in 0.1 mL of BSA/LR into the left jugular vein. Arterial blood was collected from the right carotid artery 10 min later, the mouse immediately decapitated, the brain removed, dissected into regions (olfactory bulb, frontal cortex, occipital cortex, parietal cortex, thalamus, hypothalamus, striatum, hippocampus, pons-medulla, cerebellum, midbrain), and the regions weighed. Kidney, spleen, and portions of the liver and lung were also removed and weighed. Serum was obtained by centrifuging the carotid artery blood for 10 min at 4255 ×g. Levels of radioactivity in serum, brain regions, and tissues were measured in a gamma counter. Whole brain values were calculated by summing levels of radioactivity and weight for all brain regions except for olfactory bulb. Data for brain regions from control (mice not receiving LPS) were separately analyzed for differences among brain regions and also used in comparisons with LPS treated mice. The levels of radioactivity in the arterial serum and the brain regions and tissues were determined and the results expressed as the percent of the injected I-S1 per mL for serum and delta tissue/serum ratios (μL/g) for the tissues.

Anesthetized mice were placed in the supine position and received a 1 μL injection of 2×10 5 -3×10 5 cpm I-S1 in BSA/LR administered to each naris, delivered to the level of the cribriform plate (4 mm depth), using a 10 μL Multi-flex tip (Thermo Fisher Scientific, Waltham, MA). After administration, the mouse remained in the supine position for 30 s before being placed on the left side. Arterial blood was collected from the right carotid artery 10 or 30 min later, the mouse immediately decapitated, the brain removed, dissected into regions (olfactory bulb, frontal cortex, occipital cortex, parietal cortex, thalamus, hypothalamus, striatum, hippocampus, pons-medulla, cerebellum, midbrain), and the regions weighed. Serum was obtained by centrifuging the carotid artery blood for 10 min at 4255 ×g. Levels of radioactivity in serum and brain regions were measured in a Wizard 2 gamma counter. Whole brain values were calculated by summing levels of radioactivity and weight for all brain regions except for olfactory bulb. The levels of radioactivity in the arterial serum and the tissues were determined and the results expressed as the percent of the injected I-S1 per mL for serum and the percent of the injected I-S1 per g of brain region calculated.

Human APOE targeted replacement mice expressing human apoE3 or apoE4, under control of the mouse apoE promoter and on the C57BL/6J background, were provided by Dr Patrick Sullivan for breeding 55, 56 , as described 57 ***. With targeted replacement, the resulting gene contains the mouse apoE promoter with the mouse apoE being replaced by the human apoE so that only the human gene, and not the mouse gene, is expressed. The colony is maintained by homozygous breeding. To prevent genetic drift, regularly the human apoE mice expressing different isoforms are crossed with each other and subsequently again bred to homozygosity to refresh the colony. Male and female mice that were homozygous for either apoE3 or apoE4 were studied by MTRA as described above. Two-way ANOVA used sex and APOE genotype as independent variables. As described above, T-Alb tissue/serum ratios were used to measure and correct for any BBB disruption.

The iBECs were derived from the GM25256 induced pluripotent stem cell (iPSC) line (Coriell Institute) using the method of Neal et al 58 with a seeding density of 15,000 cells per well for differentiation which was found to be optimal for this cell line. Use of this stem cell line was in accordance with an MTA between the VA Puget Sound Healthcare System and the Coriell institute, and was approved by the VA Puget Sound Healthcare System institutional biosafety committee. Briefly, iPSCs were grown to optimal density on plates coated with Matrigel (VWR cat no. 62405-134) in E8 Flex medium (Thermo Fisher Scientific cat no. A2858501), and then passaged using Accutase (Thermo Fisher Scientific cat no. A1110501) onto Matrigel-coated plates in E8 Flex medium plus 10 μM ROCK inhibitor Y-27632 (R&D Systems, cat no. 1254). The next day, the medium was changed to E6 (Thermo Fisher Scientific, cat no. A1516401) and E6 changes continued daily for 3 more days. Next, the medium was changed to human endothelial serum-free medium (HESFM, Thermo Fisher Scientific, cat no. 11111044) supplemented with 20 ng/mL bFGF (Peprotech, cat no. 100-18B), 10 μM retinoic acid (Sigma, cat no. R2625), and 1% B27 supplement (Thermo Fisher Scientific, cat no. 17504044). 48 h later, iBECs were subcultured onto 24-well transwell inserts (Corning cat no. 3470) coated with 1 mg/mL Collagen IV (Sigma, cat no. C5533) and 5mM Fibronectin (Sigma, cat no. F1141) in HESFM + 20 ng/mL bFGF, 10 uM retinoic acid, and 1% B27. 24 h after subculture, the medium was changed to HESFM + 1% B27 without bFGF or retinoic acid, and transendothelial electrical resistance (TEER) was recorded using an End EVOM2 Voltohmmeter (World Precision Instruments, Sarasota Florida) coupled to an ENDOHM cup chamber. TEER measurements occurred daily and S1 transport experiments were conducted when TEER stabilized, between 10-13 days in vitro.. TEER values in these studies exceeded 1000 Ω*cm 2 and TEER means were confirmed to be equal among groups just prior to starting the transport study.

Prior to transport studies, the medium was changed and cells were equilibrated in the incubator for 20 min. Warm HESFM + 1% B27, 1 million CPM T-Alb, and 500,000 CPM of I-S1 was then added in a volume of 100 μL to the luminal chamber. After incubation times of 10, 20, 30, and 45 min at 37 °C, 500 μL volumes of medium from the abluminal chamber were collected and replaced with fresh pre-warmed medium. Samples were then acid precipitated by adding a final concentration of 1% BSA to visualize the pellet and 15% trichloroacetic acid to precipitate the proteins in solution. The samples were centrifuged at 4255xg for 15 min at 4 °C. Radioactivity in the pellet was counted in the gamma counter and the permeability-surface area coefficients for T-Alb and I-S1 were calculated according to the method of Dehouck et al 59 . Clearance was expressed as microliters (μL) of radioactive tracer that was transported from the luminal to abluminal chamber, and was calculated from the initial level of acid-precipitable radioactivity added to the luminal chamber and the final level of radioactivity in the abluminal chamber:

Where [C] L is the initial concentration of radioactivity in the luminal chamber (in units of CPM/μL), [C] C is the concentration of radioactivity in the abluminal chamber (in units of CPM/μL) and Vc is the volume of the abluminal chamber in μL. The volume cleared was plotted vs. time, and the slope was estimated by linear regression. The slopes of clearance curves for the iBEC monolayer plus Transwell® membrane was denoted by PS app , where PS is the permeability × surface area product (in μL/min). The slope of the clearance curve for a Transwell® membrane without iBECs was denoted by PS membrane . The PS value for the iBEC monolayer (PS e ) was calculated from 1 / PS app = 1 / PS membrane + 1 / PS e . The PS e values were divided by the surface area of the Transwell® inserts (0.33 cm 2 ) to generate the endothelial permeability coefficient (Pe, in μL/min/cm 2 ).

Sample size, randomization, and blinding: Sample sizes were pre-determined based on the variance that is known from previously conducting similar experiments on BBB transport. We selected samples sizes to power at 0.8-0.9 based on 30-40% differences between means. Anesthetized mice were randomized between test groups by alternating their assignments. Transwells were assessed for TEER prior to experiments and assigned to groups in such a way that TEER values between groups had approximately equal means and variances. Blinding was not done for experiments or analysis because this knowledge is required to carry out experiments and analysis.

For multiple-time regression analysis, the method calls for exclusion of data points that contribute to non-linearity of the curve. Outliers whose exclusion improved the r 2 ≥ 0.2 were excluded from analysis. Excluded points are noted in Figures 2 and 3 by filled circles and further explained in those figure legends. For ANOVAs and comparisons of means, the Grubbs outlier exclusion test (alpha < 0.05) was applied once to detect single outliers, and any outliers that were excluded by this method are stated in the Figure legends and their values provided. Each figure was generated as a single experimental replicate, with the exception of iPSC studies where S1 and albumin transport were compared in three independent experiments. However, use of T-Alb as an internal control for all experiments allowed us to track consistency between experiments with different designs based on its predictable tissue/serum ratios. Further, the I-S1 tissue-serum ratios taken at the same time points from different experiments may be compared, and agree well with each other, demonstrating a reproducible observation of I-S1 transport into the brain and other tissues.

The Prism 8.0 statistical software package was used for all statistical calculations (GraphPad Inc, San Diego, CA). Simple linear regression analysis was used to calculate slopes and intercepts, and the error terms for the slope and Y-intercept are the standard errors. As required by multiple-time regression analysis, only the linear portion of the slope was used to calculate Ki. ANCOVA was used to determine whether slopes and intercepts of two lines differed, and two-way ANOVA was used to determine effects of sex and genotype on regression lines in the APOE mouse studies. Two-tailed t-tests were used to compare two means and one-way or two-way analysis of variance (ANOVA) followed by multiple comparisons testing were used when more than two means are compared. The statistical tests used are specified in all figure legends. Data distribution was assumed to be normal, but was not formally tested. However, all data points are shown with the mean and standard deviation. Additional information can be found in the Life Sciences Reporting Summary.

Extended Data Fig. 1 .

Gel Autoradiography of I-S1 from RayBiotech and AMSBIO. Cropped images of Fraction 9 of I-S1 from RayBiotech (left panel,1μl of approximately 300,000 CPM run under reducing and non-reducing conditions, exposed to film for 24 hours), and from AMSBIO (right panel, 1μl of approximately 600,000 CPM run under reducing and non-reducing conditions). In each case, both the reduced and non-reduced gels migrated at the molecular weights predicted by the manufacturers. The autoradiography gels to validate size were run once. The quality of subsequent I-S1 labeling was confirmed by acid precipitation, described in methods.

I-S1 is uniformly taken up by all brain regions. Mice were given an injection of I-S1 (RayBiotech) and olfactory bulb, whole brain, and blood were collected 10 min later. Brains were dissected into the 10 regions shown. One-way ANOVA with brain regions as the independent variable showed a trend for differences (p = 0.09) and post-hoc multiple comparisons tests showed no differences among the brain regions. N = 11 mice. FCtx =frontal cortex, PCtx = parietal cortex, OCtx = occipital cortex.

Capillary depletion for I-S1. Capillary depletion studies were conducted to determine partitioning of the I-S1 (RayBiotech) in the capillary lumen, in the capillary, and in the brain parenchyma over time. The compartment that contained radioactivity was plotted against clock time, and the slopes were analyzed by linear regression. The slope of the line plotted for the parenchymal fraction was 0.187 ± 0.016 μL/g-min, r2 = 0.952, n = 9 mice, and was non-zero (p < 0.0001); the slope for the capillary fraction was 0.027 ± 0.006 μL/g-min, r2 = 0.757, n = 9 mice, and was non-zero (p = 0.0023). The slope for luminal binding was 0.05041 ± 0.04555 μL/g-min and did not significantly deviate from zero (p = 0.3050, n = 9, r2 = 0.145).The mean value for luminal binding over all time points was1.75 ± 0.50 μL/g. Transport of S1 proteins across human iPSC-derived brain-like endothelial cell (iBEC) monolayers. a. RayBiotech I-S1 passage across the iBEC monolayer was not significantly faster than that of T-Alb (p = 0.1464, t = 1.527, df=16; two-tailed paired t-test). Combination of 3 independent differentiations, n = 17 transwells/group, average TEER (2578 ± 897.8 Ω*cm2). b. Transport of RayBiotech I-S1 in the presence of 5 μg/ml excess unlabeled S1.

Excess S1 had no significant effect on I-S1 or T-Albumin Pe (p > 0.5; two-way ANOVA). N = 5-6 transwells/group from a single differentiation c. Transwells were treated with WGA (0.5 mg/ml) or vehicle. There was a significant overall effect of treatment (F (1, 9) = 9.435, p = 0.0133) and analyte (F (1, 9) = 6.949, p = 0.0271) on RayBiotech I-S1 or T-Alb transport (two-way ANOVA), but there were no significant effects of WGA on I-S1 or T-Alb Pe (Sidak's multiple comparisons testing). N = 5-6 wells/group from a single differentiation. d. Comparison of RayBiotech I-S1 and AMSBIO I-S1 transport. T-Alb was included with each I-S1 and also compared to the I-S1. Two-way ANOVA showed differences between I-S1s from the two sources) (F(1,10) = 10.1, p = 0.0098], but not between analytes (T-Alb vs I-S1). Multiple comparisons using Sidak's test showed a faster transport for RayBiotech I-S1 vs AMSBIO I-S1 (p = 0.0324). N = 6 wells/group from a single differentiation *p < 0.05. Extended Data Table 2 .

The number of mice used to calculate each regression line in figure 8 is shown here. Values in parentheses were excluded based on exclusion criteria for linear regression as described in methods.

Brain (T-Alb) 11 10 10 10 Serum 11 9 (1) 9 (1) 10

Brain (I-S1) 11 9 (1) 9 (1) 10

Olfactory 

Refer to Web version on PubMed Central for supplementary material. The brain/serum ratios of I-S1 from RayBiotech (I-S1-RayBiotech, filled circles) and AMSBIO (I-S1-AMSBIO, open circles) and T-Alb that was co-injected with I-S1-RayBiotech (T-Alb-R, filled triangles) or I-S1-AMSBIO (T-Alb-A, open triangles) are plotted against exposure time. The slopes of the lines represent the unidirectional influx rate (Ki) of each compound in units of μl/g-min, as described in methods. The Y-intercept (Vi) of each compound reflects the vascular space, and is expressed in μl/g. The Ki and Vi were calculated by simple linear regression. The Ki for I-S1 (RayBiotech) was significantly non-zero (p<0.0001), indicating that there was brain uptake; The Ki for I-S1 (AMSBIO) was also significantly non-zero (p<0.0001). Statistical comparisons of I-S1 curves from each vendor showed no statistical difference between the Ki values: F(1,22)=0.05881, p=0.8106, but did show a difference between the Vi values: F(1,23) = 10.32, p = 0.0039. The Ki for T-Alb-R did not significantly deviate from zero (p= 0.3556), indicating that there was no brain uptake. The Ki for T-Alb-A also did not significantly deviate from zero, (p= 0.5792).

The apparent lack of T-Alb brain uptake indicates that T-Alb remained confined to the vascular space and did not leak Panel a. Clearance of I-S1 from RayBiotech from blood, fitted to a nonlinear one phase exponential decay model: Y = 0.617(e −0.165t )+ 1.13 with a half-life = 4.20 min, n = 15 mice, r 2 = 0.956. Clearance from blood was linear for the first 10 min, with a half-time clearance of 6.6 min. b-f. The Y-axes show the delta tissue/serum ratios for I-S1, corrected for vascular space and non-specific leakage, as described in methods. b. The I-S1 Ki for whole brain was significantly non-zero (p<0.0001), indicating that there was tissue uptake (n=15 mice). c-f. These panels show the I-S1 Ki for different tissues calculated based on the linear portions of their curves. c. The I-S1 Ki for lung was significantly non-zero (p<0.0001) (n=15 mice).

d. The I-S1 Ki for spleen Ki was significantly non-zero (p = 0.0014) (n = 8 mice; 7 mice, shown as filled circles, were excluded from regression analysis due to non-linearity which violates assumptions of MTRA). e. The I-S1 Ki for kidney was significantly non-zero (p = 0.024) (n=15 mice). f. The I-S1 Ki for liver Ki was significantly non-zero (p = 0.0001) (n = 11 mice; 4 mice, shown as filled circles, were excluded from regression analysis because their inclusion caused non-linearity). All error terms are the standard error of the mean. a. Clearance of I-S1 from AMSBIO from blood, fitted to a one phase exponential decay model, Y = 1.05(e −0.393t )+ 0.880, half life = 1.74 min, n = 11 mice, r 2 = 0.867. Clearance from blood was linear for the first 10 min, with a half-time clearance of 3.6 min. In panels b-f, the Y-axes show the delta tissue/serum ratios for I-S1, corrected for vascular space and non-specific leakage, as described in methods. b. The I-S1 Ki for whole brain was significantly non-zero (p<0.0001), indicating that there was tissue uptake (n=11 mice). c-f. These panels show the I-S1 Ki for different tissues calculated based on the linear portions of their curves. c. The I-S1 Ki for lung was significantly non-zero (p<0.0001, n=11 mice). d.

The I-S1 Ki for spleen was significantly non-zero (p = 0.002, n=11 mice). e. The I-S1 Ki for kidney spleen was significantly non-zero (p = 0.0004, n=11 mice). f. The I-S1 Ki for liver was significantly non-zero (p = 0.0005; n = 8 mice (3 mice, shown as filled circles, were excluded from regression analysis because their inclusion caused non-linearity)). All error terms are the standard error of the mean. Figure 4 . WGA treatment enhances I-S1 uptake by brain, lung, spleen, and kidney and heparin blocks uptake by liver.

Panels a-f show serum levels and tissue uptake of radioiodinated S1 from either RayBiotech or AMSBIO 10 min after intravenous injection in which WGA, a stimulator of adsorptive transcytosis, was included in the injections to half the mice. a. Serum I-S1 levels expressed as the percent of the injected dose per ml of serum. There was a main effect of S1 source (F (1, 25) = 49.79, p<0.0001) and treatment (F (1, 25) = 20.57, p<0.0001), as well as a significant effect of their interaction (F (1, 25) = 40.93, p<0.0001). WGA treatment decreased I-S1 (RayBiotech) levels in serum (p<0.0001), indicating increased clearance from blood. b. Brain I-S1 levels expressed as delta brain/serum ratios, showing significant effects (all p<0.0001) of S1 source (F (1, 25) = 92.98), treatment (F (1, 25) = 252.2), and Figure 5 . Effects of unlabeled S1, ACE2, and ACE2 substrates on I-S1 uptake into tissues.

Panels show tissue uptake of radioiodinated S1 from RayBiotech 10 min after intravenous injection with or without unlabeled S1 (from AMSBIO), ACE2 or the ACE2 substrates ghrelin or angiotensin II (Ang II). The Y-axis shows the albumin-corrected (delta) tissue/ serum ratio, and the mean and standard deviation are shown. a. Unlabeled S1 at doses 0.1 ug (n-=10 mice), 1 ug (n=10 or 10 ug (n=9) did not reduce brain I-S1 uptake compared to vehicle controls (n=10) (One-way ANOVA: F(3,33)=0.6312, p = 0.6). b. Co-injection had a significant main effect on brain I-S1 uptake (one-way ANOVA F(3,29) = 4.7, p = 0.0073), with 1μg ACE2 increasing uptake (ACE2 vs. vehicle, p=0.0109 (Dunnett's multiple Figure 6 . LPS has minimal effects on brain I-S1 uptake, but alters I-S1 clearance from blood and uptake by peripheral tissues.

We treated mice with intraperitoneal LPS using a protocol previously shown to alter BBB permeability and induce inflammation, as described in methods. Four hours after the last injection of LPS, we gave mice an intravenous injection containing I-S1 (RayBiotech) and T-Alb and collected tissue and blood samples 10 min after the intravenous injection. a. Serum levels of I-S1 (RayBiotech), expressed as % injected dose present in a ml of serum (%Inj/ml), were increased 4h after LPS injection vs control ((t = 3.66, df = 16, p = 0.0021, two-tailed t-test), indicating that LPS decreased clearance of I-S1 from blood. LPS had no significant effect on the serum levels of T-Alb (t= t=0.3739, df=16, p= 0.7134), indicating a selective effect of LPS on I-S1 clearance. b. Tissue levels of I-S1, expressed as tissue/serum ratios corrected (delta brain/tissue ratios) for vascular space and leakage by subtracting the tissue/serum ratios for T-Alb. LPS increase I-S1 uptake by lung (t = 4.58, df = 16, p = 0.0003) and decreased I-S1 uptake by spleen (t = 6.39, df = 16, p<0.0001) and liver (t = Figure 7 . I-S1 enters the brain after intranasal administration.

Brain uptake of I-S1 (RayBiotech) delivered intranasally (1 μL/naris), expressed as % administered dose taken up per g of each brain region (%Inj/g). a. Ten minutes after intranasal administration, I-S1 was detected in all dissected brain regions. One-way ANOVA showed a significant effect of brain region [F(11, 48) = 3.77, p = 0.0006], with significantly higher uptake in olfactory bulb (p = 0.031) and hypothalamus (p = 0.0041) compared to whole brain (Dunnett's multiple comparisons test). Whole-brain uptake after intranasal administration was 0.023 ± 0.0008 %Inj/g; for visual comparison, the panel also shows the whole-brain %Inj/g 10 min after IV injection as calculated from the control values for the LPS experiment in Figure 6 (filled circles, 0.22 ± 0.016 %Inj/g); N=5 mice. b. Thirty minutes after intranasal administration, I-S1 was detected in all dissected brain regions. One-way ANOVA showed a significant effect of brain region (F(11,48) = 4.33, p = 0.0002), with significantly more uptake in the olfactory bulb compared to whole brain (p = 0.0023, Dunnett's multiple comparisons test). A two-tailed t-test showed significant increases between 10 and 30 minutes for whole brain (p=0.0143, t=3.116, df=8), frontal cortex (p=0.0391, t=2.464, df=8), cerebellum (p=0.0003, t=6.226, df=8), midbrain (p= 0.0098, t=3.370, df=8) and pons (p=0.0232, t=2.801, df=8) but not the other regions. N=5 mice. * p<0.05, ** <0.01, *** <0.001; FCtx = frontal cortex, PCtx = parietal cortex, OCtx = occipital cortex. Serum levels and tissue uptake of I-S1 (RayBiotech) and brain uptake of T-Alb following intravenous injection in male and female mice that were homozygous for human APOE3 (E3) or APOE4 (E4). For panel a, multiple-time regression analysis was performed to calculate the Ki and Vi for T-Alb. Neither sex nor genotype affected clearance of I-S1 from blood (panel b). In panels c-h, the Y-axis shows delta tissue/serum ratios for I-S1, which are corrected for vascular space and non-specific leakage, as described in methods. The slopes of the regression lines that result from multiple-time regression analysis measures the rate of influx (Ki) into the tissues. The Ki values for the four groups (E3 males, E3 females,

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Vallance KL Adsorptive endocytosis of HIV-1gp120 by blood-brain barrier is enhanced by lipopolysaccharide

Cytokine and chemokine responses in serum and brain after single and repeated injections of lipopolysaccharide: Mutliplex quantification with path analysis

The olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system

ApoE e4e4 genotype and mortality with COVID-19 in UK Biobank

Impact of sex and gender on COVID-19 outcomes in Europe

COVID-19 patients' clinical characteristics, discharge rate, and fatality rate of meta-analysis

Tectonic conformational changes of a coronavirus spike glycoprotein promote membrane fusion

Lethal infection of K18-hACE2 mice infected with severe acute respiratory syndrome coronavirus

Human immunodeficiency virus-1 uses the mannose-6-phosphate receptor to cross the blood-brain barrier

Distribution of ACE2, CD147, CD26 and other SARS-CoV-2 associated molecules in tissues and immune cells in health and in asthma, COPD, obesity, hypertension, and COVID-19 risk factors

COVID-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human CD26

Elshahat ME & Elfiky AA COVID-19 spike-host cell receptor GRP78 binding site prediction

Guruprasad L Human coronavirus spike protein-host receptor recognition

Kastin AJ Characterization of lectin-mediated brain uptake of HIV-1 gp120

Considerations around the SARS-CoV-2 Spike Protein with particular attention to COVID-19 brain infection and neurological symptoms

DS A model of the ACE2 structure and function as a SARS-CoV receptor

Risk factors for severity and mortality in adult COVID-19 inpatients in Wuhan

Dysfunction in SARS-COV-2 Infection can be a Potential Cause of Respiratory Distress

AJ Comparative study of expression and activity of glucose transporters between stem cell-derived brain microvascular endothelial cells and hCMEC/D3 cells

Derivation of blood-brain barrier endothelial cells from human pluripotent stem cells

Weeber EJ & Rebeck GW Young APOE4 targeted replacement mice exhibit poor spatial learning and memory, with reduced dendritic spine density in the medial entorhinal cortex

Rueckert RR On the use of chloramine-T to iodinate specifically the surface proteins of intact enveloped viruses

Cham TM & Chung MI On-site preparation of technetium-99m labeled human serum albumin for clinical application

Patlak CS Transport of alpha-aminoisobutyric acid across brain capillary and cellular membranes

Fenstermacher JD Selection of experimental conditions for the accurate determination of blood-brain transfer constants from single-time experiments: a theoretical analysis

Capillary depletion method for quantification of bloodbrain barrier transport of circulating peptides and plasma proteins

Kastin AJ Murine tumor necrosis factor alpha is transported from blood to brain in the mouse

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Targeted replacement of the mouse apolipoprotein E gene with the common human APOE3 allele enhances diet-induced hypercholesterolemia and atherosclerosis

-dependent effects on anxiety and cognition in female TR mice

A Simplified, Fully Defined Differentiation Scheme for Producing Blood-Brain Barrier Endothelial Cells from Human iPSCs

Drug transfer across the blood-brain barrier: correlation between in vitro and in vivo models

Lung I-S1 levels show an effect of treatment (F (1, 25) = 204.6, p<0.0001). d. Spleen I-S1 levels show effects of S1 source (F (1, 25) = 43.06, p<0.0001) and treatment (F (1, 25) = 30.42, p<0.0001), and a trend for interaction (F (1, 25) = 3.657, p = 0.08). e. Kidney I-S1 levels show effects of source (F (1, 25) = 33.45, P<0.0001) and treatment (F (1, 25) = 40.77, p<0.0001). f. Liver I-S1 levels show effects of source of S1 (F (1, 25) = 55.87), treatment (F (1, 25) = 52.16) and their interaction (F (1, 25) = 32.08), all at p<0.0001. For a-f data were analyzed using two-way ANOVA with S1 source (RayBiotech vs AMSBIO) and treatment (with or without WGA) as main factors, followed by Tukey's multiple comparisons tests; figures show mean and standard deviation. *p<0.05, **p<0.01, and ***p<0.001 indicating comparisons of treatments within I-S1 group

Liver uptake of I-S1 was lower after WGA + heparin vs heparin alone (p = 0.0034) but not vs WGA alone. *p<0.05, **<0.01, ***<0.001. For g-h, data were analyzed by one-way ANOVA and Sidak's multiple comparisons test, and n=10 for each group, with the exception of liver vehicle in panel h, where one outlier was identified using a Grubbs test (77.6 ul/g) and excluded, leaving n=9 mice. comparisons test) but no effect of 1μg ghrelin or 1μg AngII. *p<0.05. N= 8, 8, 9, and 8 for the vehicle, ghrelin, AngII, and ACE2 groups, respectively. c. Co-injection had a significant main effect on lung I-S1 uptake

but no effects of 1μg AngII (p= 0.3511; all Dunnett's test). * p<0.05, ** p<0.01, *** p<0.001

The decreased I-S1 uptake by liver likely contributes to the decrease in I-S1 clearance from blood seen in panel a. c. Levels of I-S1 in brain areas, expresses as delta brain/serum ratios. Two-way ANOVA showed a significant main effect of brain region [F(11,190) = 2.72, p = 0.0028] accounting for 12.5% of the variability, and a main effect of LPS treatment

Ki values in figures are shown with their standard error of the mean. Sex or APOE genotype did not influence changes in brain uptake of T-Alb (a), levels of I-S1 in serum (b), or I-S1 uptake by brain (c) or lung (e) indicating that these factors do not influence BBB integrity or brain vascular space, clearance from blood, or uptake by brain or lung. d. Two-way ANOVA showed a significant effect of sex on I-S1 uptake in the olfactory bulb (F (1, 29) = 14.30, p = 0.0007), accounting for 29% of the variability. Post-hoc Tukey's multiple comparisons showed a significantly larger Ki for E3 males compared to E3 females (p = 0.0056) and to E4 females (p = 0.0015). APOE genotype had a trend-level effect on uptake in the olfactory bulb (F (1, 29) = 3.838, p = 0.06). f. APOE genotype had a significant main effect on I-S1 uptake in the liver (F (1, 24) = 28.44, p<0.0001) accounting for 50% of the variability, with a lower uptake rate for E4 females compared to E3 females (p = 0.0057) and to E3 males (p = 0.0001), and a lower uptake rate for E4 males vs E3 males (p = 0.0039). g: There was a significant main effect of sex on I-S1 uptake in the spleen (p = 0.0004) accounting for 20% of the variability, a main effect of ApoE genotype (p<0.0001) accounting for 43% of variability, and a significant effect of their interaction (p= 0.0029) accounting for 13% of variability. Multiple comparisons showed faster uptake rates for E3 females compared to E4 females (p<0.0001) to E3 males (p = 0.0003), and to E4 males (p<0.0001). h: APOE genotype had a significant effect on I-S1 uptake in the kidney (p = 0.0046) accounting for 25% of variability

Supported by the VA (WAB, MAE), NIH R21 AG065928-01 (MAE), and NIH RF-1 AG059088 (WAB, JR).We wish to thank Kristen Baumann, Riley Weaver, Sarah Pemberton, and Danny Quaranta for technical assistance.

The data that support the findings of this study are available from the corresponding author upon reasonable request.