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.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the coronavirus disease 2019 (COVID-19) pandemic. In addition to pneumonia and acute respiratory distress, COVID-19 is associated with a host of symptoms that relate to the CNS, including loss of taste and smell, headaches, twitching, seizures, confusion, vision impairment, nerve pain, dizziness, impaired consciousness, nausea and vomiting, hemiplegia, ataxia, stroke and cerebral hemorrhage^1,2. It has been postulated that some of the symptoms of COVID-19 may be due to direct actions of the virus on the CNS; for example, respiratory symptoms could be in part due to SARS-CoV-2 invading the respiratory centers of the brain^1,3. Encephalitis has also been reported in COVID-19, and could be a result of virus or viral proteins having entered the brain^4,5. SARS-CoV-2 mRNA has been recovered from the cerebrospinal fluid⁴, suggesting it can cross the blood–brain barrier (BBB). Other coronaviruses, including the closely related SARS virus that caused the 2003–2004 outbreak, are able to cross the BBB^6,7,8, and SARS-CoV-2 can infect neurons in a BrainSphere model⁹. However, SARS-CoV-2 could induce changes in the CNS without directly crossing the BBB, as COVID-19 is associated with a cytokine storm, and many cytokines cross the BBB to affect CNS function¹⁰.

Here we assess whether one viral protein of SARS-CoV-2, the spike 1 protein (S1), can cross the BBB. This question is important and clinically relevant for two reasons. First, some proteins shed from viruses have been shown to cross the BBB, inducing neuroinflammation and otherwise impairing CNS functions^{11,12,13,14,15,16,17}. Second, the viral protein that binds to cells can be used to model the activity of the virus; in other words, if the viral binding protein crosses the BBB, it is likely that protein enables the virus to cross the BBB as well^18,19. S1 is the binding protein for SARS-CoV-2 (ref. ²⁰); it binds to angiotensin-converting enzyme 2 (ACE2)^21,22,23 and probably other proteins as well.

In this study, we show that I-S1 readily crossed the murine BBB, entered the parenchymal tissue of the brain and, to a lesser degree, was sequestered by brain endothelial cells and associated with the brain capillary glycocalyx. We describe I-S1 rate of entry into the brain after intravenous (i.v.) and intranasal administration, determine its uptake in 11 different brain regions, examine the effect of inflammation, APOE genotype and sex on I-S1 transport, and compare I-S1 uptake in the brain to the uptake in the liver, kidney, spleen and lung. Based on experiments with the glycoprotein WGA, we found that brain entry of I-S1 likely involves the vesicular-dependent mechanism of adsorptive transcytosis.

I-S1 protein is transported across the mouse blood–brain barrier

We obtained S1 proteins from two commercial sources: RayBiotech and AMSBIO. The S1 proteins were radiolabeled in-house, and verified to be intact after labeling by autoradiography gels (Extended Data Fig. 1 and Supplementary Fig. 1). We determined whether intravenously injected I-S1 could cross the BBB in mice, by measuring its blood-to-brain influx constant (K_i) 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 K_i, that is, the unidirectional influx constant for I-S1.

We co-injected ^99mTc-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 K_i values of the I-SI proteins from the two sources differed by about 3% (Fig. 1). These results show that unlike T-Alb, I-S1 readily crosses the mouse BBB.