Non-neural expression of SARS-CoV-2 entry genes in the olfactory epithelium suggests mechanisms underlying anosmia in COVID-19 patients

David Brann; Tatsuya Tsukahara; Caleb Weinreb; Darren W. Logan; Sandeep Robert Datta

The presence of Ace2 and Tmprss2 transcripts in mouse WOM and their (near total) absence in purified OSNs suggest that the molecular components that enable CoV-2 entry into cells are expressed in non-neuronal cell types in the mouse nasal epithelium. To identify the specific cell types that express Ace2 and Tmprss2, we performed scSeq (using the 10x Chromium platform, see Methods) on the WOM; olfactory sensory neurons did not express Ace2 in this de novo analysis (2 of 28769 mature OSNs were positive for Ace2), while expression was observed in Bowman's gland cells and HBCs in a small percentage of cells (Figures 2C-E and S1B-C, see methods). Because WOM scSeq de-enriches certain cell types (e.g., sustentacular cells), to complement these data we employed a previously established lineage tracing protocol to perform scSeq on HBCs and their descendants during injury-induced regeneration (27) . This analysis revealed that Ace2 and Tmprss2 are expressed in subsets of sustentacular cells and HBCs, as well as activated HBCs that serve to regenerate the epithelium ( Figures 2F-H and S2A ). To validate these results, we reanalyzed a similar lineage tracing dataset in which identified HBCs and their progeny were subject to Smart-seq2-based deep sequencing (27) . In this dataset, Ace2 was detected in more than 0.7% of GBCs, nearly 2% of HBCs and nearly 3% of sustentacular cells but was not detected in OSNs (Figures 2I and S2B) . Furthermore, larger percentages of HBCs, GBCs and sustentacular cells expressed Tmprss2. Taken together, these bulk RNA-Seq and scSeq mouse data demonstrate that CoV-2 cell entry-related genes are expressed in support and stem cells in the mouse OE. (39) . Normalized counts for each gene in the whole olfactory mucosa (WOM) and olfactory sensory neurons (OSNs) are shown. Each circle represents a biological replicate and each color indicates the category of the gene shown on the right (CoV-2 and other CoVs: genes involved in the entry of these viruses, other categories: marker genes for specific cell types such as horizontal basal cells (HBC), and sustentacular cells (SUS)). Mean ± SD normalized counts in OSNs -Ace2: 8 To address whether there are similar patterns of gene expression in human OE, we queried previously published bulk WOM RNA-Seq data derived from macaque, marmoset and human (43) , and found expression of almost all CoV-entry-related genes in all WOM samples, consistent with our observations in mouse WOM ( Figure S3A ). To identify the specific cell types in human OE that express ACE2, we quantified gene expression in scSeq derived from four human nasal biopsy samples recently reported by Durante et al (44) . As in the mouse scSeq datasets, neither ACE2 nor TMPRSS2 were detected in mature OSNs, whereas these genes were detected in both sustentacular cells and HBCs (Figures 3A-E and S3B-C). In contrast, genes relevant to cell entry of other CoVs were expressed in OSNs, as well as in other OE cell types. We confirmed the expression of ACE2 proteins via immunohistochemical staining of human olfactory epithelium biopsy tissue, which revealed expression in sustentacular and basal cells, and an absence of ACE2 protein in OSNs ( Figure 3F ). Together, these results demonstrate that sustentacular and olfactory stem cells, but not mature OSNs, are potentially direct targets of CoV-2 in the human OE.