SARS-CoV-2 Receptors and Entry Genes Are Expressed in the Human Olfactory Neuroepithelium and Brain

Abstract

Reports indicate an association between COVID-19 and anosmia, as well as the presence of SARS-CoV-2 virions in the olfactory bulb. To test whether the olfactory neuroepithelium may represent a target of the virus, we generated RNA-seq libraries from human olfactory neuroepithelia, in which we found substantial expression of the genes coding for the virus receptor angiotensin-converting enzyme-2 (ACE2) and for the virus internalization enhancer TMPRSS2. We analyzed a human olfactory single-cell RNA-seq dataset and determined that sustentacular cells, which maintain the integrity of olfactory sensory neurons, express ACE2 and TMPRSS2. ACE2 protein was highly expressed in a subset of sustentacular cells in human and mouse olfactory tissues. Finally, we found ACE2 transcripts in specific brain cell types, both in mice and humans. Sustentacular cells thus represent a potential entry door for SARS-CoV-2 in a neuronal sensory system that is in direct connection with the brain.

Keywords: Bioinformatics; Biological Sciences; Cell Biology; Microbiology; Omics; Transcriptomics; Virology.

Graphical abstract

Figure 1

ACE2 and TMPRSS2 Expression in Olfactory and Respiratory Nasal Epithelia (A) Schematic of the human nasal respiratory and olfactory epithelia, with their respective cell types. (B) Design of the bulk tissue transcriptome analysis. OE biopsies: n = 3, RE biopsies: n = 3. (C) Volcano plot corresponding to the differential expression analysis, displaying the expression fold change and the associated adjusted p value (∗∗∗∗: p value ≤ 0.0001; ∗: 0.01 < p value ≤ 0.05; ns: non-significant, p value > 0.05) between olfactory and respiratory epithelia for each gene. OR genes, olfactory transduction cascade specific genes, and SARS-CoV-2 entry genes ACE2 and TMPRSS2 are highlighted with a color code. (D–G) Transcript quantifications corresponding to (D) OR genes, (E) olfactory sensory epithelium markers and (F and G) SARS-CoV-2 entry proteins in olfactory and respiratory biopsies. A different color was attributed to the data of each patient (n = 4). Olfactory and respiratory epithelium sample data points are shown on a white and gray background, respectively. OE, olfactory epithelium; RE, respiratory epithelium.

Figure 2

Coexpression of ACE2 and TMPRSS2 in Human Sustentacular and Respiratory Ciliated Cells (A) Violin plots displaying ACE2 and TMPRSS2 normalized expression levels in all ACE2-positive (n = 169) and ACE2-negative (n = 28,557) cells. (B) Visualization of the clustering results reported in Durante et al.(2020) on a uniform manifold approximation and projection (UMAP) plot. “Respiratory columnar cells” was renamed as “Respiratory early secretory cells” based on the expression of SERPINB3. a.u., arbitrary units. (C) Normalized expression levels of ACE2, TMPRSS2, ERMN (a sustentacular cell gene marker), and GSTA2 (a respiratory ciliated cell gene marker), shown on the UMAP plot. norm. UMI, normalized unique molecular identifier counts. (D) ACE2 and TMPRSS2 coexpressing cells, highlighted in black on the UMAP plot. (E) Coexpression of ACE2 and TMPRSS2 in the different cell clusters (n = 26 clusters). Means of ACE2 and TMPRSS2 normalized expression levels are plotted. (F and G) Violin plots displaying ACE2 and TMPRSS2 normalized expression levels in (F) ACE2-positive (n = 71) and ACE2-negative (n = 3,011) sustentacular cells, and in (G) ACE2 positive (n = 19) and ACE2 negative (n = 567) respiratory ciliated cells. (H) Number of cells per cluster expressing ACE2 and/or TMPRSS2. (I) Percentage of cells per cluster expressing ACE2 and/or TMPRSS2 in all coexpressing clusters (i.e., clusters with double-positive cells). Color gradient scales in (C) and y axis scales in (A), (F) and (G) are in log₁₀.

Figure 3

ACE2 expression in the Mouse Olfactory Neuroepithelium (A) Schematic representation of the mouse olfactory system. The sensory tissue and the brain are highlighted in green. The blue frame indicates the coronal section level corresponding to the next panels. OB, olfactory bulb; OE, olfactory epithelium. (B) Schematic representation of a coronal section of the mouse olfactory system. The OE is highlighted in green, and the brain, including the olfactory bulb, in light green. (C–F) Immunostainings on coronal sections of mouse olfactory neuroepithelium with three different anti-ACE2 antibodies (red) and a negative control. (C) ACE2 Ab1, polyclonal antibody against the extracellular domain of mouse ACE2. (D) ACE2 Ab2, polyclonal antibody against the intracellular domain of human ACE2 (that cross reacts with mouse ACE2). (E) ACE2 Ab3, monoclonal antibody against the extracellular domain of human ACE2 (that cross reacts with mouse ACE2). (F) Negative control immunostaining without primary antibody against ACE2. (G) Immunostaining for ACE2 (red) on a coronal section of the mouse olfactory neuroepithelium. Scale bar, 0.2 mm. Squares indicate zones magnified in (H) and (I). The schematic on the lower left indicates that ACE2 expression is observed in the dorsal (H), but not in the ventral part (I) of the olfactory epithelium. (J) Double immunostaining for ACE2 (red) and the neuronal marker TUJ1 (green) in the ACE2-positive part of the mouse olfactory neuroepithelium. The square indicates the portion of the MOE that is magnified in (K)–(R). Scale bar, 20 μm. (K–N) Double immunostaining for ACE2 (red) together with (K) the neuronal marker TUJ1 (green), (L) the marker of mature olfactory sensory neurons OMP (green), (M) the marker for sustentacular cells ERMN (green) and (N) TMPRSS2 (green). (O and P) Negative control immunostaining without primary antibody against (O) ERMN and (P) TMPRSS2. (Q and R) Double immunostaining for the sustentacular cell marker ERMN (red) together with (Q) OMP and (R) TUJ1. (S) Schematic of the external part of the olfactory epithelium, highlighting the relative positions of ACE2, TUJ1, ERMN, and OMP according to the immunostainings shown in (K)–(R). TUJ1 is present in the dendrites, axons, and somata of olfactory sensory neurons, OMP more concentrated in the cell body of olfactory sensory neurons, and ACE2 and ERMN in the apical portion of sustentacular cells. All sections with immunostaining were counterstained with DAPI (blue). When not indicated, scale bars are 10 μm. See also Figures S1–S3.

Figure 4

ACE2 Expression in the Human Olfactory Neuroepithelium (A and B) Section of human respiratory epithelium with (A) immunostaining for ERMN (red) and the neuronal marker TUJ1 (green), and (B) the same section stained with Alcian blue and counterstained with nuclear fast red, highlighting a transition between sensory (left) and respiratory (right, note the goblet cells) epithelium. ERMN is expressed in the apical part of sustentacular cells within the olfactory neuroepithelium (arrowheads). (C, E) A portion of human olfactory neuroepithelium immunostained for ACE2 (red) and the neuronal marker TUJ1 (green), and (D) with Alcian blue (note the absence of goblet cells). (F, G) Higher magnification of the external border of the human olfactory neuroepithelium. The same region is shown with (F) and without (G) TUJ1 staining. Nuclei were counterstained with DAPI (blue) or FastRed (red). Scale bars, 10 μm. See also Figures S4 and S5.

Figure 5

Ace2 and Tmprss2 Expression in the Mouse Brain (A) Mouse brain cell type classification from Zeisel et al. (2018) was visualized via http://mousebrain.org/. Rows with shaded boxes below the tree indicate the gene mean expression levels for each cell type, relative to its maximum. Rbfox3, encoding the neuronal marker NEUN, and Rgs5, a marker of pericytes, are shown in comparison with Ace2 and Tmprss2. (B) Details of the gene expression levels in pericytes (blue edges clade of the classification dendrogram in (A)). Discs represent mean relative expression levels. Maximum values of mean normalized expression levels for Rgs5 and Ace2 are connected to the corresponding circles. (C) Ace2 and Tmprss2 expression levels in the top 10 Ace2-expressing cell populations from the http://dropviz.org/ dataset (Saunders et al., 2018). Abbreviations in parentheses denote the origin brain structure. STR, striatum; HC, hippocampus; PC, posterior cortex; FC, frontal cortex; GP, globus pallidus; CB, cerebellum; TH, thalamus; SN, substantia nigra; ENT, entopeduncular. (D) Similar to Figure 3, subsequent panels show coronal sections (blue area) crossing the nasal cavity and the olfactory bulb. As represented in this schematic, olfactory sensory neurons from the olfactory epithelium send axonal projections to the olfactory bulb through the cribriform plate of the ethmoid bone, along the nerve layer. OB, olfactory bulb; OE, olfactory epithelium; nl, nerve layer; gl, glomerular layer; ml, mitral cell layer; gr, granule cell layer. (E–G) Immunostaining for ACE2 (red) and for the neuronal marker TUJ1 (green) on sections of the mouse olfactory bulb. White arrowheads indicate the presence of ACE2 in the pericytes of capillaries. Scale bars, 20 μm. (H) Magnification of capillary ACE2 immunostaining (red). Scale bar, 5 μm. All sections were counterstained with DAPI (blue).

Figure 6

Coexpression of ACE2 and TMPRSS2 in the Human Brain (A) Visualization of the human brain cell-type subclasses on a t-distributed stochastic neighbor embedding (t-SNE) plot. a.u., arbitrary units. (B and C) Normalized expression levels of (B) RBFOX3 (which encodes for the neuronal marker NEUN), (C) ACE2 and TMPRSS2, shown on the t-SNE plot. norm. expr, normalized expression levels. (D) ACE2 and TMPRSS2 coexpressing cells, highlighted in black on the t-SNE plot. (E) Coexpression of ACE2 and TMPRSS2 in the different subclasses (n = 19 subclasses). Means of ACE2 and TMPRSS2 normalized expression levels are plotted. (F) Number of cells per subclass expressing ACE2 and/or TMPRSS2. (G) Percentage of cells per subclass expressing ACE2 and/or TMPRSS2 in all coexpressing subclasses (i.e., subclasses with double-positive cells). Color gradient scales in (B) and (C) are in log₁₀. L, cortical layer; IT, intratelencephalic; CT, corticothalamic; ET, extratelencephalic; NP, near-projecting; LAMP5, lysosomal-associated membrane protein family member 5; PAX6, paired box 6; VIP, vasoactive intestinal polypeptide; SST, somatostatin; PVALB, parvalbumin; OPC, oligodendrocyte precursor cell; VLMC, vascular leptomeningeal cell. See also Figure S6.