Immediately after cervical dislocation, the mice were exsanguinated and tissues were dissected. and non-neuronal expression of TMPRSS2 in the OE,12,13 except for one study which did not find TMPRSS2 expressed in OE.14 The levels of TMPRSS2 expression in non-neuronal OE cells seem to be higher than that in ORNs,10,13 but different subpopulations of mature ORNs appear to differ ML167 in their levels of TMPRSS2;13 such a mosaic expression pattern is not typical for the majority of other ORN genes. Our own RNAseq profiling in murine OE readily detected expression of TMPRSS2, and its levels were higher as compared to ACE2 (GEO accession number “type”:”entrez-geo”,”attrs”:”text”:”GSE147771″,”term_id”:”147771″GSE147771). TMPRSS2 expression increased with old age (Table 1). In summary, our RNAseq and other available expression profiling data for murine OE indicate that ACE2 is mainly expressed in non-neuronal cells and TMPRSS2 is usually widely expressed in both neuronal ML167 and non-neuronal cells, likely with higher expression levels in non-neuronal cells.17 Since gene expression in murine and human OE is highly conserved,14 these results suggest that non-neuronal cells rather than ORNs in the human OE are the most likely site of SARS-CoV-2 virus entry to the olfactory epithelium. Since transcriptome data should be validated by additional gene and protein expression studies,15,18,21 we next used RT-PCR to examine gene expression. Our analysis of expression performed by real-time RT-PCR showed a slight decrease of ACE2 in ML167 the OE in juvenile mice, while levels of expression subsequently remained constant during aging (Figure ?Physique11A). Because of recent reports discussing the possibility of SARS-CoV-2 contamination in the brain,4,5 we also examined gene expression in the brain. Expression of ACE2 was significantly lower in the brain as compared to OE, and low levels in the olfactory bulb and frontal cortex did not change with age (Figure ?Physique11B, C). TMPRSS2 expression in the OE increased from young adult to old age mice (Physique ?Figure11D), and this is consistent with our RNAseq data (Table 1). To validate our real-time RT-PCR ML167 approach, we examined expression of another protease, TMPRSS4, which is also known to be expressed in non-neuronal cells, as is the case for TMPRSS2.12,14 Expression of TMPRSS4 has an opposite trend in OE with age (decreasing, Figure ?Physique11G) as compared to TMPRSS2 (increasing in the OE with old age) (Physique ?Figure11D). Open in a separate window Physique 1 Gene expression analysis by RT-PCR for ACE2 and TMPRSS2 in murine olfactory epithelium (OE), olfactory bulb (OB), and frontal cortex (FC) at different ages. Relative expression levels were normalized to expression in 2 month old OE (100%) using GAPDH as housekeeping gene. Comparable results were obtained with -actin as the housekeeping gene. (A) Relative expression of Mouse monoclonal to FUK ACE2 in OE decreases from juvenile 1 month old (1MO) to 2 month old (2MO), but it is usually stable in 2 year old (2YO). (B, C) ACE2 levels observed in OB and FC are lower as compared to OE and stable during aging. Note that OE has higher expression level than OB and FC at each stage analyzed. (D) For TMPRSS2, much higher expression was detected in OE as compared to OB (E) and FC (F). Note that with aging the level of TMPRSS2 increases significantly in OE but not in OB or FC. (G) Expression of another protease, TMPRSS4, in OE significantly decreases during aging. (H) Lower expression of ACE2 was noted in OE of female as compared to male 2 month old mice. (I) TMPRSS2 expression in OE did not show gender-specific differences. All graphs give the mean values, and error bars represent SEM. * 0.01, ** 0.005, *** 0.001. Comparison of adult male and female mice showed lower levels of ACE2 expression in the OE of females (Physique ?Figure11H) but not in the olfactory bulb or in frontal cortex (data not.