The Molecular Regulation of Horizontal Basal Cell Activation in the Olfactory Epithelium

Project SummaryOlfactory dysfunction, including anosmia, is an unexpected and unexplained consequence of infection by the b-coronavirus SARS-CoV-2 and can be the sole manifestation of COVID-19. Existing bulk and single cell RNAseq datasets demonstrate that a very low percentage of nonneuronal cells - sustentacular (Sus) cells, horizontal basal cells (HBCs), and Bowman's gland and duct (BG/D) cells - of the olfactory epithelium (OE) express ACE2, the high affinity receptor for SARS-CoV-2, and the viral relevant proteases, TMPRSS2, FURIN and CATHEPSINS B and L. However, the lack of protein expression data prevents definition of which types and what percentage of cells are susceptible to infection. Specific Aim 1 proposes immunohistochemical evaluation of our library of human and mouse olfactory mucosa (OM) for the presence of the corresponding proteins using validated antibody markers for OE cell types and viral-relevant proteins. The inclusion of mouse allows us, first, to assess protein expression during OE regeneration, and, second, makes feasible subsequent experiments examining the interaction of virus with the olfactory periphery, whether in normal mice or ones that are made transgenic for hACE2, offering a humanized animal model and greatly enhancing our understanding of the disease. For example, other b-coronaviruses reach and decimate the CNS after intranasal inoculation even though olfactory sensory neurons (OSNs) do not express the relevant receptors (Schwob et al, 2001). Even if Aim 1 demonstrates that a substantial percentage of non-neuronal cells express the machinery necessary to support infection and replication, the mechanism underlying the diminution/elimination of the sense of smell would remain obscure. Hence, Specific Aim 2 proposes the pathological examination of the impact of COVID-19 on the human OM and olfactory bulb (OB) in patients that die of the disease and come to autopsy at Massachusetts General Hospital, which Autopsy Service has agreed to supply us with these tissues. This Aim takes advantage of the PI's expertise in general anatomic pathology, and more specifically that of the diseased olfactory epithelium and bulb as well as the contributions of Dr. Eric Holbrook, co-I of this competitive revision, who will harvest the mucosa and assist in the analysis of the tissue. Besides conventional pathological assessment, the tissue will be interrogated for the presence of active virus, the type and extent of inflammatory infiltrate, and the loss of the various cell populations of the epithelium and bulb. The results will clarify whether and to what extent SARS-CoV-2 transits the OSNs and the olfactory nerve and damage the CNS as do other b-coronaviruses. Finally, Specific Aim 3 proposes high risk-high reward experiments based on our ability to differentiate the OE cell types from activated HBCs in vitro, which will then be assayed for the protein apparatus underlying infection and compared with the in vivo results obtained in Aims 1 and 2. When the Aims are completed successfully, the field will then be poised to begin the virological investigations in vitro and in vivo that will truly explain why olfactory dysfunction is such a hallmark of COVID-19.