Novel role of cystic fibrosis transmembrane conductance regulator in maintaining adult mouse olfactory neuronal homeostasis

Abstract

The olfactory epithelium (OE) of mice deficient in cystic fibrosis transmembrane conductance regulator (CFTR) exhibits ion transport deficiencies reported in human CF airways, as well as progressive neuronal loss, suggesting defects in olfactory neuron homeostasis. Microvillar cells, a specialized OE cell-subtype, have been implicated in maintaining tissue homeostasis. These cells are endowed with a PLCβ2/IP3 R3/TRPC6 signal transduction pathway modulating release of neuropeptide Y (NPY), which stimulates OE stem cell activity. It is unknown, however, whether microvillar cells also mediate the deficits observed in CFTR-null mice. Here we show that Cftr mRNA in mouse OE is exclusively localized in microvillar cells and CFTR immunofluorescence is coassociated with the scaffolding protein NHERF-1 and PLCβ2 in microvilli. In CFTR-null mice, PLCβ2 was undetectable, NHERF-1 mislocalized, and IP3 R3 more intensely stained, along with increased levels of NPY, suggesting profound alteration of the PLCβ2/IP3 R3 signaling pathway. In addition, basal olfactory neuron homeostasis was altered, shown by increased progenitor cell proliferation, differentiation, and apoptosis and by reduced regenerative capacity following methimazole-induced neurodegeneration. The importance of CFTR in microvillar cells was further underscored by decreased thickness of the OE mucus layer and increased numbers of immune cells within this tissue in CFTR-KO mice. Finally, we observed enhanced immune responses to an acute viral-like infection, as well as hyper-responsiveness to chemical and physical stimuli applied intranasally. Taken together, these data strengthen the notion that microvillar cells in the OE play a key role in maintaining tissue homeostasis and identify several mechanisms underlying this regulation through the multiple functions of CFTR.

Ascl3 transcription factor marks a distinct progenitor lineage for non-neuronal support cells in the olfactory epithelium

“…Both microvillar cells and Bowman’s glands are important for the maintenance of OE homeostasis391113. Our data suggest that all microvillar cells and Bowman’s glands are derived from Ascl3-expressing progenitors.…”

“…5B,K). Knockouts of CFTR, IP3R3 and NPY in microvillar cells have shown defects in the OE91113, but the phenotype in the Ascl3-DTA mice appeared more severe. As expected, we rarely observed cells labeled with PLC β2, Trpm5 or AQP5 in OE sections from Ascl3-DTA mice, confirming the specific ablation of Ascl3 lineages (Fig.…”

“…A role for microvillar cells in regulating or maintaining the OE has been demonstrated through analysis of several gene knockouts, including CFTR13, NPY9 and IP3R311. In all cases, loss of gene function in the microvillar cells affected proliferation and regenerative ability of the basal progenitor cells.…”

“…Knockout of NPY, or its receptor, results in reduced stem cell proliferation and decreased production of OSNs910. Numerous lines of evidence have indicated that the microvillar cells play an important role in OE homeostasis9111213.…”

Deconstructing Olfactory Stem Cell Trajectories at Single-Cell Resolution

“…Cells in two clusters (immature and mature sustentacular cells, iSus and mSus, respectively) express increasing levels of Cyp2g1 , a marker of sustentacular cells (Gu et al, 1998). We also identified a cluster of microvillous cells (MV2) based on their expression of Ascl3 and Cftr (Pfister et al, 2015). The identity of one small cluster of cells could not be readily classified due to the heterogeneous expression of a few identifying marker genes.…”