CFTR HIGH EXPRESSER CELLS EXPRESS NEUROPOD GENES AND RESPOND TO ENVIRONMENTAL CUES IN THE INTESTINE

CFTR High Expresser cells (CHEs) are a rare (1-2%) enterocyte subtype in rat and human proximal small intestine that express very high levels of CFTR. CHEs traffic CFTR from subapical endosomes into the brush border following second messenger agonists, supporting its role in cystic fibrosis (CF) and diarrheal diseases. Published single cell (sc) RNA-seq data in human intestine and preliminary data in rat jejunum identified CHE-specific genes for transcription factors, ion channels, acid sensors, bicarbonate secretion, trafficking, and neuropod cell function. Neuropod cells are a subtype of enteroendocrine cells that express pre- and post-synaptic proteins, engaging in bidirectional signal transduction with the enteric nervous system in response to luminal conditions. Unpublished data from our laboratory also confirmed that CHEs are selectively innervated by axons expressing choline acetyltransferase (ChAT), critical for synthesizing acetylcholine. Validating that CHEs are neuropod cells would provide a novel avenue through which they sense and respond to their environment. This study used normal rat intestine to (1) examine neuropod proteins enriched in CHEs and (2) elucidate changes in CHE-specific proteins and function in disease. scRNA-seq of normal rat jejunum was examined for CHE-specific neuropod genes that could regulate CHE cell fate and function. CHE-specific proteins were visualized in rat jejunum cryosections through triple-labeling immunofluorescence (IF) of bestrophin 4 (BEST4), a calcium-activated Cl^-/HCO3 channel distinctly enriched in CHEs, synaptotagmin 3 (SYT3), a neurotransmitter receptor that mediates postsynaptic transmission by calcium signaling, and beta tubulin 3 (TUBB3), a critical microtubule component in neurons. Additional neuropod proteins were localized through double-labeling IF of BEST4 with beta tubulin 2b (TUBB2B), a key microtubule component in vesicle trafficking, and S100A6, a calcium-binding protein involved in cytoskeletal dynamics and vesicular transport, with Meis homeobox 1 (MEIS1), a CHE-specific transcription factor. Double-labeling IF of MEIS1 and the proton channel otopetrin 2 (OTOP2) was performed in jejunum cryosections following luminal exposure of rats to normal or low pH consistent with CF. BEST4+ CHEs were identified with basal processes in close proximity to neurons expressing SYT3 and TUBB3 characteristic of neuropod cells. CHEs were further enriched for TUBB2B and S100A6 in the apical domain. scRNA-seq data revealed CHE-specific enrichment of the acid sensing receptor GPR4. Furthermore, OTOP2 was highly upregulated in the apical domain of CHEs in low luminal pH conditions consistent with CF. Our findings reveal CHEs to be definitive neuropod cells that sense and respond to their environment, especially to luminal pH, supporting a critical role in CF intestinal disease pathogenesis.