Visceral pain (VP) is a global problem with complex etiologies and limited therapeutic options.Guanylyl cyclase C (GUCY2C), an intestinal receptor producing cyclic GMP which regulates luminal fluid secretion, has emerged as a therapeutic target for VP. Indeed, FDA-approved GUCY2C agonists ameliorate VP in patients with chronic constipation syndromes, although analgesic mechanisms remain obscure. Here, we reveal that intestinal GUCY2C is selectively enriched in neuropod cells, a type of enteroendocrine cell that synapses with submucosal neurons in mice and humans. GUCY2C High neuropod cells associate with co-cultured dorsal root ganglia neurons and induce hyperexcitability, reducing the rheobase and increasing the resulting number of evoked action potentials. Conversely, the GUCY2C agonist linaclotide eliminated neuronal hyperexcitability produced by GUCY2C-sufficient, but not GUCY2C-deficient, neuropod cells, an effect independent of bulk epithelial cells or extracellular cGMP. Genetic elimination of intestinal GUCY2C amplified nociceptive signaling and VP that was comparable to chemicallyinduced VP but refractory to linaclotide. Importantly, eliminating GUCY2C selectively in neuropod cells also increased nociceptive signaling and VP that was refractory to linaclotide. In the context of loss of GUCY2C hormones in patients with VP, these observations suggest a specific role for neuropod GUCY2C signaling in the pathophysiology and treatment of these pain syndromes.

Most sporadic colorectal cancer reflects acquired mutations in the adenomatous polyposis coli (APC) tumor suppressor gene, while germline heterozygosity for mutant APC produces the autosomal dominant disorder Familial Adenomatous Polyposis (FAP) with a predisposition to colorectal cancer. In these syndromes, loss of heterozygosity (LOH) silences the remaining normal allele of APC, through an unknown mechanism, as the initiating step in transformation. Guanylyl cyclase C receptor (GUCY2C) and its hormones, uroguanylin and guanylin, have emerged as a key signaling axis opposing mutations driving intestinal tumorigenesis. Indeed, uroguanylin and guanylin are among the most commonly repressed genes in colorectal cancer. Here, we explored the role of APC heterozygosity in mechanisms repressing hormone expression which could contribute to LOH. In genetic mouse models of APC loss, uroguanylin and guanylin expression were quantified following monoallelic or biallelic deletion of the Apc gene. Induced biallelic loss of APC repressed uroguanylin and guanylin expression. However, monoallelic APC loss in Apc min/+ mice did not alter hormone expression. Similarly, in FAP patients, normal colonic mucosa (monoallelic APC loss) expressed guanylin while adenomas and an invasive carcinoma (biallelic APC loss) were devoid of hormone expression. Thus, uroguanylin and guanylin expression by normal intestinal epithelial cells persists in the context of APC heterozygosity and is lost only after tumor initiation by APC LOH. These observations reveal a role for loss of the hormones silencing the GUCY2C axis in tumor progression following biallelic APC loss, but not in mechanisms creating the genetic vulnerability in epithelial cells underlying APC LOH initiating tumorigenesis.

Silencing receptor GUCY3C tumor suppressor signaling occurs widely in colorectal tumors, reflecting loss of GUCY2C ligand expression. This study identifies a novel b-catenin/ TCF-sensitive locus control region that mediates ligand loss and can be targeted for gene reactivation with CRISPR activation.

Background: Monogenic protein aggregation diseases, in addition to cell selectivity, exhibit clinical variation in the age of onset and progression, driven in part by inter-individual genetic variation. While natural genetic variants may pinpoint plastic networks amenable to intervention, the mechanisms by which they impact individual susceptibility to proteotoxicity are still largely unknown. Results: We have previously shown that natural variation modifies polyglutamine (polyQ) aggregation phenotypes in C. elegans muscle cells. Here, we find that a genomic locus from C. elegans wild isolate DR1350 causes two genetically separable aggregation phenotypes, without changing the basal activity of muscle proteostasis pathways known to affect polyQ aggregation. We find that the increased aggregation phenotype was due to regulatory variants in the gene encoding a conserved autophagy protein ATG-5. The atg-5 gene itself conferred dosagedependent enhancement of aggregation, with the DR1350-derived allele behaving as hypermorph. Surprisingly, increased aggregation in animals carrying the modifier locus was accompanied by enhanced autophagy activation in response to activating treatment. Because autophagy is expected to clear, not increase, protein aggregates, we activated autophagy in three different polyQ models and found a striking tissue-dependent effect: activation of autophagy decreased polyQ aggregation in neurons and intestine, but increased it in the muscle cells. Conclusions: Our data show that cryptic natural variants in genes encoding proteostasis components, although not causing detectable phenotypes in wild-type individuals, can have profound effects on aggregation-prone proteins. Clinical applications of autophagy activators for aggregation diseases may need to consider the unexpected divergent effects of autophagy in different cell types.