Beyder, Arthur

The intestinal epithelium is exposed to diverse combinations of microbiota-derived compounds; however, the mechanisms by which the host integrates these signals remain poorly defined. Studying two highly abundant microbial metabolites, we identified the purine metabolite hypoxanthine as an effector metabolite that directly drives signaling and the short-chain fatty acid butyrate as a regulatory metabolite that conditions host responsiveness. Specifically, hypoxanthine activates the adenosine A1 receptor–TRPC4 axis in enterochromaffin (EC) cells, triggering calcium influx and serotonin release resulting in accelerated gastrointestinal transit locally and increased platelet activation systemically. In contrast, butyrate epigenetically upregulates specific G protein–coupled receptors and ion channels to enhance response to hypoxanthine and the neurotransmitters norepinephrine and dopamine. These findings define a cooperative signaling framework and highlight the role of EC cells as a distinct epithelial signaling hub that senses and integrates microbial metabolite signals to drive physiological responses. Our findings provide a mechanistic foundation for therapeutic strategies that leverage combinatorial microbial signaling.

ABSTRACT The enteric nervous system (ENS) is a complex peripheral neural network that coordinates gastrointestinal motility through highly organized synaptic communication. Although tissue-resident muscularis macrophages (MMs) closely associate with enteric neurons, whether they regulate enteric synaptic organization remains unknown. In the central nervous system (CNS), microglia sculpt neural circuits through complement-dependent synaptic remodeling, raising the possibility that analogous neuroimmune mechanisms operate in the gut. Here, we identify a previously unrecognized role for C1qa□ MMs in regulating enteric synaptic homeostasis and gastrointestinal motility. Using macrophage-specific constitutive and inducible C1qa deletion models, single-cell RNA sequencing, enteric synaptosome proteomics, physiology, and advanced imaging, we demonstrate that loss of MMs-derived C1qa increases enteric synaptic density without altering neuronal numbers. C1qa deficiency induced broad transcriptional changes in enteric neurons and macrophages, including altered synapse-associated, lysosomal, and endocytic programs. Proteomic analysis revealed that enteric synapses share a conserved molecular architecture with brain synapses while exhibiting distinct gastrointestinal-specific complement-associated synaptic networks enriched for structural and receptor-localization pathways. Functionally, macrophage-specific C1qa deletion altered excitatory and inhibitory enteric neurotransmission, enhanced cholinergic signaling, reduced nitrergic responses, and accelerated gastrointestinal transit, while smooth muscle responsiveness remained preserved. C1qa□ MMs displayed transcriptional and functional features consistent with a phagocytic synapse-remodeling phenotype, including enrichment of complement, lysosomal, and engulfment pathways. Loss of C1qa impaired macrophage phagocytic activity both in vitro and in vivo and was associated with synapse accumulation and altered macrophage morphology. Importantly, inducible deletion of C1qa in adulthood recapitulated the synaptic and motility phenotypes, demonstrating that C1qa□ MMs continuously regulate enteric synaptic organization beyond development. Together, these findings identify a complement-dependent neuroimmune mechanism that regulates enteric circuit organization and gut motility, establishing MMs as active modulators of adult ENS synaptic homeostasis. Abstract Figure

SUMMARY Aging is accompanied by profound changes in bladder function, leading to increased urinary frequency and incontinence that impair quality of life in humans and are recapitulated in mouse models. Bladder filling and emptying rely on precise mechanosensory feedback, yet how aging alters this sensory control remains unclear. PIEZO ion channels convert mechanical forces into cellular signals essential for bladder fullness sensation. Using inducible smooth-muscle specific Piezo1 deletion, we find that loss of Piezo1 attenuates aging-related bladder dysfunction. Dietary enrichment with margaric acid, a membrane-active fatty acid previously shown to inhibit PIEZO channels, reduced urinary dysfunction in aged mice. In humans, genotype–phenotype analyses reveal an association between a PIEZO1 gain-of-function variant and early-onset neuromuscular bladder dysfunction. Together, these findings define a smooth-muscle, PIEZO1-mediated mechanosensory basis for aging-related bladder dysfunction and introduce a non-invasive strategy for targeting PIEZO channels in vivo .

ABSTRACT Background The circadian rhythm regulates gastrointestinal motility. In humans and preclinical models, such as rodents, whole gut transit (WGT) is slower during the rest phase compared to the active phase. Investigators typically study GI transit in rodents during the day, which is their rest phase, rather than during the night, which is their active phase. A circadian rhythm reversal in which mice are in a dark room during the working day (reverse light) allows studies on nocturnal animals during their active phase and has been previously shown to reduce WGT time. GI motility is often disrupted in individuals with disorders of gut–brain interaction (DGBI), which are female predominant. However, the effect of circadian rhythm on regional transit and sex dependence of the differences is not known, as most motility studies looking at circadian rhythm reversal are done in male mice. Methods We tested C57BL/6 wild‐type male and female mice in rest (12 h of light during the day) and active (reverse cycle for 2 weeks: 12 h of dark during the day) phases. We noted female estrous cycle by visual inspection. We performed carmine WGT by monitoring time‐lapse videos of pellet production. We performed fluorescence imaging of excised intestines 30 min after gavage to assess percent fluorescence for each GI region and then examined small intestinal transit (SIT) by measuring geometric center and leading edge. For colonic transit, we monitored bead expulsion time from distal colon to anus. Key Results Compared to rest phase, in the active phase, like male mice, female mice had (1) faster WGT, (2) increased frequency of pellet expulsion in the first 3 h of transit, (3) and greater total pellet production. Both male and female mice in their active phase exhibited (4) more contrast emptied from the stomach and they had (5) further leading edge of fluorescence and (6) geometric center, in SIT, and (7) faster colonic bead expulsion times. There were no significant sex differences in the active phase of WGT. In SIT, male mice had further leading edge in the rest phase than female mice, but this difference was not seen in the active phase, and in colonic transit, male mice in both the active and rest phases had faster bead expulsion than female mice. Conclusions Mice in the active phase have faster regional transit in small and large bowel than mice in the rest phase that collectively contributes to faster WGT times in the active phase of both male and female mice. These findings highlight the importance of circadian biology in sex‐dependent rodent GI transit.

The gastrointestinal (GI) tract plays a crucial role in nutrient absorption, motility, and metabolism, while serving as a barrier against pathogens. The intestinal epithelial layer, particularly enteroendocrine cells (EEC), is pivotal in sensing luminal stimuli due to its strategic position. Although comprising only 1% of the GI epithelium, in total EEC comprises perhaps the largest endocrine organ that couples GI luminal stimuli with release of hormones and neurotransmitters such as serotonin, glucagon-like peptide-1 (GLP-1), and peptide–tyrosine–tyrosine (PYY), which regulate local and systemic physiology. In this review, we use EECs as a model to explore critical intracellular signaling pathways—centering around calcium (Ca²⁺) and cyclic adenosine monophosphate (cAMP). EECs utilize ionotropic and metabotropic receptors to detect luminal stimuli, leading to increased intracellular cytoplasmic Ca²⁺ and altered intracellular cAMP dynamics. Ca²⁺ influx is critical for the exocytosis of signaling molecules. The interplay between Ca²⁺ and cAMP signaling pathways enhances the release of these molecules, affecting both local and systemic responses. We make a point to discuss the conventional and emerging methodologies for measuring Ca²⁺ and cAMP dynamics in EECs, which is a rapidly expanding toolbox, containing genetically encoded indicators and resonance energy transfer techniques. Understanding these signaling cascades is essential for deciphering the complex roles of EECs in GI physiology and their impact on systemic health.

Key Points PIEZO1 activation reduces cystogenesis: Yoda1 activates PIEZO1, raising calcium and lowering cAMP, reducing cyst growth in autosomal dominant polycystic kidney disease models. Context-dependent role of PIEZO1: PIEZO1 knockout in mice with or without polycystic kidneys does not affect cyst formation, suggesting redundancy in mechanosensitive pathways. Therapeutic potential: Findings support PIEZO1 activation as a part of combination therapy to slow cyst growth in autosomal dominant polycystic kidney disease, needing more in vivo studies. Background The disruption of calcium signaling associated with polycystin deficiency is a key factor in abnormal epithelial growth in autosomal dominant polycystic kidney disease. Calcium homeostasis can be influenced by mechanotransduction. The mechanosensitive cation channel PIEZO1 has been implicated in sensing intrarenal pressure and regulating urinary osmoregulation, but its role in kidney cystogenesis is unclear. Methods We hypothesized that altered mechanotransduction contributes to cystogenesis in autosomal dominant polycystic kidney disease and that activation of mechanosensitive cation channels could be a therapeutic strategy. Results We demonstrate that Yoda1, a PIEZO1 activator, increases intracellular calcium and reduces forskolin-induced cAMP levels in mouse inner medullary collecting duct (mIMCD3) cells. Notably, knockout of polycystin-2 attenuated the efficacy of Yoda1 in reducing cAMP levels in mIMCD3 cells. Yoda1 also reduced forskolin-induced mIMCD3 cyst surface area in vitro and cystic index in mouse metanephros ex vivo in a dose-dependent manner. However, collecting duct–specific PIEZO1 knockout neither induced cystogenesis in wild-type mice nor altered cystogenesis in the Pkd1 RC/RC mouse model. Conclusions These findings support the potential role of PIEZO1 agonists in mitigating cystogenesis by increasing intracellular calcium and reducing cAMP levels, but the unaltered in vivo cystic phenotype after PIEZO1 knockout in the collecting duct suggests possible redundancy in mechanotransductive pathways.

Piezo2 is a mechanically gated ion channel most commonly expressed by specialized mechanoreceptors, such as the enteroendocrine cells (EECs) of the gastrointestinal epithelium. A subpopulation of EECs expresses Piezo2 and functionally resembles the skin’s touch sensors, called Merkel cells. Low-magnitude mechanical stimuli delivered to the mucosal layer are primarily sensed by mechanosensitive EECs in a process we term “gut touch.” Piezo2 transduces cellular forces into ionic currents, a process that is sensitive to bilayer tension and cytoskeletal depolymerization. E-cadherin is a widely expressed protein that mediates cell–cell adhesion in epithelia and interacts with scaffold proteins that anchor it to actin fibers. E-cadherin was shown to interact with Piezo2 in immortalized cell models. We hypothesized that the Piezo2–E-cadherin interaction is important for EEC mechanosensitivity. To test this, we used super-resolution imaging, co-immunoprecipitation, and functional assays in primary tissues from mice and gut organoids. In tissue EECs and intestinal organoids, we observed multiple Piezo2 cellular pools, including one that overlaps with actin and E-cadherin at the cells’ lateral walls. Further, E-cadherin co-immunoprecipitated with Piezo2 in the primary colonic epithelium. We found that E-cadherin knockdown decreases mechanosensitive calcium responses in mechanically stimulated primary EECs. In all, our results demonstrate that Piezo2 localizes to the lateral wall of EECs, where it physically interacts with E-cadherin and actin. They suggest that the Piezo2–E-cadherin–actin interaction is important for mechanosensitivity in the gut epithelium and possibly in tissues where E-cadherin and Piezo2 are co-expressed in epithelial mechanoreceptors, such as skin, lung, and bladder.

AbstractBackgroundIn preclinical studies whole gut transit (WGT) in mice is a gold‐standard “leading‐edge” approach that measures the time between orogastric gavage of carmine red and defecation of the first carmine red pellet. Transit studies in humans are performed during the active day because GI motility and transit are suppressed during the night. Since mice are nocturnal, WGT studies traditionally done during the day occur during their rest phase. How circadian rhythm affects WGT in mice is not known.MethodsWe used an automated approach for high temporal resolution uninterrupted testing of mouse WGT and activity. We housed wild‐type Bl6/C57 mice under the standard 12 h light–dark cycles. At 8 weeks, we performed carmine red orogastric gavage and assessed WGT during Light (rest) conditions. Then, we exposed mice to a reverse 12 h light–dark cycle for 2 weeks and tested them in the Dark (active) under red light conditions. Timelapse videos were analyzed to quantify activity and to timestamp all pellets, and multiple parameters were analyzed.Key ResultWhen complementary light cycle reversal experiments were performed, we found a significant increase in mouse activity when mice were tested during their Dark (active) phase, compared to their Light (rest) phase. In mice tested in the Active phase compared to the Rest phase, we found a significant acceleration in WGT, increased rate and total number of pellets produced, and more pellet clustering. These data show that the mice tested in the Active phase have important differences in activity that correlate with multiple alterations in gastrointestinal transit.Conclusion & InferencesDuring the Active phase mice have faster WGT, produce more pellets, and cluster their output compared to testing in the Rest phase. Like in humans, circadian rhythm is an important consideration for transit studies in mice, and a simple reverse light cycle approach facilitates further studies on the role of circadian rhythm in GI motility.

TMEM63A has biophysical features distinctive for mechano-gated cation channels, but some properties are less clear, including no functional information in whole cells. We report that pressure-dependent yet voltage-independent TMEM63A currents in cell membrane patches correlated with cell size. In addition, radial stretch of whole cells on flexible membranes increased the [Ca2+]i responses more in TMEM63A-transfected cells. Inward TMEM63A currents in response to high stretch can depolarize plasma membranes and contribute to a sustained [Ca2+]i increase.

Abstract Enteroendocrine cells (EECs) are specialized sensors of luminal forces and chemicals in the gastrointestinal (GI) epithelium that respond to stimulation with a release of signalling molecules such as serotonin (5‐HT). For mechanosensitive EECs, force activates Piezo2 channels, which generate a very rapidly activating and inactivating (∼10 ms) cationic (Na + , K + , Ca 2+ ) receptor current. Piezo2 receptor currents lead to a large and persistent increase in intracellular calcium (Ca 2+ ) that lasts many seconds to sometimes minutes, suggesting signal amplification. However, intracellular calcium dynamics in EEC mechanotransduction remain poorly understood. The aim of this study was to determine the role of Ca 2+ stores in EEC mechanotransduction. Mechanical stimulation of a human EEC cell model (QGP‐1) resulted in a rapid increase in cytoplasmic Ca 2+ and a slower decrease in ER stores Ca 2+ , suggesting the involvement of intracellular Ca 2+ stores. Comparing murine primary colonic EECs with colonocytes showed expression of intercellular Ca 2+ store receptors, a similar expression of IP 3 receptors, but a >30‐fold enriched expression of Ryr3 in EECs. In mechanically stimulated primary EECs, Ca 2+ responses decreased dramatically by emptying stores and pharmacologically blocking IP 3 and RyR1/3 receptors. RyR3 genetic knockdown by siRNA led to a significant decrease in mechanosensitive Ca 2+ responses and 5‐HT release. In tissue, pressure‐induced increase in the Ussing short circuit current was significantly decreased by ryanodine receptor blockade. Our data show that mechanosensitive EECs use intracellular Ca 2+ stores to amplify mechanically induced Ca 2+ entry, with RyR3 receptors selectively expressed in EECs and involved in Ca 2+ signalling, 5‐HT release and epithelial secretion. image Key points A population of enteroendocrine cells (EECs) are specialized mechanosensors of the gastrointestinal (GI) epithelium that respond to mechanical stimulation with the release of important signalling molecules such as serotonin. Mechanical activation of these EECs leads to an increase in intracellular calcium (Ca 2+ ) with a longer duration than the stimulus, suggesting intracellular Ca 2+ signal amplification. In this study, we profiled the expression of intracellular Ca 2+ store receptors and found an enriched expression of the intracellular Ca 2+ receptor Ryr3, which contributed to the mechanically evoked increases in intracellular calcium, 5‐HT release and epithelial secretion. Our data suggest that mechanosensitive EECs rely on intracellular Ca 2+ stores and are selective in their use of Ryr3 for amplification of intracellular Ca 2+ . This work advances our understanding of EEC mechanotransduction and may provide novel diagnostic and therapeutic targets for GI motility disorders.