. 2023 Aug 3;186(16):3386-3399.e15.

doi: 10.1016/j.cell.2023.07.006.

PIEZO2 in somatosensory neurons controls gastrointestinal transit

Affiliations

¹ Department of Neuroscience, Dorris Neuroscience Center, Scripps Research, San Diego, CA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA.
² National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; NIH-Brown University Graduate Program in Neuroscience, Providence, RI, USA; National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, MD, USA.
³ National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
⁴ Division of Gastroenterology and Hepatology, Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, MN, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.
⁵ Department of Neuroscience, Baylor College of Medicine, Jan and Dan Duncan Neurological Research Institute, Houston, TX, USA.
⁶ National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, MD, USA. Electronic address: alexander.chesler@nih.gov.
⁷ Department of Neuroscience, Dorris Neuroscience Center, Scripps Research, San Diego, CA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA. Electronic address: ardem@scripps.edu.

PMID: 37541196
PMCID: PMC10501318
DOI: 10.1016/j.cell.2023.07.006

PIEZO2 in somatosensory neurons controls gastrointestinal transit

M Rocio Servin-Vences et al. Cell. 2023.

. 2023 Aug 3;186(16):3386-3399.e15.

doi: 10.1016/j.cell.2023.07.006.

Authors

Affiliations

¹ Department of Neuroscience, Dorris Neuroscience Center, Scripps Research, San Diego, CA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA.
² National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; NIH-Brown University Graduate Program in Neuroscience, Providence, RI, USA; National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, MD, USA.
³ National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA.
⁴ Division of Gastroenterology and Hepatology, Enteric Neuroscience Program (ENSP), Mayo Clinic, Rochester, MN, USA; Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN, USA.
⁵ Department of Neuroscience, Baylor College of Medicine, Jan and Dan Duncan Neurological Research Institute, Houston, TX, USA.
⁶ National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD, USA; National Center for Complementary and Integrative Health, National Institutes of Health, Bethesda, MD, USA. Electronic address: alexander.chesler@nih.gov.
⁷ Department of Neuroscience, Dorris Neuroscience Center, Scripps Research, San Diego, CA, USA; Howard Hughes Medical Institute, Chevy Chase, MD, USA. Electronic address: ardem@scripps.edu.

PMID: 37541196
PMCID: PMC10501318
DOI: 10.1016/j.cell.2023.07.006

Abstract

The gastrointestinal tract is in a state of constant motion. These movements are tightly regulated by the presence of food and help digestion by mechanically breaking down and propelling gut content. Mechanical sensing in the gut is thought to be essential for regulating motility; however, the identity of the neuronal populations, the molecules involved, and the functional consequences of this sensation are unknown. Here, we show that humans lacking PIEZO2 exhibit impaired bowel sensation and motility. Piezo2 in mouse dorsal root, but not nodose ganglia is required to sense gut content, and this activity slows down food transit rates in the stomach, small intestine, and colon. Indeed, Piezo2 is directly required to detect colon distension in vivo. Our study unveils the mechanosensory mechanisms that regulate the transit of luminal contents throughout the gut, which is a critical process to ensure proper digestion, nutrient absorption, and waste removal.

Keywords: PIEZO2 deficiency; Piezo2; dorsal root ganglia; gastrointestinal tract; gut motility; gut transit; interoception; mechanosensation; sensory neurons; spinal innervation.

PubMed Disclaimer

Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

**Figure 1.. Gastrointestinal dysfunction in individuals deficient in *PIEZO2*.**
Summary of responses obtained from *PIEZO2*-deficient individuals to GI-PROMIS questionnaires. Data indicates the subject identifier, age at which the questionnaires were answered and gender. Data is organized by ascending age (top set of rows) and symptoms are categorized in sensory deficits and GI problems, which span constipation and diarrhea. Each question assessed symptoms from the seven days prior to the survey. Unless otherwise noted, the color code indicates the following: grey represents the average response from 1,177 healthy control participants, which indicates no pathology and is typically close to never experience or lacking the particular symptom in the past 7 days; blue: rarely; yellow: sometimes; orange: often, red: always. Therefore, every color except for gray indicates a deviation from the average. Blank indicates unanswered questions. Individual identifier corresponds to those published in our previous urinary function study . The symbol “*” denotes those subjects who experienced neonatal and childhood constipation, ”+“ indicates lack of medical history concerning their GI behaviors in childhood. NA: not answered, No path: no pathology.

**Figure 2.. Piezo2 in sensory neurons is required for gastrointestinal function in mice.**
A) Illustration of the *SNS^Cre;Piezo2*-targeting coverage of extrinsic neurons that innervate the GI tract. Blue designates *Piezo2* deletion in nodose (NG) and DRG neurons, but not ENS. B) Total GI transit time measured after gavaging carmine red dye into *SNS*^Cre+/−*;Piezo2^fl/fl* (WT; n=14) and *SNS^Cre+/−;Piezo2^fl/fl* (KO; n=10) mice (unpaired two-tailed t-test: ****P<0.0001, t(22)=9.301). C) Number of stools expelled per mouse during 1 hour of collection from *SNS^Cre−/−;Piezo2^fl/fl* (WT; n=13 mice) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO; n=13 mice) (unpaired two-tailed t-test: **P=0.0076, t(24)=2.916). D) Water content present in the stool samples from panel (C) as a percent of the total composition (unpaired two-tailed t-test: ****P<0.0001, t(24)=7.418). E) All the stool samples collected in panel (C) were dried, individually weighted and averaged per mouse (Mann-Whitney test: ****P<0.0001 two-tailed, U=16). F) Quantification of individual stool width and length (G) from fresh samples collected during one hour from *SNS^Cre*^−/−*;Piezo2^fl/fl* (WT; N=3 mice, n=19 stools) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO; N=3 mice, n=37 stools) mice. Unpaired two-tailed t-test: ****P<0.0001, t(54)=7.037) and **P=0.0021, t(54)=3.236). H) Representative images of dried stools collected during one hour from *SNS^Cre*^−/−*;Piezo2^fl/fl* (WT) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO) mice. Scale bar indicates 1 cm. I) Total GI transit time measured after gavaging carmine red dye in mice fasted for 12 hours or with *ad libitum* food access. *SNS^Cre*^−/−*;Piezo2^fl/fl* (WT; n=10) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO; n=8) mice (two-way ANOVA: ****P_genotype=0.0009, F(1,16)=16.32: Sidak’s P_adjusted: P_Fasted=0.7697; ****P_Fed<0.0001).

**Figure 3.. Piezo2 in DRG neurons is required for gastrointestinal transit in mice.**
A) Illustration of the *Phox2b^Cre;Piezo2* targeting coverage in neurons innervating the GI tract, green designates *Piezo2* deletion in nodose, but not in DRG and enteric neurons (left panel). Total GI transit time after gavaging carmine red into *Phox2b*^Cre^−/−;*Piezo2^fl/fl* (WT; n=18) and *Phox2b^Cre*^+/−*;Piezo2^fl/fl* (KO; n=12) mice (unpaired two-tailed t-test: P=0.8735, t(28)=0.1607; not statistically significant) (middle left panel). Number of stools expelled during one hour of collection from *Phox2b^Cre*^−/−;*Piezo2^fl/fl* (WT; n=15) and *Phox2b^Cre*^+/−;*Piezo2^fl/fl* (KO; n=13) mice (unpaired two-tailed t-test: P=0.9548, t(26)=0.05727; not statistically significant) (middle right panel). Representative images of dried stools collected during one hour from *Phox2b^Cre*^−/−;*Piezo2^fl/fl* (WT) and *Phox2b^Cre*^+/−;*Piezo2^fl/fl* (KO) mice (right panel). Scale bar represents 1 cm. B) Illustration of the *Hoxb8^Cre;Piezo2* targeting coverage in the GI epithelium and neurons innervating the GI tract, teal color designates *Piezo2* deletion in DRG neurons and enterochromaffin cells of intestinal epithelia, but not in enteric and nodose neurons (left panel). Total GI transit time after gavaging carmine red into *Hoxb8^Cre*^−/−*;Piezo2^fl/fl* (WT; n=21) and *Hoxb8^Cre*^+/−*;Piezo2^fl/fl* (KO; n=15) mice (unpaired two-tailed t-test: ***P=0.0003, t(34)=4.004) (middle left panel). Number of stools expelled during one hour of collection from *Hoxb8^Cre*^−/−*;Piezo2^fl/fl* (WT; n=26) and *Hoxb8^Cre*^+/−*;Piezo2^fl/fl* (KO; n=14) mice (unpaired two-tailed t-test: ***P=0.0001, t(38)=4.316) (middle right panel). Representative images of dried stools collected during one hour from *Hoxb8^Cre*^−/−*;Piezo2^fl/fl* (WT) and *Hoxb8-^Cre*^+/−*;Piezo2^fl/fl* (KO) mice (right panel). Scale bar represents 1 cm. C) Total GI transit time after carmine red gavage into *Vil1^Cre*^−/−*;Piezo2^fl/fl* (WT; n = 14) and Vil1Cre^+/−;Piezo2fl/fl (KO; n = 9) mice (unpaired two-tailed t test: p = 0.1980, t(21) = 1.329; ns, not statistically significant) (middle left). Number of stools expelled during 1 h of collection from Vil1Cre^−/−;Piezo2fl/fl (WT; n = 17) and *Vil1^Cre*^+/−*;Piezo2^fl/fl* (KO; n = 13) mice (unpaired two-tailed t test: p = 0.1622, t(28) = 1.436; ns, not statistically significant) (middle right). Representative images of dried stools collected during 1 h from *Vil1^Cre*^−/−;Piezo2^fl/fl (WT) and *Vil1^Cre*^+/−;Piezo2^fl/fl (KO) mice (right). Scale bar represents 1 cm. D) *Piezo2^fl/fl*::PHP.s-*tdTomato* (Control; n = 10) and *Piezo2^fl/fl*::PHP.s-*iCre* (Cre; n = 10) mice (Mann-Whitney test: ***p = 0.0005 two-tailed, U = 7) (middle left). Number of stools expelled during 1 h of collection from *Piezo2^fl/fl*::PHP.s-*tdTomato* (Control; n = 7) and *Piezo2^fl/fl*::PHP.s-*iCre* (Cre; n = 11) mice (Mann-Whitney test: *p = 0.0208 two-tailed, U = 13.5) (middle right). Representative images of dried stools collected during 1 h from *Piezo2^fl/fl*::PHP.s-*tdTomato* (Control) and *Piezo2^fl/fl*::PHP.s-*iCre* (Cre) mice (right). Scale bar represents 1 cm.

**Figure 4.. Neuronal Piezo2 mediates gastric emptying, intestinal and colonic transit in mice.**
A) Illustration of the strategy to test gastric emptying in *Piezo2*^SNS mice. B) Quantification of the percentage of gastric emptying observed after gavaging the far-red dye GastroSense-750 at different time points in *SNS*^Cre^−/−*;Piezo2^fl/fl* (*Piezo2^WT*; n = 3-4 mice per time point) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (*Piezo2^SNS*; n = 4 per time point) mice (two-way ANOVA: ***p_genotype = 0.0005, F(1,17) = 18.40; Sidak’s p_adjusted: *p_{30 min} = 0.0122; **p_{45 min} = 0.0022; p_{90 min} = 0.9970) (left). Representative images of dye release from stomachs (right panel) 45 min after gavaging *SNS^Cre*^−/−*;Piezo2^fl/fl* (WT) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO) mice. The stomach is outlined by a white dashed line, scale bar represents 5 mm and pseudocolor scale indicates the dye intensity. C) Schematic of the duodenal infusion in *Piezo2^SNS* mice through an implanted catheter. D) Quantification of intestinal transit time measured after infusing carmine red into the duodenum of *SNS^Cre*^−/−*;Piezo2^fl/fl* (WT; n = 8) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO; n = 6) (Mann-Whitney test: **P=0.0051 two-tailed, U=1). E) Schematic of the colonic infusion in *Piezo2*^SNS mice through an implanted catheter. F) Quantification of colonic transit time measured after infusing carmine red into the cecum of of *SNS^Cre*^−/−*;Piezo2^fl/fl* (WT; n=10) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO; n=8) (Mann-Whitney test: **P=0.001 two-tailed, U=9). G) Schematic of the celiac ganglia denervation (CGX) in *Piezo2*^SNS mice. S-Ch: sympathetic chain, CSC: celiac superior complex. H) Quantification of GI transit time measured before and after CGX in *SNS*^Cre^−/−*;Piezo2^fl/fl* (WT; n=7) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO; n=10) (two-way ANOVA: ****P_genotype<0.0001, F(1,15)=46.80; Sidak’s P_adjusted: ****P_Piezo2-WT<0.0001).

**Figure 5.. Piezo2 dorsal-root-ganglion neurons innervate the gastrointestinal tract.**
A) Illustration of the strategy to assess DRG neuronal innervation by intrathecally injecting AAV9-*flex-GFP* particles into *Piezo2*^Cre^+/+ mice. B) Quantification of the IGVE density, defined as the number of enteric ganglia innervated by IGVE in the total area across the whole GI tract. C) Quantification of total innervation density, defined as innervated nerve area by the total area across the GI tract. D) Representative images of stomach, small intestine, and colon. The enteric neuron nuclei were labeled with HuD/HuC antibody and represented in red. Piezo2-positive nerve endings are shown in cyan. Scale bar values are shown in each picture.

**Figure 6.. Piezo2 -expressing DRG neurons detect colon distention.**
A) Illustration of the Cre line used for the glass bead expulsion test. **B-E**) Measurement of expulsion time in *SNS^Cre*^−/−*;Piezo2^fl/fl* (WT) and *SNS^Cre*^+/−*;Piezo2^fl/fl* (KO) mice following the insertion of a glass bead into their colons. Representative pictures of used beads are shown above each plot. B) 1 mm bead (unpaired two-tailed t-test: P=0.2592. t(20)=1.161; ns, not statistically significant). C) 2 mm bead (Mann-Whitney test: P=0.9900 two-tailed, U=84.5; not statistically significant). D) 3 mm bead (unpaired two-tailed t-test: *P=0.0196, t(24)=2.500). E) 4 mm bead (unpaired two-tailed t-test: ****P<0.0001, t(22)=5.910). F) Illustration of *in vivo* calcium imaging recording in anesthetized mice focused on Sacral DRGs. G) Comparison of calcium responses of DRG neurons obtained after stimulating Control (*Hoxb8^Cre*^+/−*;GCaMP6f*^+/+, n=594 cells ; N=6 mice) and *Piezo2^cKO* (*Hoxb8*^Cre^+/−*;Piezo2^fl/fl;GcaMP6f*^+/+, n=376 cells; N=6 mice) mice (Chi-square test: ****P<0.0001, df=501.5, 2). The responses are classified in three categories: **Internal**: corresponds to the colonic stimulation with the soft brush and balloon; **External noxious**: response to only anal skin pinch; **External gentle**: cells that responded to air puff and/or brush on the surface of the anal skin, if they additionally responded to pinch, they were included in this category. The insets represent the numbers of recorded cells per category. H) Heatmap showing calcium responses (as ΔF/F) recorded from Control (*Hoxb8*^Cre^+/−*;GCaMP6f*^+/+) DRG neurons. Neurons were functionally classified based on their response to stimuli and sorted by ΔF/F. External (air puff, brush, and pinch) and internal (brush insertion and extraction, balloon insertion and inflation) stimulations are shown on top of heatmap. I) Calcium representative traces from individual neurons are shown and color coded for the categories showed on (G), blue LTMRs, purple HTMRs and teal for gut responding neurons. J) Heatmap showing Calcium responses recorded from *Piezo2^cKO* (*Hoxb8^Cre*^+/−*;Piezo2^fl/fl;GCaMP6f*^+/+) DRG neurons. External and internal stimulations are shown. K) Calcium representative traces from individual neurons are shown and color coded as in the categories showed on panel (G).

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Comment in

Interoception: Spinal sensory neurons that innervate the intestines.
Ly T, Knight ZA. Ly T, et al. Curr Biol. 2023 Sep 25;33(18):R945-R947. doi: 10.1016/j.cub.2023.07.066. Curr Biol. 2023. PMID: 37751704

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PIEZO2 in somatosensory neurons controls gastrointestinal transit

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PIEZO2 in somatosensory neurons controls gastrointestinal transit

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Conflict of interest statement

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