Regulation of GI Motility and Intestinal Barrier Function by LPA1 in the Enteric Nervous System Restricted; Files Only

Abstract

Lysophosphatidic acid (LPA) is known to influence diverse physiological processes through its receptor family (LPA1-6). Among these receptors, Lysophosphatidic Acid Receptor 1 (LPA1) is most expressed in the intestinal tract and plays a role in the regulation of gastrointestinal (GI) functions. The enteric nervous system (ENS), a complex neuronal network embedded within the intestinal wall, significantly influences GI motility, secretion, and barrier integrity, especially under stress conditions, which can exacerbate GI dysfunction. However, the precise mechanisms by which LPA1 affects GI homeostasis through the enteric nervous system (ENS) remain largely unclear. This study aimed to elucidate the regulatory functions of LPA1 within the ENS, focusing specifically on its influence on GI motility and intestinal epithelial barrier integrity.

Transgenic mice with targeted deletion of LPA1 in ENS-specific cells (Lpar1f/f; Wnt1-Cre2 and PLP1-Cre) were used in this study. Gastrointestinal motility assays revealed significantly impaired transit times in Lpar1^ΔENS mice, indicative of compromised intestinal motility. Western blot analysis accompanied with IF staining of the Na+/H+ exchanger NHE3, a critical protein for fluid absorption, showed increased brush border membrane localization, suggesting enhanced functional activity, despite unchanged total protein expression.

In addition, the intestinal epithelial barrier function is investigated in this study. Intestinal barrier prevents the entry of harmful substances from the gut lumen into underlying tissue, and its function is maintained by junctional proteins. Lpar1^ΔENS mouse exhibited increased intestinal paracellular permeability, correlating with significantly decreased expression of key tight junction proteins, including Claudin 4, Claudin 7, and adherens junction protein E-cadherin.

Conclusively, these results showed that LPA1 signaling within the ENS is essential for maintaining normal GI motility and intestinal epithelial barrier function. The findings could also have broader implications for treating various GI disorders characterized by barrier dysfunction and motility problems, such as inflammatory bowel disease and irritable bowel syndrome.

Table of Contents

INTRODUCTION 8

MATERIALS AND METHODS 11

1. Mouse 11

2. Motility Assay 12

2.1. GI Motility Assay 12

2.2 Colonic Motility Assay 12

3. Immunofluorescent Staining 12

3.1 Genotype Validation by Staining Myenteric Plexus 12

3.2 Staining for NHE3 and Junctional Proteins 13

4. Western Blot 14

5. Paracellular Permeability Test 14

6. Statistical Analysis 15

RESULTS 15

1. Validation of Mice Genotypes 15

2. Gastrointestinal and Colonic Transit Time 18

3. Expression and Activity of NHE3 20

4. Assessment of in vivo Epithelial Paracellular Permeability 23

5. Expression Analysis of Junctional Proteins 25

DISCUSSION 28

CONCLUSION 29

REFERENCES 31

About this Honors Thesis

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School	Emory College
Department	Biology
Degree	B.S.
Submission	Honors Thesis
Language	English
Research Field	Biology, Physiology Biology, Molecular Biology, Cell
Keyword	LPA1 Gastrointestinal motility Intestinal Epithelial Barrier Gastroenterology Enteric Nervous System NHE3 Lysophosphatidic acid
Committee Chair / Thesis Advisor	Yun, Chris, Emory University
Committee Members	Thakur, Jitendra, Emory University Srinivasan, Shanthi, Emory University

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