PROJECT SUMMARY. Down syndrome (DS), caused by Trisomy 21 (T21), occurs in ~1 in 700 live births, making the most commonly occurring chromosomal abnormality. Individuals with DS experience a unique disease spectrum, whereby they are protected from some conditions, including solid tumors, and predisposed to others, such as Alzheimer’s disease and autoimmunity. Among the conditions more common in people with DS are gastrointestinal (GI) abnormalities, including esophageal motility disorders, gastro-esophageal reflux, irritable bowel syndrome, small bowel motility disorders, colonic dysmotility, slow transit constipation, and others. However, the molecular mechanisms underlying these conditions remain unclear, creating challenges for their clinical management. Recent work has established that many of these conditions can be caused by damage to the enteric nervous system (ENS). Furthermore, a mouse model of DS was recently shown to have fewer ENS neurons than its wild- type counterparts. Here, we propose that progressive injury to the enteric nervous system (ENS) drives GI motor and sensory abnormalities in DS. The transformative hypothesis of this proposal is that progressive injury to the ENS drives colonic secreto-motor and permeability (SMP) abnormalities in DS leading clinically to chronic constipation. This proposal could illuminate novel aspects of the pathophysiology of GI diseases, which affect more than 50% of individuals with DS. To address these key research gaps and define the mechanisms underlying ENS dysfunction in DS-associated GI disease, we propose a two-part approach: deep-phenotyping in a cohort study of individuals with DS and cause-effect animal research using mouse models of DS. Our Specific Aims are: Specific Aim 1: To expand our ongoing pan-omics cohort study to define associations between markers of inflammation, metabolic dysregulation, and altered GI microbiota, with chronic constipation. Specific Aim 2: To determine the effects of experiment