Project Summary/Abstract Individuals with multiple sclerosis (MS) frequently suffer from autonomic dysfunction in addition to the neuromuscular ailments that represent their hallmark symptoms, and one of the most common and debilitating of their autonomic problems is constipation. Our theory is that constipation develops as a form of collateral damage from the central nervous system (CNS) inflammatory response that occurs in MS. During this inflammatory response, the patient's immune system generates antibodies that target proteins in the cellular debris of dying neurons and glial cells in affected areas, and these autoantibodies could have actions at distant sites. The enteric nervous system (ENS) is unique in that it contains intrinsic reflex circuitry that regulates motility, secretion and vascular tone in the gut, independently of CNS innervation. The ENS contains glial cells that share transcriptome features of oligodendrocytes and astrocytes, including proteolipid protein and myelin basic protein. These are two known antigenic targets in MS that could be incidentally targeted where they are also expressed in the ENS. This grant proposal is designed to test the hypothesis that autoantibodies generated in MS patients can target proteins on neuronal and glial membranes in the ENS, resulting in altered enteric neural reflex activity leading to constipation. In support of our hypothesis and the feasibility of our proposed studies, we have demonstrated that GI function is altered in mice with experimental autoimmune encephalomyelitis (EAE), the predominant mouse model of MS, in ways that are consistent with the development of constipation. Furthermore, we have found that blood samples from MS patients and EAE mice contain antibodies that bind to neurons and glial cells in the GI tract. In our proposed studies, we will examine the features of GI dysfunction in EAE mice, and confirm that the changes in gut motility involve circulating autoantibodies. We will determine the ENS cell types that MS autoantibodies bind to, and what effects these antibodies have on GI function in nave mice. We will also directly examine the physiological activities of glial cells and neurons in the GI tract, and we will elucidate how these activities are altered in EAE mice, and in response to treatment with autoantibodies from MS patients. In these studies, we will also use colons from mice in which a calcium indicator protein is expressed by enteric glial cells or select populations of enteric neurons, and we will use intracellular recording to evaluate the strength of excitatory and inhibitory neuromuscular transmission. Finally, we will test potential therapeutic approaches for their effectiveness in alleviating constipation in the MS mouse model. Understanding the mechanisms responsible for constipation in MS is a clinically relevant goal since it is a critical step toward developing an effective therapeutic solution for the GI ailments of affected MS patients and others with autoimmune-mediated dysmotility.