This proposal is for an Underrepresented Minority Supplement for Wilmarie Morales- Soto, a second year predoctoral student in the Neuroscience Graduate Program, to the funded parent RO1 (RO1DK103723). Wilmarie?s research project will build upon studies described in specific aim 1 of the parent grant.
Aim 1 is designed to identify how the activation of glial signaling pathways that couple to fluxes in intracellular Ca2+ contributes to reactive gliosis and how reactive gliosis contributes to neuroinflammation in the gut. These studies test the hypothesis that glial Ca2+ responses driven by ADP cause reactive gliosis, neuron death and gut dysfunction. Ongoing studies performed by the candidate have discovered that communication between nociceptors, enteric neurons, and enteric glia is required for the development of neuroinflammation in the gut. Further, her preliminary studies indicate that proinflammatory mediators act on glial release mechanism to alter intercellular communication between glia and neurons. These findings are important because they identify novel mechanisms that contribute to neuroplasticity in the gut and these mechanisms could be targeted by therapies to improve gut motility and visceral pain. This body of work will be the focus of her dissertation and she will complete this work over the two years of requested supplemental funding. She will also develop and submit her own F31 fellowship as part of her training under this supplemental support and will transition to this funding if successful. Wilmarie has already begun studies that utilize GFAP::hM3Dq mice to drive glial Ca2+ signaling as described in specific aim 1a to study how this affects nociceptor activity.
She aims to complete these studies during the first year of support. She is also working to complete studies that test how inflammatory mediators affect gliotransmitter release mechanisms and aims to complete these experiments in the next year as well. These experiments build upon the cellular physiology studies described in specific aims 1a and 1b. The abstract of the funded parent grant is pasted below for reference: Reflex behaviors of the intestine including peristalsis are orchestrated by the enteric nervous system (ENS); a complex neural network embedded in the gut wall. Inflammation profoundly alters ENS circuits controlling motility by promoting enteric ganglionitis; an inflammatory neuropathy characterized by the death of enteric neurons. Neuropathy is increasingly recognized as a trigger for persistent gut dysfunction in gastrointestinal (GI) motility and functional bowel disorders but the mechanisms that regulate neuropathy are not understood. This proposal investigates the role of enteric glial cells, astrocyte-like cells that surround neurons in the ENS, in the regulation of enteric neuropathy. The proposed studies will use in vivo models of GI inflammation, transgenic mice, immunohistochemistry, live-cell imaging with fluorescent probes, biosensing assays and functional tests to study neuron-glia interactions. The central hypothesis is that purinergic activation of enteric glial cells differentially regulates neuron survival depending on glial activation by ADP or adenosine. There are 2 specific aims in this proposal, each with three sub-aims.
Each aim will link in vitro mechanistic studies in tissue from humans and mice with in vivo functional studies in transgenic mice.
Aim 1 will test the hypothesis that glial Ca2+ responses driven by ADP cause reactive gliosis, neuron death and gut dysfunction.
Specific aim 1 A will test how activation of glial Ca2+ responses in GFAP::hM3Dq mice or human tissue transduced with glial-specific vectors affects the induction of reactive gliosis and neuron death.
Aim 1 B will test whether glial cells directly drive neuron death by releasing neurotoxic substances or if glial-driven neuron death requires immune cell recruitment. Mice with an inducible ablation of connexin-43 or MHC-II in glia will be used to specifically interfere with gliotransmitter release or immune cell recruitment, respectively.
Aim 1 C will test how manipulation of gliosis using the transgenic mice listed above affects in vivo and ex vivo intestinal function.
Aim 2 will test the hypothesis that adenosine inhibits reactive gliosis and stimulates protective mechanisms in glia to preserve ENS function.
Aim 2 A will use drugs and CD73 null mice to test if activation of glial adenosine receptors is necessary and/or sufficient to reverse reactive gliosis.
Aim 2 B will test whether the neuroprotective actions of glial A2BR activation are mediated by altering the release of glial mediators or by decreasing the inflammatory infiltrate following in vivo inflammation in CD73 null mice.
Aim 2 C will use in vivo inflammation, drugs and CD73 null mice to determine how manipulation of glial adenosine signaling impacts in vivo and ex vivo assays of gut function following acute inflammation. Significance: Intestinal inflammation can drive enteric neuropathy, leading to persistent gut dysfunction in GI motility disorders. Understanding how glial mechanisms both promote, and limit enteric neuropathies is important because it could lead to the discovery of novel therapeutic targets and a common causative mechanism of neuron death in GI motility disorders, functional bowel disorders and inflammatory bowel disease.
(RELEVANCE) The manifestation of gastrointestinal (GI) motility and functional bowel disorders is caused, in part, by alterations to the function or survival of neurons that control GI muscle. The proposed studies investigate how the glial cells that surround these neurons drive neuronal dysfunction. A detailed understanding of the role of enteric glia will lead to new therapies to treat GI motility and functional bowel disorders.
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