Enteric neuronal loss is responsible for intestinal dysmotility in several conditions (e.g. aging, diabetes mellitus, and slow transit constipation). Neuronal nitric oxide synthase (nNOS)-expressing neurons are critical to proper gastrointestinal motility. The mechanism underlying nNOS neuronal susceptibility to injury is largely unknown. Saturated fatty acids (SFA) are incorporated in cell membrane inducing the formation of lipid rafts, that regulate signaling from membrane-bound proteins such as Toll like receptors. Our preliminary studies show that (i) WD feeding for 12 weeks leads to loss of nitrergic enteric neurons and reduction of colonic motility in conventional, but not germ free or TLR4-/- mice; (ii) Less nitrergic neurons correlates with delayed colonic motility; (iii) In vitro palmitate and LPS enhance nitrergic neuronal loss in a lipid rafts dependent fashion; (iv) Palmitate and LPS can lead to activation of NLRP3 inflammasome and caspase-11, and subsequently pyroptotic nitrergic neuronal loss; NF?B over activation contributes to nNOS neuronal loss. We hypothesize that palmitate enhance LPS action through the TLR4 dimerization in lipid rafts, facilitating TLR4 signaling and NF?B activation in myenteric neurons in an ROS dependent fashion. This leads to activation of NLRP3 inflammasomes through canonical and non-canonical pathways and subsequent nitrergic enteric neuronal damage and colonic dysmotility. To test this hypothesis, we propose the following inter-related but independently achievable aims:
Specific Aim 1 : To determine the role of lipid rafts and ROS in SFA and TLR4/NF?B signaling in enteric neurons. We will determine whether TLR4 recruitment into lipid rafts is necessary and sufficient for SFA/LPS-induced TLR4 activation and signaling and if this is dependent on ROS production. Using inhibitors and gene silencing we will dissect out the role of ROS in SFA-mediated TLR4 activation of NF?B in enteric neurons Specific Aim 2: To understand the role of NLRP3 inflammasomes in nitrergic neuronal loss. We will establish the critical role of NLRP3 inflammasomes in mediating SFA/LPS- induced TLR4/NF?B activation and enteric neuroinflammation using both In vitro and in-vivo models. We will examine the effect of SFA and LPS on oligomerization of NLRP3 inflammasome components leading to activation of caspase-1/caspase-11 and pyroptotic neuronal cell death. In conditional nitrergic IKK2-/-, NLRP3-/-, Caspase 1-/- and Caspase 11-/- mice, we will determine their effects on enteric neurons and motility fed a regular diet (RD) or WD. For the gain-of-function studies, we will determine the effect of inducible nitrergic NLRP3 or IKK2 overexpression on enteric neurons and motility using nNOS-Cre-ERT/Nlrp3A350VneoR mice or nNOS-Cre- ERT/Ikk2CA mice. These studies will elucidate a novel mechanism in the pathogenesis of enteric neuronal dysfunction as well as provide ?proof of principle? for targeted therapies to prevent or treat gastrointestinal motility disorders.

Public Health Relevance

Degeneration of enteric neurons can lead to intestinal motility disorders as seen in diabetes, aging, slow transit constipation, Hirschsprung?s disease and high fat diet intake. In this proposal, using our model of high fat diet induced neuronal loss, we will examine the mechanism of how gut microbial products and saturated fatty acids work together leading to damage of a specific type of enteric neuron called nitrergic neurons. Experiments outlined in this proposal may also lead to new therapeutic targets for treating the altered gastrointestinal motility in humans with a high fat diet intake as well as in aging and diseases such as diabetes and slow transit constipation.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK080684-10
Application #
9891980
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Shea-Donohue, Terez
Project Start
2009-08-01
Project End
2023-03-31
Budget Start
2020-04-01
Budget End
2021-03-31
Support Year
10
Fiscal Year
2020
Total Cost
Indirect Cost
Name
Emory University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Reichardt, François; Chassaing, Benoit; Nezami, Behtash Ghazi et al. (2017) Western diet induces colonic nitrergic myenteric neuropathy and dysmotility in mice via saturated fatty acid- and lipopolysaccharide-induced TLR4 signalling. J Physiol 595:1831-1846
Chassaing, Benoit; Raja, Shreya M; Lewis, James D et al. (2017) Colonic Microbiota Encroachment Correlates With Dysglycemia in Humans. Cell Mol Gastroenterol Hepatol 4:205-221
Anitha, Mallappa; Reichardt, François; Tabatabavakili, Sahar et al. (2016) Intestinal dysbiosis contributes to the delayed gastrointestinal transit in high-fat diet fed mice. Cell Mol Gastroenterol Hepatol 2:328-339
Taba Taba Vakili, Sahar; Kailar, Roshni; Rahman, Khalidur et al. (2016) Glial cell line-derived neurotrophic factor-induced mice liver defatting: A novel strategy to enable transplantation of steatotic livers. Liver Transpl 22:459-67
Mwangi, Simon Musyoka; Peng, Sophia; Nezami, Behtash Ghazi et al. (2016) Glial cell line-derived neurotrophic factor protects against high-fat diet-induced hepatic steatosis by suppressing hepatic PPAR-? expression. Am J Physiol Gastrointest Liver Physiol 310:G103-16
Taba Taba Vakili, S; Nezami, B G; Shetty, A et al. (2015) Association of high dietary saturated fat intake and uncontrolled diabetes with constipation: evidence from the National Health and Nutrition Examination Survey. Neurogastroenterol Motil 27:1389-97
You, Shuo; Anitha, Mallappa; deSouza, Sean Md et al. (2015) Hepatic insulin gene therapy prevents diabetic enteropathy in STZ-treated CD-1 mice. Mol Ther Methods Clin Dev 2:15028
Pimentel, Mark; Morales, Walter; Pokkunuri, Venkata et al. (2015) Autoimmunity Links Vinculin to the Pathophysiology of Chronic Functional Bowel Changes Following Campylobacter jejuni Infection in a Rat Model. Dig Dis Sci 60:1195-205
He, Peijian; Zhao, Luqing; Zhu, Lixin et al. (2015) Restoration of Na+/H+ exchanger NHE3-containing macrocomplexes ameliorates diabetes-associated fluid loss. J Clin Invest 125:3519-31
Yarandi, S S; Srinivasan, S (2014) Diabetic gastrointestinal motility disorders and the role of enteric nervous system: current status and future directions. Neurogastroenterol Motil 26:611-24

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