Srinivasan Digestive Diseases Lab
Shanthi Srinivasan, MD
Associate Professor of Gastroenterology and Liver disease
Whitehead Biomedical Research Building
615 Michael Street, Suite 201A
Atlanta GA 30322
• Simon Mwangi, PhD
• Francois Reichardt, PhD
• Behtash Ghazi Nezami, MD,
• Sahar Tabatabavakili, MD,MPH
Sahar Tabatabavakili - firstname.lastname@example.org
Association of Fecal metabolites with enteric neuronal survival/impairment and gastrointestinal motility disorders
High fat diet (HFD) intake can lead to gastrointestinal complications such as constipation which results in morbidity and mortality. Excessive dietary fat intake correlates with constipation and prolonged colonic transit times. Our previous research demonstrates that constipation due to HFD results from loss of enteric neurons due to apoptosis. My laboratory focus is on the mechanism of enteric neuronal damage in mice with high fat diet intake with a careful look at colonic microbiota. We focus on a novel concept that high fat diet induced alteration in microbial flora resulting in the altered gastrointestinal motility. We aim to understand the role of intestinal dysbiosis (a complex of microorganism species living in the digestive tracts) in high fat diet-induced delayed gastrointestinal motility and elucidate the mechanisms involved. The hypothesis is that high fat diet induces intestinal dysbiosis leading to endotoxemia and subsequent enteric neuronal damage. Our preliminary data showed evidence that in the presence of prebiotics there was improvement in high fat diet induced delayed gastrointestinal motility. To understand the involvement of fecal microbiota in the mechanism of high fat diet-induced delayed gastrointestinal motility, we perform experiments using germ free mice as well as WT mice. We have designed a unique “Western-diet” for mice (similar to a typical American nutrition) and will assess its impact on the enteric nervous system survival and intestinal motility.
Our future projects will involve assessing the effects of fecal transplant from HFD fed mice to WT mice and germ free mice on gastrointestinal motility and enteric neuronal survival. Also to establish the role of prebiotics in prevention or treatment of high fat diet-induced endotoxemia and delayed gastrointestinal motility and neuronal apoptosis. We will evaluate for the presence of specific bacteria known to be associated with constipation. These studies are funded by R01 grant.
Factors affecting survival and differentiation of the enteric nervous system
Signal transduction mechanisms of growth factor induced proliferation and survival of enteric neurons My laboratory focuses on the factors affecting the survival and differentiation of the Enteric Nervous System (ENS). The ENS is an integrated network of neurons and glia within the wall of the gut that consists of the myenteric and submucosal plexus. The myenteric plexus directly controls intestinal motility. The submucosal plexus controls mucosal secretion and responds to sensory stimuli from the intestinal surface. The ENS is created from cells that originate in the vagal and sacral regions of the neural crest. The microenvironment of the gut provides signals critical for the development and maturation of these ENS precursors.
Studies in my laboratory are performed in primary culture system of isolated mouse embryonic enteric neuronal cells. The enteric nervous system develops from the neural crest cells. These cells have migrated into the intestine by embryonic day 14. We use mouse embryos at day E14 and dissect the embryos to obtain the stomach, small intestine and colon. These tissues are digested with enzymes and the enteric neuronal cells are isolated from this cell suspension using a process of magnetic immunoselection. The isolated enteric neurons can be cultured for up to 96 hours. During normal development, signaling through the Ret transmembrane tyrosine kinase is essential for the survival, proliferation and the extension of neuronal processes. Glial Derived Neurotrophic factor (GDNF) is a growth factor that acts through Ret. While Ret is known to activate many signal transduction pathways including MAP kinase and PI-3 kinase, the contribution of these individual signaling molecules to enteric neuron survival, proliferation and axonal extension remains poorly understood. My initial results demonstrate that GDNF mediates survival of enteric neurons through a PI-3-kinase/Akt dependent pathway. We plan to dissect out the signal transduction involved in GDNF mediated enteric neuronal survival by studying the downstream targets of Akt including forkheads, GSK3 beta and Bad. We have cultured enteric neurons in the presence and absence of GDNF and performed a mircorarray analysis to determine the genes up regulated and down regulated by GDNF. These experiments have given us clues to the new genes that are involved in GDNF mediated enteric neuronal survival and proliferation. We plan to pursue the mechanism of each of these genes on enteric neuronal survival and proliferation.
Long noncoding RNAs (lncRNAs) have been reported to play a critical role in regulation of diverse cellular processes such as stem cell pluripotency, development, and cell growth. They are characterized by lack of protein-coding sequence, often located proximal to genes encoding regulatory proteins, which have been shown to modulate cell cycle distribution, differentiation and apoptosis. Previously, we found that a high-fat diet (HFD) can cause delayed gastrointestinal transit in mice. HFD induced increased apoptosis and loss of colonic myenteric neurons. Mice fed a low-palmitate HFD did not develop a similar phenotype. Palmitate caused apoptosis of enteric neuronal cells associated with mitochondrial dysfunction and endoplasmic reticulum stress. It also significantly increased the expression of microRNA-375 (miR-375), in vitro. Mir375 expression was increased in myenteric ganglia of mice fed HFD, and associated with decreased levels of Mir375 target mRNAs, including Pdk1. Systemic injection of a Mir375 inhibitor for 5 weeks prevented HFD-induced delays in intestinal transit and morphologic changes. Our studies suggest the Mir375 could be a target for prevention of enteric neuronal loss.
To define the changes in long non-coding RNAs (lncRNAs) in enteric neurons of mice fed a high fat diet compared to a regular diet. In addition the effect of palmitate on enteric neurons on changes in lncRNA involved in neuronal apoptosis will be assessed.
In the current project my laboratory want to understand the changes in long-non coding RNAs under conditions of high fat in vitro and in vivo including changes in lnc-RNA in enteric ganglia of mice fed a regular diet or high-fat diet. We are hoping to find a novel treatment for motility disorders by targeting Lnc-RNA with higher efficiency and less side effects.Effect of Diabetes on the enteric nervous system
The other main focus of my laboratory is how diabetes affects the enteric nervous system and thereby altering gastrointestinal motility. Chronic diabetes causes gastroparesis and altered colonic motility that result in significant morbidity and mortality. Long standing diabetes has been associated with a reduction in enteric neural inhibitory transmitters. Injury to enteric neurons in the setting of diabetes may include neuronal cell death as well as injury to axonal processes. The mechanisms by which diabetes causes neuronal injury are, however, poorly understood. The effects of diabetes on the ENS are examined in vivo using streptozotocin-induced diabetic rats and in vitro using primary cultures of enteric neurons.
In animal models of diabetes such as the streptozotocin-induced diabetic rat we have evidence for enteric neuronal degeneration in diabetic rats compared to control rats. We plan to examine enteric neuronal apoptosis in diabetic and control rats and study the PI-3-kinase pathway in these animal models. We will use tools such as confocal microscopy and laser capture microscopy to study this.
To correlate the changes seen in animal models of diabetes we will examine the effects of glucose on primary culture of enteric neurons. Using primary enteric neurons isolated from rat embryos, we study the effects of high levels of glucose in vitro on enteric neuronal apoptosis and proliferation, with a focus on the role of the phosphotidyl-inositol-3-kinase pathway. Our preliminary studies demonstrate that elevated glucose levels also cause increased apoptosis of enteric neurons in culture. We plan to dissect out the molecular mechanisms by which elevated glucose levels injure enteric neurons. Significance identifying the mechanism of changes in the enteric nervous system seen in diabetes can help us develop new targets for the treatment of the gastrointestinal motility disorders seen in this disease.
Effect of neurotrophic factors on High Fat Diet induced Obesity
We are recently studying the effects of neurotrophic factors like GDNF on the regulation of high fat diet induced obesity. Our recent findings show effects of GDNF on adipocyte differentiation and function. Using our recently acquired indirect calorimetry chambers we are beginning to assess metabolic rates in mice with alteration of the GDNF and related genes.
Role of GDNF in inducing Liver Defatting
The goal of this project is to examine the role of Glial derived neurotrophic factors on reduction of liver fat content. Our research has previously shown that GDNF can protect against high-fat diet-induced hepatic steatosis in mice. Knowing that macrovesicular steatosis of hepatocytes increases the risk for primary graft dysfunction in almost 50% of livers for transplantation; my laboratory tries to investigate if GDNF can defat steatotic livers making them amenable for transplantation. We are proposing that GDNF could be used to clear excess lipid storage in fatty livers providing a new means to recondition donor livers considered unacceptable for transplantation. These studies are supported by a VA-MERIT award.