John R. Grider - US grants

DESCRIPTION (provided by applicant): Peristalsis is the main propulsive motility of the intestine and colon. The long term goal of this project has been to identify the components of the underlying reflex, the peristaltic reflex, and understand the complex interplay between luminal stimulants, paracrine agents released from mucosal enteroendocrine cells, and the neural elements which make up the sensory (afferent), interneuronal, and motor circuits of the reflex. The objective of this renewal application is to characterize the role of the neurotrophin, Brain Derived Neurotrophic Factor (BDNF), in the physiology and pathophysiology of the enteric nervous system (ENS) with regard to the regulation of the peristaltic reflex. Our preliminary studies show that proBDNF and mature BDNF (mBDNF) are present in mucosal enteroendocrine cells that contain serotonin (5-HT) and sensory neurons containing calcitonin gene-related peptide (CGRP) and substance P (SP), and that BDNF acts to enhance 5-HT and CGRP release in response to mucosal stimulation thereby enhancing peristalsis. Preliminary data also show that BDNF inhibits neurite outgrowth in adult enteric neurons. Thus, we hypothesize that the BDNF system plays an integral role in the peristaltic reflex by strengthening and enhancing the sensory limb of the peristaltic reflex circuit (Hypothesis 1) and that the BDNF system plays an integral role in the remodeling of the enteric nervous system in inflammatory states (Hypothesis 2). We will examine these hypotheses in a variety of in vitro (intact whole colonic segments, full-thickness muscle strips, variously dissected muscle strips), and culture (pure neuronal, pure smooth muscle, and nerve/muscle co-cultures) preparations. In the studies outlined in AIM 1, we will characterize the differential localization (mucosal enteroendocrine cells versus enteric and extrinsic neurons) and release of the precursor proBDNF, which has been shown to be secreted from neurons in the CNS, and the processed mature form, mBDNF. Release of BDNF will be determined in response to physiological stimuli (mucosal stroking, free fatty acids, and bile salts), to neurotransmitters (CGRP and SP), and to paracrine agents (5-HT and endocannabinoids) known to mediate or modulate the peristaltic reflex. In AIM 2, we will use pharmacological, biochemical, and molecular tools to identify the receptors (TrkB, p75, and sortilin) and intracellular signaling pathways (PLC-gamma, PI-3-K/AKT, ERK1/2, Rho/Rock) activated by proBDNF and mBDNF, that are involved in the enhancement of peristalsis and/or inhibition of neurite growth. In AIM 3, we will examine the production and role of proBDNF and mBDNF in mediating changes in peristalsis and remodeling of the enteric nervous system during and following recovery from colitis in animal models. As the role of neurotrophins in the physiology of the adult gut is virtually unknown, we anticipate that these studies will launch a new field of investigation and yield insight into new avenues for development of therapeutic agents for the treatment of motility disorders. PUBLIC HEALTH RELEVANCE: Peristalsis, the main propulsive motility of the gut, and the underlying peristaltic reflex are regulated by the complex interplay between luminal stimulants, agents released from mucosal enteroendocrine cells, and the neural elements of the enteric nervous system. Disorders of any of these components result in abnormal motility patterns that cause diarrhea or constipation and associated disturbances in absorption of nutrients and water from the gut. Based on our preliminary data, we propose to characterize the effects of a new type of agent, the neurotrophin Brain Derived Neurotrophic Factor (BDNF), on peristalsis in normal animals and an animal model of colitis. The results would open a new field of study and point the way to new avenues of investigation for pharmacological agents to treat diarrhea and/or constipation.

The objective of this proposal is to identify the direct measurement the enteric sensory, modulatory and motor neurotransmitters that regulate athe peristaltic reflex elicited by muscle stretch and mucosal stimulation in rat colon and human intestine. Our previous studies and preliminary experiments have led us to identify neuronal circuits consisting of modulatory neurons coupled to motor neurons that account for reciprocal circular and longitudinal muscle activity during the ascending and descending phases of the reflex. The experimental approach exploits novel preparations that enable identification of various components of the circuits: (i) flat-sheet compartmented intestinal segments with which to measure specifically the release of sensory transmitters; (ii) longitudinal muscle strips with adherent myenteric plexus with which to identify the coupling of modulatory neurons to motor neurons innervating longitudinal muscle; (iii) circular muscle st rips devoid of myenteric plexus with which to measure presynaptic regulation of transmitter release, and from which to prepare synaptosomes to characterize the binding of transmitters to presynaptic receptors; and (iv) freshly dispersed myenteric ganglia with which to examine feedback regulation of modulatory interneurons within the plexus. In Specific Aim I, studies in rat colonic and human intestinal segments will identify (a) the role of an integrated modulatory circuit consisting of somatostatin, enkephalin and GABA neurons that regulates motor neurons innervating circular and longitudinal muscle during the ascending and descending phases of the reflex, and (b) the role of 5-HT acting via neuronal 5-HT4 receptors in regulating release of the sensory transmitters, CGRP and the tachykinins, substance P (SP) and neurokinin A (NKA). In Specific Aim II, studies in myenteric plexus-longitudinal muscle strips will demonstrate that the modulatory circuit acts via VIP/PACAP/NOS neurons to regulate cholinergic/tachykinin motor neurons that innervate longitudinal muscle. In Specific Aim III, studies on circular muscle strips containing only axonal terminals will identify the presence of regulatory presynaptic (i.e., axo-axonal) interactions between modulatory nerve terminals and excitatory and inhibitory motor nerve terminals. The existence of these interactions will be corroborated by radioligand binding studies on synaptosomes derived from these terminals. In Specific Aim IV, studies on isolated ganglia devoid of axonal projections to muscle will characterize the interplay of modulatory interneurons and demonstrate the role of somatostatin interneurons as the neuronal switch between the two phases of the reflex. The combined approach embodied by these aims should advance our understanding of a major neuromuscular function.

The objective of this proposal is to characterize the neuronal circuits that mediate the peristaltic reflex in normal and inflamed colon, and determine the effect of inflammatory cytokines (IL-1beta and IL-6) and neurotrophins (GDNF and BDNF) on neurotransmitter expression and neuronal remodeling. Previous studies and preliminary experiments have lead us to identify neuronal circuits within the myenteric plexus consisting of modulatory neurons coupled to motor neurons that mediate the reciprocal activity of circular and longitudinal muscle. The experimental approach exploits novel preparations that enable identification of the neural pathways: (1) compartmented intestinal segments to measure release of sensory (CGRP), modulatory neural pathways: (1) compartmented intestinal segments to measure release of sensory (CGRP), modulatory (somatostatin, GABA, [Met]enkephalin], and excitatory (ACh and SP) or inhibitory (IP, PACAP and NO) motor neurotransmitters in animals and humans; (2) longitudinal muscle strips with adherent myenteric plexus to identify the coupling of interneurons to each other and to excitatory motor neurons; (3) circular muscle strips with axonal remnants, and synaptosomes derived from these strips to identify pre-synaptic regulation of neurotransmitter release; (4) freshly dispersed and cultured myentric ganglia to examine feedback regulation of somatostatin interneurons and neuronal remodeling by neurotrophins; (5) organotypic cultures of smooth muscle to identify the interplay of cytokines and neurotrophins; (5) organotypic cultures of smooth muscle to identify the interplay of cytokines and neurotrophins in normal and TNBS-induced colitis. The Specific Aims of the proposal are: (1) to characterized the modulatory roles of somatostatin, GRP and NPY in the ascending and descending phases of the peristaltic reflex, and identify the pre- synaptic interactions between motor neurons; (2) to characterize the sensory pathways activated by chemical stimuli and identify the 5-HT4 receptor splice variant mediating desensitization of the reflex; (3) to characterize the roles of the cytokines, IL-1beta and IL-6, and trophic (GDNF) and anti-trophic (BDNF) neurotrophins in mediating changes in peristaltic neurotransmission and neuronal remodeling in TNBS-induced colitis. The aims are supported by preliminary studies and will provide a deeper understanding of neurotransmission in health and disease.

Enteric glial cells (EGCs) regulate intestinal homeostasis and may be involved in purinergic signaling and motility, but their precise role is not understood, especially in humans. In the inflamed state, they convert to a `more reactive phenotype', releasing inflammatory mediators that could contribute to pathophysiology. PROBLEM: The role of human EGCs (hEGCs) in the physiology and pathophysiology of the GI tract is not known, and addressing this critical gap in knowledge is important in determining how hEGCs modulate GI functions in health and disease. Our preliminary data support the concept that purinergic signals in hEGCs regulate cellular communication, synaptic physiology, motor behavior, and inflammatory signals. Significance of these translational studies is underscored by significant species differences in glial responses, mechanisms and a 7 fold higher glial to neuron ratio in human ENS (versus mouse). Groundbreaking ? mechanistic, translational studies are proposed on `mechanosensitivity', `modulation of motility' and postoperative ileus. It became necessary to develop and apply sophisticated new technologies to study i.e. mechanical stimulation (pressure, shear stress, radial stretch) of hEGCs, currents, Vm, Ca2+waves, monitor ATP release using a `sniffer cell', spaciotemporal imaging of motility, and gene expression (i.e. RiboTag mouse & a 107-custom gene readout of `reactive glia'). Studies on surgical trauma/intestinal manipulation, IL1? or conditioned media from hEGCs provide precise ways to study `reactive glia' in human gut. OVERALL HYPOTHESIS: ?Human EGCs are critical regulators of mechanosensitivity, cell-to-cell communication, synaptic physiology, neuromuscular transmission and motility in the human intestinal tract and inflammation disrupts their normal activity. Purinergic signaling plays a pivotal role in `function and dysfunction'. EGCs exert inhibitory modulation of neural network activity. EGCs may exert differential (opposing) effects in the two muscle layers by acting in the ENS to modulate ascending and descending phases of the peristaltic reflex and coordinate or synchronize motility. Glial activation disrupts motility in postoperative ileus. Studies are supported by a dream team of investigators with a record of collaboration, a superb environment, strong preliminary data (Figs 1-16), 2 new publications in `Inflammatory Bowel Diseases' in 2016 and one revised publication to `GLIA' on the `basic hEGC model'. Our overall hypothesis is tested in 4 original Aims. AIM 1: To investigate mechanotransduction pathways of ATP release in hEGCs. AIM 2: To evaluate mechanosensory transduction and glial communication in the ENS of intact neural plexus networks in human intestine. AIM 3: To determine the role of hEGCs in the modulation of motility. AIM 4: To determine the impact of inflammation in postoperative ileus on reactive glia and motility and use human tissue to the extent it is possible. IMPACT: Translational studies will fill a critical gap in knowledge of glia in the `little brain' of the human gut in health and disease, with implications for motility disorders, gut inflammation and therapeutics.