Xinzhong Dong - US grants

[unreadable] DESCRIPTION (provided by applicant): In mammals, the initial detection of sensory information, such as tactile, proprioceptive, thermal, and pain stimuli from both external objects and the body, is carried out by primary sensory neurons. The cell bodies of these neurons locate in dorsal root ganglia (DRG). Their axons are able to innervate specific targets in the periphery and the spinal cord. The long-term objective of our research is to understand the molecular mechanism of how the projection patterns of primary sensory neurons are established and to determine the neural circuits of different subpopulation of sensory neurons. Recently, we have identified a large family of G protein coupled receptors (GPCRs), called Mrgs, expressed specifically in primary sensory neurons in DRG. Interestingly, different Mrgs are expressed in different subpopulations of sensory neurons. We have generated several knock-in mouse lines, in which different axonal tracers including farnesylated green fluorescent protein and human placenta alkaline phosphatase were inserted into different Mrg loci. By analyzing these mice, we found that sensory neurons expressing different Mrgs have highly distinct and specific projection patterns both in the periphery and the spinal cord. One striking example is that the axons of MrgB4+ neurons only innervate hairy skin but not glabrous skin. To our knowledge, MrgB4 is the first marker which can segregate hairy skin innervating neurons from glabrous skin innervating neurons. Interestingly, most of MrgB4+ fibers terminate at a specific region of hair follicles. Furthermore, the projection of MrgB4+ axons in the dorsal horn of the spinal cords is also very unique. The MrgB4+ fibers terminations in the dorsal horn lamina layer form an unusual discontinuous band, suggesting that MrgB4+ fibers converge to specific regions within the lamina layer. The high specificity of MrgB4+ axon projection pattern raises the question of whether this GPCR play a role in the establishment of such specific pattern in the periphery and the spinal cord. Here we propose to study the function of MrgB4 in axon guidance of sensory neurons and their neural circuits in the higher order of the CNS. Aim I is to analyze the projection patterns of MrgB4+ fibers in hairy skin and spinal cord of MrgB4 homozygous mutant mice, from which we would like to determine whether MrgB4 plays roles in axon guidance or target recognition. In Aim II, we will determine whether hair growth is involved in establishment and maintenance of sensory innervations in hairy skin. To achieve this goal, we will study the projection patterns of MrgB4+ fibers in hairless knockout mouse background. Aim III we would like to map the neural circuit of MrgB4+ neurons using a trans-neuronal anterograde tracer, wheat germ agglutinin (WGA). We will generate MrgB4-WGA knock-in mice and we will carry out a detailed analysis of the pattern of WGA expression to identify the second- (possibly third-) order neurons to MrgB4+ primary sensory neurons labeled by WGA. We believe that studying the molecular mechanism of how sensory nerves establish proper projections in the skin and the spinal cord will have broad implications for understanding neuropathological diseases associated with abnormal wiring in the somatosensory system such as those in certain chronic conditions. In addition, the study of sensory neural circuits will greatly facilitate our understanding of how sensory information is transmitted and processed, which will be essential for developing novel drugs to treat diseases with abnormal sensory function such as chronic pain. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The long-term objective of our research is to understand how TRP activities are regulated. Recently, we identified a novel gene, called phosphoinositide interacting regulator of TRP (Pirt), which is highly expressed in pain-sensing, or nociceptive neurons in dorsal root ganglia (DRG) and absent in the central nervous system. Pirt encodes a two-transmembrane domain protein. Behavioral and electrophysiological studies of Pirt null mice suggest that Pirt functions as a positive regulator of TRPV1. Biochemical analysis shows that Pirt binds both TRPs and phosphatidylinositol-4,5-bisphosphate (PIP2). Importantly, Pirt enhances TRPV1 activity via PIP2. Our preliminary data suggest that Pirt can also bind and modulate TRPM8, a molecular sensor for cold sensation and menthol. In addition, we identified another structurally related PIP2 binding protein, called Pirt2. Interestingly, Pirt2 also binds certain TRPs including TRPA1 and TRPV1. In this proposal, we will take behavioral, electrophysiological, and biochemical approaches to further study the role of Pirt and Pirt2 in regulating these PIP2-sensitive TRP channels. Aim I is to assess whether the positive effect of Pirt on TRPM8 is also found in cultured DRG neurons by comparing neurons from wild-type and Pirt null mice. We will study how Pirt affects channel properties of TRPM8 at the single channel level. To determine whether Pirt affects TRPM8-mediated cold sensation in vivo, we will perform previously described behavioral tests on Pirt null mice and their wild-type littermates. In Aim II, we will study the molecular mechanism of how Pirt regulates TRPM8. We will employ biochemical approach to determine whether TRPM8 and Pirt form a complex in DRG neurons and which domains in Pirt and TRPM8 are required for their binding. Then we will assess whether Pirt and PIP2 require each other to enhance TRPM8. Aim III is to determine whether Pirt2 plays role in modulation of TRPA1 via PIP2. We will test the hypothesis that Pirt2 is involved in the PIP2 inhibitory effect on TRPV1. By analyzing the behavioral phenotypes of Pirt2 knockout mice, we will know whether Pirt2 plays role in TRPA1- and TRPV1-mediated pain sensation. The proposed analysis of Pirt and Pirt2 will facilitate our understanding of TRP regulation. PUBLIC HEALTH RELEVANCE: Transient receptor potential (TRP) channels are involved in many fundamental biological processes including pain sensation. Our studies suggest that Pirt and Pirt2 represent a novel family of TRP channel regulators. Therefore, functional analysis of Pirt and Pirt2 should facilitate our understanding of TRP modulation and thereby open the door for developing novel drugs to treat conditions like chronic pain.

Project Summary The goal of our research is to understand the cellular and molecular mechanisms of mast cell-mediated itch and how mast cells interact with sensory nerves to induce itch. Primary sensory neurons in dorsal root ganglia (DRG) play an essential role in generating itch. We showed that several Mrgprs, a large family of G protein-coupled receptors (GPCRs) are specifically expressed in the DRG and function as novel itch receptors by detecting various pruritogens. Although sensory neurons are pivotal in facilitating itch sensation, other cell types in the skin such as mast cells and keratinocytes also play a major role in generating itch. Mast cells are innate immune cells embedded in most tissues of the body and secrete a wide range of substances such as histamine and serotonin. Inappropriate mast cell activation has been linked to an increasing number of serious diseases, including skin diseases, chronic itch, gastrointestinal disorders, and asthma. Besides the classical IgE-dependent pathway, a variety of cationic substances, collectively called basic secretagogues, can also activate mast cells. Recently we identified that MrgprB2, the orthologue of human MrgprX2, is the mast cell receptor for basic secretagogues in mice. Unlike Mrgprs found in sensory neurons, both MrgprB2 and MrgprX2 are exclusively expressed in mast cells and activated by various basic secretagogues (e.g. compound 48/80 and substance P). Although mast cells have been implicated in many itch associated skin diseases, the underlying cellular and molecular mechanisms have not been fully characterized. In this proposal, we will take molecular, genetic, behavioral, and imaging approaches to dissect the functions of MrgprB2 and properties of Mrgpr-expressing mast cells in itch. Our preliminary data showed that skin anti-microbial peptide PAMP, which induces itch in humans, is an MrgprB2/X2 ligand. We also found that PAMP-induced mast cell activation and itch are mediated by MrgprB2. In Aim I, we will determine whether MrgprB2 is involved in acute and chronic itch. Furthermore, we will test the hypothesis that PAMP and substance P are candidates for the endogenous ligands of MrgprB2 in itch. Finally we will examine what mediators are released from mast cells activated by MrgprB2 in itch. Since MrgprB2 is expressed in ~100% of mast cells, it provides us with a great genetic tool to specifically study mast cells. In Aim II, we will study how mast cells contribute to acute and chronic itch by MrgprB2-Cre dependent genetic manipulation of mast cells (i.e. labeling, ablation, and activation). Using Pirt- GCaMP3 mice we successfully imaged DRG neuron activation in response to pruritogens at a populational level in live mice. In Aim III, we will use this powerful in vivo DRG imaging technique to answer important questions on itch: whether activation of mast cells can activate sensory neurons under acute and chronic itch conditions, what types of sensory neurons, and what mediators are involved. The results of the project will provide insight into key itch mechanisms and open the door for the development of novel itch therapeutics.

? DESCRIPTION (provided by applicant): We seek to renew our postdoctoral Interdisciplinary Training Program in Biobehavioral Pain Research at the Johns Hopkins Medical Institutions (JHMI). Chronic pain is one of the most common and disabling symptoms in our society. Pain is a highly complex phenomenon that involves, genetic, molecular, neurophysiologic, cognitive-emotional, and sociocultural determinants. The treatment of pain remains inadequate in almost every clinical situation and consequently demands programs to create specialized, interdisciplinary training in pain research that addresses the tremendous challenge of developing, evaluating, disseminating, and integrating effective pain treatments into clinical care. The overarching goal of the proposed postdoctoral program is to prepare the next generation of innovative research leaders to work cooperatively within an interdisciplinary team to address the complex problem of pain. To this end, we are again requesting support for four postdoctoral fellows. We expect each of these fellows to stay in the program for two years, unless they receive independent funding (e.g. an F32 grant). To encourage a diverse program, we are requesting support for two fellows (PhDs) who are in the first year of post-graduate research (PGY0), as well as two fellows who have completed their residency following medical school (PGY5). This renewal proposes to emphasize the neuroscience of pain throughout the training. Each faculty mentor is actively funded, engaged in the education of young investigators, and committed to interdisciplinary collaboration. The Program incorporates coursework and mentored research experiences in at least two scientific domains that are synthesized by: 1) an integrated research project, 2) an extramural grant application, and 3) the writing and publishing of papers. The training objectives are to: 1) develop an enhanced foundation in the neuroscience of pain; 2) engender a broad conceptualization of pain that includes, but is not limited to, neurobiologic, cognitive, emotional, behavioral, and social processes; 3) develop skills for communicating, networking and collaborating with scientists in other disciplines; and 4) design and conduct an integrative pain research project. Each fellow is to be collaboratively mentored by two core faculty with distinct domains of pain research expertise in either: 1) neuroscience, 2) clinical research and 3) behavioral or social science. Program faculty cut across the Schools of Medicine, Nursing, and Public Health. Our goal is to prepare the next generation of pain scientists to lead interdisciplinary research teams that will innovatively address the problem of pain using transformative research paradigms.

Project Summary The goal of our research is to understand the cellular and molecular mechanisms behind the development of Stevens-Johnson syndrome. Adverse drug reactions (ADRs) are a serious drug safety concern that can be hard to predict and treat. Stevens-Johnson syndrome (SJS) is one of the most serious and life- threatening ADRs characterized by keratinocyte cell death and mucosal breakdown; patients often form lesions on their palms of hands and soles of their feet and blistering and necrosis of their eyelids, conjunctiva and cornea. The exact cellular and molecular mechanisms behind drugs triggering SJS is unknown. Using a Ca2+ imaging assay, we found certain members of the Mrgpr GPCR family are activated by multiple known SJS- causing drugs. To examine the immune effects of these SJS drugs, we developed a novel mouse treatment where daily oral ingestion of a SJS-causing drug resulted in WT mice forming mucosal secretion in their eyes and blister bleeding in their hindpaws, similar to symptoms seen in patients suffering from SJS. We generated a mouse line where the specific Mrgpr's open reading frame was deleted and replaced with GFP and this genetic removal prevented formation of the SJS-like phenotype. Strikingly, heterozygous Mrgpr +/GFP mice, mice retaining 1 functional copy of the certain Mrgpr allele, developed the SJS-like phenotype, implying an important role for this Mrgpr in drug-caused SJS. Further experiments revealed the Mrgpr was expressed in a subset of dendritic cells, cells known to play an important role in antigen-presentation and immune response initiation. In this proposal, we will use molecular, cellular, genetic approaches and a potential novel in vivo animal model to uncover the role of the Mrgpr-expressing dendritic cells in drug-activated SJS. Aim I will focus on establishing our novel drug-induced SJS mouse model. We will examine what immune changes occur as mice are dosed with specific SJS-causing drugs and then we will determine what role the specific Mrgprs play in these immune responses. In Aim II, we will examine the specific characteristics of Mrgpr-expressing dendritic cells. We will find what type of dendritic cell expresses the Mrgprs and the role they play in the immune system. We will then examine how the cells change upon exposure to SJS-causing drugs and how these changes cause a cytotoxic immune response and SJS phenotype. We will also confirm the pivotal role these Mrgpr-expressing cells play in the development of SJS by rescuing the phenotype in the Mrgpr KO mouse. In Aim III, we will continue examining the immune cascade triggered by the SJS drugs. Dendritic cells are known for their important role of presenting antigens to T cells, which activate and in turn cause an immune response. We will determine what T cells are being activated by the Mrgpr-expressing dendritic cells and what cellular effects and mediators are released by these activated T cells. We will establish the necessity of T cells in continuing the immune response that is initiated and maintained by activation of the Mrgpr-expressing dendritic cell. Data collected from this project will help reveal some of the cellular and molecular reasons behind certain drugs causing SJS; these new cellular and molecular targets could lead to new treatments and drug therapies for SJS and adverse drug reactions.

Project Summary Marked increases in esophageal mast cells (MCs) have been identified not only in allergic but also in non-allergic esophageal disorders. At present, their roles in the pathogenesis of those disorders are still less clear. Unlike intestinal mucosal MCs, esophageal MCs are predominantly distributed in the lamina propria of the mucosa, whereas they are matured in non-keratinized stratified squamous epithelium and developed into distinctive phenotype. Recently, the Immunologic Genome (ImmGen) Project Consortium has classified esophageal MC as one of the typical connective tissue MCs. Moreover, our new study has identified that MrgprB2 (the orthologue of human MrgprX2) is a GPCR and exclusively expressed in connective tissue MCs. Many basic secretagogues (substance P, VIP, PAMP, defensins, et al) and eosinophil cationic proteins are now known to activate mast cells exclusively via MrgprB2/X2 mechanisms. Our published and preliminary studies are supporting the novel hypothesis that MrgprB2 (MrgprX2 in human) mediates esophageal inflammation-induced MC activation and directly contributes to esophageal epithelial barrier dysfunction and esophageal afferent nociceptive nerve hyperexcitability. We will address this hypothesis in the mouse and human esophagus with following aims. In Aim 1, we will characterize MrgprB2-positive MCs in healthy and inflamed esophagus with respects to their distribution, phenotype, and activation response (mediators/cytokines release) to basic secrectagogues. We will then address the hypothesis that MrgprB2 mediates non-IgE-dependent MC activation by comparing MC mediator release in inflamed esophagus in wild type and MrgprB2mut mice. Lastly, we will explore, in an eosinophilic esophagitis model, whether eosinophil granule basic proteins (MBP, EPO, END,) directly activate MrgprB2. In Aim 2, we will continue to advance our interesting preliminary data demonstrating that reflux-induced esophageal epithelial barrier dysfunction (increased permeability) is significantly attenuated in MrgprB2 mut mice. To determine if non-MrgprB2 mast cell activation mechanisms also contribute to barrier breakdown we will compare permeability changes in our reflux vs allergic esophagitis models in three groups of mice, wild type mice; mice where the mast cells do not express functional MrgprB2 mut; and thirdly mice that are mast cell deficient. In Aim 3, we will compare the effect of MrgprB2 vs allergen-evoked mast cell activation on esophageal nociceptive C-fiber terminal activities using our well-established extra-cellular recording techniques along with our newly-developed two-photon neuron imaging methodology. In Aim 4, we will compare the expression and function of MrgprX2 in human esophageal biopsy specimens from reflux and eosinophilic esophagitis and then determine their correlations with esophageal histology/symptoms. Translationally, we will briefly characterize MrgprX2 expression and function in mouse esophagus by using our newly-established humanized MrgprX2 mouse line. Clarifying MrgprB2/X2-mediated esophageal mast cell activation may motivate investigation of novel targeted therapeutic strategies for esophageal inflammatory disorders.