Brij B. Singh - US grants

A grant has been awarded to the University of North Dakota under the direction of Dr. Van Doze to acquire a stereology microscope system for quantitative microscopic analysis of nerve cells. The instrument will allow researchers to make detailed observations of cell structure to study nerve cell development, plasticity, and cellular interactions in the nervous system. Using fluorescent probes and imaging montage software, the investigators will be able to construct three-dimensional images on computers at several workstations. The equipment will be used in courses and in student research, including several programs aimed at rural students and students from Native American tribes.

Calcium ion (Ca2+) influx is an important fundamental player in how cytosolic molecules regulate cellular functions. Little is known about how the levels of cytosolic Ca2+ are regulated or about how Ca2+ enters the cell. Ca2+ influx in most cells occurs via the store/receptor-mediated Ca2+ entry mechanism, which is activated by depletion of Ca2+ from the internal Ca2+ stores. Recently, Drosophila homologues of a Ca2+ influx protein, the transient receptor potential (TRP) gene, have been identified in mammals and have been suggested to encode Ca2+ influx channels. Preliminary results suggests that one homolog, TRPC1, plays a critical role in Ca2+ influx across the plasma membrane and regulates several critical processes such as secretion, motility, growth, differentiation, and apoptosis. However, the mechanism of regulation of the activity of TRPC1 channel has not yet been established. The central hypothesis for this project is that TRPC1 function and regulation is mediated via its interaction with other proteins through specific domain(s) present on TRPC1. To test this hypothesis an interdisciplinary approach combining electrophysiological, molecular, and biochemical techniques, which will evaluate, characterize, and confirm the vital role of the TRPC1 multimerization domain will be used. Additionally, the role of a classical, calcium binding and regulatory protein, calmodulin, in activation/ inactivation of the TRPC1 channel, and in determining the role of another protein PICK1 in the regulation of TRPC1 will be investigated and validated. These studies will not only extend the basic understanding of how TRPC1 is regulated but will also provide important insights into the mechanisms underlying certain fundamental processes which are dependent on cytosolic Ca2+. This project integrates education with research efforts through development of relevant undergraduate- and graduate-level courses. These courses will address scientific literacy and will involve undergraduates and high school students (especially women and minorities) in research. The objectives are to help students develop critical thinking and problem solving skills, as well as enhance their potential as future scientists. Further, these educational experiences are being provided in a state that is demographically under-represented by biological research scientists.

DESCRIPTION (provided by applicant): Saliva performs a number of extremely important biological functions that are instrumental in maintaining oral health. It has been estimated that more than 2 million people in the U.S. is compromised by from salivary gland dysfunction. Secretion of saliva is driven by concerted activities of a number of ion channels and transporters. Although, it is well established that calcium is the primary intracellular factor which regulates fluid secretion, the molecular mechanism involved in the regulation of cytosolic calcium is not clearly understood. This is primarily due to the absence of information regarding the calcium channels present in salivary glands. Moreover, in Sjogren's syndrome patients, although the acinar tissues appear to be normal, they do not function properly and have a decreased calcium response to agonist-stimulation. This observation raises the possibility that calcium channels might be altered in this pathological condition. Members of the Transient Receptor Potential (TRP) superfamily have been identified as calcium channels, which could be important in agonist-stimulated fluid secretion. Therefore, this study is designed to thoroughly characterize the role of cytosolic calcium in salivary gland function and to determine the relationship between transient receptor potential (TRPC1) protein-1 and saliva secretion. Our preliminary data indicates that TRP proteins are expressed in salivary glands and are involved in salivary secretion. The hypothesis of this study is that since calcium plays a pivotal role in the physiological function of salivary glands, characterization of calcium channels in salivary glands will be important to understand the mechanism of saliva secretion, which could represent as drug targets in salivary gland dysfunction. We will coordinate our efforts in order to determine the functional significance of TRPC1 channel protein by examining its effect by gene disruption using TRPC1 knockout mice. In Aim 2, we will investigate the localization and biochemical characterization of TRPC1 protein in mouse submandibular gland cells. Aim 3 will identify the mechanism involved in the regulation of TRPC1 protein. The results of our studies are expected to provide new insights into the role of calcium channels and the molecular mechanism involved in saliva secretion. Greater understanding of these events responsible for saliva secretion will be important in elucidating new therapy for salivary gland dysfunction.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with selective loss of dopaminergic neurons in the substantia nigra pars compacta. A number of pathogenic factors have been implicated in the degeneration of dopaminergic neurons in the substantia nigra including generation of free radicals, impairment of mitochondrial function, disturbances of calcium homeostasis, and apoptosis. It could be hypothesized that a common factor should be involved in the pathogenesis of PD. Calcium being a common factor in all the above processes raises the possibility that this could be the possible link. Also calcium homeostasis plays an important role in stimulating and inhibiting neuronal cell death, and calcium mediates apoptosis. Thus, depending on spatial and temporal factors, calcium channels may have a salutary effect on conditions such as PD in which apoptosis may be the ultimate mode of cell death. Calcium enters the cytoplasm from two sources;it is either released from the intracellular stores, or it enters through the plasma membrane. Depletion of the intracellular stores leads to the opening of plasma membrane calcium channels which are known as store-operated calcium entry (SOCE) channels. Recently, a mammalian homologue of the Drosophila trp gene, TRPC1, has been suggested as a SOCE channel. Moreover, our recent data indicate that TRPC1 protein levels and its plasma membrane localization is significantly decreased after treatment with drugs known to cause PD (MPP+ or salsolinol). Importantly, overexpression of TRPC1 protected SH-SY5Y neuronal cells against the cellular toxicity elicited by MPP+ and salsolinol. The protection exhibited by TRPC1 was dependent on its calcium influx properties and the translocation of pro-apoptotic proteins from the endoplasmic reticulum to mitochondria. These data demonstrate, for the first time, that TRPC1 has a role in protecting dopaminergic neurons. Nevertheless, the role of TRPC1 in vivo has yet to be elucidated and the mechanisms by which TRPC1 protects dopaminergic neurons are not known. Thus, we propose to extend our knowledge in animal models (TRPC1 knockout mice, and PD mouse models) and determine the role of TRPC1 in PD by identifying mechanism(s) by which TRPC1 protects dopaminergic neurons.

DESCRIPTION (provided by applicant): Saliva performs a number of extremely important biological functions that are instrumental in maintaining oral health. It has been estimated that more than 5 million people in the US suffers from salivary gland dysfunction. Secretion of saliva is driven by concerted activities of a number of ion channels and transporters. Although, it is believed that calcium is the primary intracellular factor that regulates fluid secretion, the molecular mechanism involved in the regulation of cytosolic calcium is not clearly understood. This is primarily due to the lack of information regarding the mechanism of regulation of calcium channels present in salivary glands. Furthermore, no information is available as how increase in cytosolic calcium modulates saliva secretion. Moreover, in Sj"gren's syndrome patients, although the acinar tissues appear to be normal, they do not function properly and have a decreased calcium response to agonist-stimulation. This observation raises the possibility that calcium channels might be altered in this pathological condition. Results obtained from our awarded grant indicate that TRPC1 is the primary calcium channel in salivary glands and is intimately involved saliva secretion. To understand the regulation of TRPC1 channel we have shown that in human submandibular gland cells, TRPC1 interaction with STIM1, Cav1, and Orai1 dictates TRPC1 mediated calcium entry. Furthermore, these protein-protein interactions were confined to specific domains in the plasma membrane, however nothing is known if similar mechanisms are also present in vivo in salivary gland tissues. Therefore, in this renewal we intend to thoroughly characterize the role of cytosolic calcium in salivary gland function and to determine the relationship between transient receptor potential canonical (TRPC1) -1 and saliva secretion. The hypothesis of this study is that because calcium influx via TRPC1 plays a pivotal role in the physiological function of salivary glands, characterization of calcium channels in salivary glands will be important to understand the mechanism of saliva secretion, which could represent as drug targets in salivary gland dysfunction. We will coordinate our efforts in order to determine the functional significance of TRPC1 channel in regulating saliva secretion. We will also investigate the role of lipid rafts in the assembly/activation of the TRPC1 channel in mouse submandibular gland cells and will identify the mechanism involved in the regulation of TRPC1 via STIM1 and Orai1. The results of our studies are expected to provide new insights into the role of calcium channels and the molecular mechanism involved in saliva secretion. Greater understanding of these events responsible for saliva secretion will be important in elucidating new therapy for salivary gland dysfunctions. PUBLIC HEALTH RELEVANCE: It has been estimated that more than 5 million people in the US suffer from salivary gland dysfunctions. Loss of salivary gland function is a limiting side-effect of numerous drugs, serious consequences of head and neck cancer treatment using radiation therapy, and also a frequent outcome of autoimmune diseases such as Sj"gren's syndrome. Our current studies are focused on the function of calcium influx via the Transient Receptor Potential canonical-1 in modulating saliva secretion. We anticipate that elucidating the mechanism of calcium entry-mediated regulation of saliva secretion;will be important to understand salivary gland dysfunction. Furthermore, we will identify the signalplex necessary for TRPC1-mediated calcium entry and define the mechanism involved in regulating TRPC1 channel per se. Greater understanding of these events will not only be important for understanding saliva secretion, but will be important in elucidating new therapies for salivary gland dysfunctions.

DESCRIPTION (provided by applicant): Although alveolar epithelial type II cells (AECII) form the barrier of alveolar spaces and produce surfactants to maintain lung integrity, the unique AECII population in the lung may also play a critical role in anti-microbial immunity by secreting cytokines, such as monocyte chemoattractant protein (MCP-1) against P. aeruginosa (PA) infection. However, the mechanism of cytokine secretion is largely unknown. Our long-term goal is to understand mechanisms of lung host defense, thereby identifying new strategies for treating bacterial infection. Our objective of this application is to characterize the mechanism of cytokine secretion by AECII. We hypothesize that AECII play immune roles by secreting cytokines, which is regulated through a mechanism of membrane microdomain reorganization. We have formulated this hypothesis based on our recent studies and two separate lines of evidence. First, in a high purity cell population, we found that a conditioned medium from PA-infected primary AECII enhanced AM immunity. On the other hand, the importance of lipid rafts (membrane microdomains) for innate immunity has been indicated by the fact that CF patients, who are particularly at risk for PA infection, may often suffer from abnormal lipid raft function due to ceramide deficiency and fatty-acid imbalance. Consistent with this, we showed that lipid rafts may be involved in the secretion of the cytokine MCP-1, which is localized in ceramide-rich rafts. Our rationale is that elucidating the relevant mechanism in ceramide-rich microdomains will define a general mechanism for cytokine secretion. Our laboratory is ideally suited for this research since we have the requisite expertise in AECII isolation, PA infection, and advanced biochemical techniques. To test our hypothesis, we propose the following two specific aims: Specific Aim 1: To characterize dynamic reorganization of membrane microdomains in regulating cytokine secretion in AECII and in mice using imaging tools. Our working hypothesis is that PA infection initiates dynamic changes in membrane microdomains that impact cytokine production. Specific Aim 2: To define the mechanism by which membrane microdomains regulate key cytokines required for PA defense. Our hypothesis is that ceramide is generated by hydrolysis and translocated to specialized domains, where it initiates signaling for production of MCP-1. Due to the important role of cytokines in various physiological and pathological conditions, these novel mechanisms will improve understanding of cytokine secretion for other types of cells, pathogens, and inflammatory situations, and thereby identifying new therapeutic targets. PUBLIC HEALTH RELEVANCE: Although alveolar epithelial type II cells (AECII) are structural and progenitor cells in the lung, they also provide immune defense by secreting cytokines. This unique immunity may critically increase host defense against P. aeruginosa (PA), a bacterium that causes severe infections in immunodeficient individuals, such as AIDS, tuberculosis and cystic fibrosis. We hypothesize that AECII secrete cytokines through changes in cell membrane microdomains, a novel mechanism that may impact many disease processes. Thus, our research will provide new insights into the development of novel treatment for this recurrent infection.

DESCRIPTION (provided by applicant): Sj?gren's syndrome (SS) is an autoimmune disease of the oral system that leads to salivary gland destruction. Almost 3% of the U.S. population is affected by salivary gland hypofunction, a complex disorder resulting from disease etiologies, as well as being a common side effect of drug therapy. Saliva is an essential oral component needed for normal speech, taste, mastication, swallowing, and in protecting the hard and soft tissues of the oral cavity, however the etiology as why salivary glands are destroyed in diseases such as SS is still not known. Recent findings have provided strong evidence that like many inflammatory disorders, SS have its origin in an overactive immune response against salivary tissue and causing glandular destruction. However, the mechanisms underlying infiltration of immune cells and development of inflammatory response in salivary glands remain ill-understood. Thus, our goal is to understand the molecular events that contribute to the development of chronic salivary gland inflammation. Emerging evidence suggests that during inflammation, dead or dying host cells can release endogenous host factors called alarmins. They perform homeostatic functions when contained in intracellular compartments under normal conditions, but exhibit chemotactic and immune activating properties once released in extracellular milieu. Current evidence indicates that alarmins not only initiate but also amplify and sustain on-going inflammation. This underscores that identification and characterization of alarmins can direct the development of effective therapies against inflammatory disorders such as SS. Our ongoing studies have identified that galectin-3 and -9, host C-type lectins, normally found intracellularly, are extracellularly released and act as alarmins to exacerbate inflammatory responses during bacterial infection. Our preliminary studies revealed that both galectin-3 and -9 are abundantly expressed in salivary tissue of transgenic female mice overexpressing IL14? (IL14? mice) exhibiting SS associated phenotypes (mouse model of SS). Our central hypothesis is that galectin-3 and -9, once secreted into the extracellular milieu of salivary tissu, act as alarmins to exacerbate inflammatory responses by recruitment and activation of immune cells resulting in development of SS. To test our hypothesis we will: characterize galactin- 3 and -9 as novel alarmins in SS (AIM1), and determine the role of galectin-3 and -9 in salivary gland inflammation and degeneration using galectin-/- or IL14a/galectin-/- mice (AIM2). We expect that the proposed experiments will provide novel insights into the contribution of galectins as novel alarmins towards the development of pathogenesis of autoimmune diseases, including, but not limited to SS.

? DESCRIPTION (provided by applicant): The objective of this grant application is to establish a Center of Biomedical Excellence in Host- Pathogen interactions that is highly interactive and consists of a dynamic group of junior investigators whose research focus is on understanding the mechanisms that underline the host respose to various infectious agents. We have proposed five new independent, but cohesive research projects, all led by junior investigators, that will use molecular, biochemical, immunological, and microbiological approaches to study host-pathogen interactions in different infectious disease models. We will define the role that certain host factors such as C-type lectins, Toll-like receptors, and other cell surface receptors that play a major role in the initiation of the host response, and we propose cutting edge, critica research in identifying molecules and signaling pathways that can regulate immune response and inhibit bacterial/pathogen growth. These goals will be accomplished utilizing an integrative approach to link several investigators to understand various infections. To enhance the program further, we have included a translational component as a key facet of this COBRE, which is consistent with our global, strategic mission to address the needs of North Dakota's aging, largely rural population that are prone to infectious diseases such as pulmonary infections, sepsis, neuroinflammation and vector-borne disease. We will also identify novel parasite-induced immune suppressive mechanisms, which could lead to new treatment paradigms for debilitating autoimmune diseases. The rationale for this center is that each individual brings unique expertise and key directions that together can help elucidate the role of immunomodulatory changes in various infectious disease models. This center will nurture a focused group of investigators, and successful completion of our specific aims will result in the establishment of a collaborative and sustainable Center of Excellence in Host-Pathogen interactions capable of attracting the brightest and most talented faculty, students and fellows to conduct world-class research in the burgeoning field of infectious disease. UND is fully committed to ensuring the growth and sustainability of this group even after the COBRE grant ends, which will establish and maintain UND as the emerging infectious disease center in the US.

Project Summary Saliva performs a number of extremely important biological functions that are instrumental in maintaining oral health. It has been estimated that more than 5 million people in the US suffers from salivary gland dysfunction (Sjogren's syndrome). Although no genes mutations have been identified that could explain the pathogenesis of Sjogren's syndrome (SS), recent evidence have suggested that T17-cell infiltration and induction of apoptosis in salivary gland acinar cells could be the two major events that could lead to salivary gland destruction. However, the molecular mechanism involved in the activation of T cells and apoptosis of salivary acinar cells is not known. Interestingly, similar to other autoimmune diseases, females have been shown to be affected with SS more than their male counterparts, with greater than 90% of SS cases being diagnosed in women. One hypothesis to explain this gender difference is that loss of random X-chromosome inactivation could be the cause of this disease (since many genes involved in immune function are expressed on the X- chromosome); however, the reason for the loss of X-chromosome inactivation is not known in any autoimmune disease, including SS. Results obtained from our ongoing studies indicate that a series of key epigenetic changes are observed in SS patients. As a result transcription of a set of genes that are essential for controlling proper immune response may be decreased. In addition, loss of expression of XIST1 (that is critical for random X-chromosome inactivation) may lead to the activation of certain genes on the X- chromosome that increases T cell activation, and initiates apoptosis. Furthermore, most of the loss of methylation on the X-chromosome was found in the CpG islands, which could lead to chromosomal instability and loss of imprinting. To further understand the mechanism, we performed a global RNA seq analysis on control and SS samples and have identified that a master regulator gene ELF4 that is present on the X- chromosome was upregulated (due to loss of X-chromosome inactivation) and could assist in the pathology of SS. These results are novel, and suggest a strong epigenetic origin for SS, but they need to be further validated. Therefore, in this grant proposal we intend to thoroughly characterize the role of epigenetic changes in salivary gland destruction and to determine the relationship between abnormal methylation and X- chromosome inactivation. The hypothesis of this study is that epigenetic changes along with the loss of X- chromosome inactivation alters ELF4 that increases susceptibility to immune changes and promote apoptosis of acinar cells, thereby leading to salivary gland destruction. Thus, identification of the mechanism as well as the pathways that lead to salivary gland destruction could represent as drug targets in salivary gland dysfunction. We will coordinate our efforts in order to determine the functional significance of inhibiting epigenetic changes in order to protect against salivary gland destruction. The results of our studies are expected to provide new insights into the role of epigenetic changes and the molecular mechanism involved in salivary gland destruction. Greater understanding of these events will be important in elucidating new therapy for salivary gland dysfunctions and Sjögerns patients.

Project Summary Saliva performs a number of extremely important biological functions that are instrumental in maintaining oral health. It has been estimated that more than 5 million people in the US suffers from salivary gland dysfunctions that occurs as a results of various pathological conditions such as radiation therapy for head and neck cancer and Sjögerns syndrome. Loss of saliva in these patients causes mastication and swallowing difficulties, burning sensation to the mouth and dysgeusia. Although current treatment options reduce the severity of the symptoms, they cannot restore acini function. Thus, efforts to restore salivary gland tissues are of utmost importance for these patients. Importantly, stem cells have been suggested as possible source for tissue regeneration; it has not been used for salivary gland regeneration. One of the major issues in using stem cells for regeneration is that soon after introduction of these stem cells in an organ system, they start differentiating, thereby not providing enough stem cells that can restore the organ function. Results obtained from our ongoing studies indicate that loss of calcium entry induces stem cell differentiation, thereby inhibiting stem cell proliferation. Furthermore we have identified that Ca2+ entry in mesenchymal stem cells is mediated via the store-operated Ca2+ entry (SOCE) mechanism that is dependent on TRPC1-STIM1 interaction. Importantly, overexpression of a mutant STIM1 construct (STIM1D76A) functionally interacts with TRPC1-Orai1 complex (plasma membrane Ca2+ channels that function as SOCE channels) and constitutively activates Ca2+ entry that is essential for cell proliferation. These results are novel, and suggest a strong relationship with regard to changes in calcium handling that can promote proliferation, but they need to be further validated. Therefore, in this grant proposal we intend to thoroughly characterize the role of changes in calcium signaling and to determine if manipulation of these calcium signaling modulators can restore radiation induced damaged salivary glands. The hypothesis of this study is that regulated Ca2+ entry can support c-kit positive mesenchymal stem cell proliferation that can regenerate salivary gland cells and restore salivary secretion. Thus, identification of the mechanism as well as the factors that lead to salivary gland restoration could represent as new therapeutic options for patients that suffer from salivary gland dysfunction. We have initiated collaboration with a group, who have developed this technique and will coordinate our efforts in order to determine the functional significance of calcium channel in regulating stem cell proliferation and differentiation to restore saliva secretion. We will coordinate our efforts in order to determine the functional significance of mesenchymal stem cells in restoring salivary gland function. We will also investigate the mechanism and the ion channels involved in stem cell proliferation/differentiation. The results of our studies are expected to provide new insights into the role of stem cells as possible reagents for salivary gland regeneration. Greater understanding of these events will be important in elucidating new therapy for patients that suffer from salivary gland dysfunctions.