Reinhold Penner - US grants

DESCRIPTION: Microglia cells are the major fully immunocompetent cells of the brain, where they serve similar functions as macrophages in peripheral tissues. It is becoming apparent that most chronic neurodegenerative diseases, such as Alzheimer's, and even acute events such as trauma and stroke have strong immunological components involving microglia cells. Other immunocompetent cells use calcium signaling as central mechanism for numerous cellular functions, including proliferation. However, the involvement of calcium and the role of different calcium signaling mechanisms in microglia activation is largely unknown. Indicated in the literature and supported by our experimental observations, we hypothesize that calcium mobilization plays a pivotal role in microglia activation. We therefore propose experiments that will: 1) Identify criteria do discern activated microglia, evaluating ion channel expression levels, cytokine production, morphological changes and immunocytochemistry; 2) Explore the dependence of microglia activation on the magnitude, duration and temporal patterns of calcium signals; 3) Investigate the role of CRAC channels in microglia activation; 4) Assess the role of ATP-gated channels in microglia activation; 5) Assess the relative roles and efficacies of CRAC and ATP-gated channels in activating microglia; 6) Identify physiological and pathological microglia activators involving calcium signaling and 7) Investigate the cross-talk of calcium signaling mechanisms initiated by various microglia activators. The laboratory's approach to study these questions is at the single-cell level and employs a combination of biophysical techniques (patch-clamp electrophysiology, video microscopy, and dual-wavelength fluorometry). We will assess physiological parameters such as ionic fluxes, membrane potential, and intracellular calcium levels. These techniques are complemented by immunofluorescence and pharmacological tools to investigate calcium signaling in microglia cells. Answers to the questions above will enlighten the understanding of the calcium signaling events in microglia cells and may yield information to identify future targets for therapeutic intervention in the serious CNS pathologies that are attributable to microglia activation.

DESCRIPTION (provided by applicant): Inositol 1,4,5-trisphosphate (InsP3) ligation of its receptor (InsP3R) results in release of stored intracellular calcium. This store-depletion couples to subsequent calcium influx via the store-operated calcium current Icrac. Calcium, derived from both intracellular store release and influx via Icrac, regulates a remarkably diverse range of cellular processes including growth, differentiation, death, and the acute effector responses of immune system cells to antigen. InsP3 levels are tightly controlled, and calcium release and influx responses can be dissociated by their differential sensitivity to cytosolic InsP3 levels. Modulation of either of these calcium signals for therapeutic purposes may be achieved via manipulation of the threshold for their generation. Here we propose to investigate the mechanisms that set the response threshold for InsP3-driven calcium release and influx. We hypothesize that both intrinsic properties of the InsP3R, and the environment provided by enzymes that metabolize InsP3, may be critical determinants of the specific activity of a given concentration of InsP3. Moreover, we will investigate the contribution of these factors to the generation of specific calcium-store subcompartments with different response thresholds, such as the calcium store that couples to Icrac activation (the CRAC store). This compartment is notable for its low sensitivity (i.e., high threshold) to InsP3. Finally, we will explore the biology of phosphoinositides other than Ins (1,4,5) P3 in the control of calcium release and influx responses in immune system cells.

DESCRIPTION (provided by applicant): Sustained calcium entry, in many cells, is fundamental to the initiation and maintenance of specific cellular responses. In addition to the widespread store-operated calcium influx mechanism, whose molecular nature remains elusive, other putative calcium influx pathways have emerged through identification of a growing number of genes coding for calcium-permeable cation channels. Our preliminary data describe the molecular characterization of a novel calcium-entry pathway controlled by ADP-ribose (ADPR). This characterization includes the identification of a novel highly specific ADP-ribose hydrolase, NUDT9, which shares high homology with the C-terminal region of a previously identified gene, LTRPC2, and the functional demonstration that LTRPC2 encodes a protein product that is an ADPR-gated calcium-permeable cation channel. We also identified natively expressed ADPR-dependent conductances in pancreatic beta cells and human monocytes. Based on these data, we propose to explore the mechanisms that regulate ADPRmediated calcium entry in recombinant and physiological systems. In Specific Aim 1, we will analyze the biophysical and molecular aspects of LTRPC2 ion channel function by using a combination of calcium imaging and electrophysiological analysis of cells that express recombinant LTRPC2. To identify the structural basis for ADPR-dependent gating of LTRPC2, we will express and characterize truncated or chimeric LTRPC2 constructs directed towards the C-terminal nudix domain, the putative ADPR binding region. We also propose to systematically alter amino acids within this domain to assess structure-function relationships that confer selectivity for ADPR gating. In Specific Aim 2, we will address the functional and physiological role of ADPR-gated channels in the regulation of calcium homeostasis of beta cells. We will investigate the specific properties of native ADPR-gated channels and compare them with those of recombinant LTRPC2. We will assess their functional role in the cellular responses of the above cells by comparing the relative contributions of ADPR-gated Ca2+ signals to those of store-operated Ca2+ influx and voltage-dependent Ca2+ channels. Finally, we will seek to identify the mechanisms responsible for ADPR production by investigating the major enzymes and pathways involved in its metabolism.

DESCRIPTION (provided by applicant): Inositol 1,4,5-trisphosphate (InsP3) ligation of its receptor (InsP3R) results in release of stored intracellular calcium. This store-depletion couples to subsequent calcium influx via the store-operated calcium current ICRAC. Calcium, derived from both intracellular store release and influx via ICRAC, regulates a remarkably diverse range of cellular processes including growth, differentiation, death, and the acute effector responses of immune system cells to antigen. InsP3 levels are tightly controlled, and calcium release and influx responses can be dissociated by their differential sensitivity to cytosolic InsP3 levels. Modulation of either of these calcium signals for therapeutic purposes may be achieved via manipulation of the threshold for their generation. Here we propose to investigate the mechanisms that set the response threshold for InsP3- driven calcium release and influx. We hypothesize that both intrinsic properties of the InsP3R, and the environment provided by enzymes that metabolize InsP3, may be critical determinants of the specific activity of a given concentration of InsP3. Moreover, we will investigate the contribution of these factors to the generation of specific calcium-store sub-compartments with different response thresholds, such as the calcium store that couples to ICRAC activation (the 'CRAC store'). This compartment is notable for its low sensitivity (i.e., high threshold) to InsP3.

[unreadable] DESCRIPTION (provided by applicant): The transient receptor potential (TRP) ion channel family recently emerged as harboring crucial channel proteins involved in processes such as sensory perception, magnesium homeostasis or apoptosis. We identified TRPM2, a member of the TRP family, as a calcium-permeable cation channel gated by the novel second messenger ADP-ribose (ADPR) through the channel's intrinsic pyrophosphatase domain. TRPM2 is expressed in a variety of cell types, including pancreatic beta cells and neutrophils. Recently, the role of the molecules in the metabolic network surrounding ADPR on the functional properties of TRPM2 have emerged. Here, the novel calcium-release compound cADPR and other adenine dinucleotides materialize as crucial factors in TRPM2 physiology and hence processes involving cellular calcium homeostasis. Based on its primary activation by ADPR and reactive oxygen species, TRPM2 may have a function in cellular stress and remains a prime candidate for being involved in the apoptotic process. To further our understanding of TRPM2 in cell physiology, we propose experiments in Specific Aim 1 that will focus on the function of TRPM2 in events initiated by adenine-nucleotides. We hypothesize that TRPM2 may function as dual calcium influx and calcium release channel by taking advantage of a TRPM2 overexpressing cell system and a TRPM2 knock-out mouse model. We will explore agonist-receptor interactions engaging the main candidate in generating adenine dinucleotides, namely CD38. We will investigate the involvement of TRPM2 in cell death using a streptozotocin-based mouse model for diabetes type-1. These experiments will be performed in an expression system and mouse neutrophils and beta cells isolated from wild-type, CD38, PARP and TRPM2 knock-out mice models using a combination of patch-clamp, imaging and apoptotic studies. In Specific Aim 2 we will investigate the molecular determinants of TRPM2 agonist binding and address the question whether calmodulin is the mediator of the calcium-dependent facilitation of TRPM2. Understanding TRPM2 and adenine-dinucleotide function in calcium signaling will refine our knowledge of calcium regulation in tissues expressing this ion channel and may provide new targets for therapeutic interference in processes involving TRPM2 function, including diabetes-causing cell death of pancreatic beta cells. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): The prototypical store-operated calcium-influx pathway ICRAC (for "Ca2+-release activated Ca2+ current") was identified in 1992. Since then, substantial information has been acquired about ICRAC's physiological and clinical importance, however, its molecular composition has remained elusive. Only recently did break-through findings identify two proteins that are essential in store-operated Ca2+ influx, namely stromal interaction molecule (STIM1) and CRAC Modulator 1 ((CRACM1) or Orai1). The combined overexpression of STIM1 and CRACM1 greatly amplifies store-operated currents and these possess the most defining characteristics of ICRAC. Subsequently, it was demonstrated that CRACM1 is a pore-forming subunit of the CRAC channel. Our preliminary data further suggest that the CRACM1 homologs CRACM2 and CRACM3 also form store-operated channels with distinct properties and that STIM2 can transiently activate CRACM proteins in store-dependent and store-independent manners. We therefore hypothesize that CRACM and STIM homologs represent a group of proteins that mediate store-operated Ca2+ entry with distinct functional properties. Specific Aim 1 tests the hypothesis that specific structural features of STIM1 and CRACM1 determine their function and interaction. We will assess the properties of the three CRACM homologs by modifying molecular sites that have been identified as possible structural determinants of specific, known functional characteristics of ICRAC. The CRACM mutant constructs will be co-expressed with STIM1 in HEK293 cells and functionally analyzed using biophysical approaches, including whole-cell patch-clamp and calcium imaging. In a next step, the molecular coupling elements of STIM1/2 and CRACM1/2/3 that result in CRAC channel activation will be investigated. The proposed CRACM mutant constructs will be co-expressed with wt STIM1/2 in HEK293 cells and STIM1/2 mutants will be co-expressed with wt CRACM1/2/3. Specific Aim 2 tests the hypothesis that STIM1 and STIM2, as well as CRACM1, CRACM2 and CRACM3 are alternate molecular components of the CRAC channel conferring different channel characteristics and Ca2+ signals in native cell systems. To this end, the human cell systems Jurkat T, HEK293, and HeLa cells will be used to correlate store-operated Ca2+ channel complements with agonist-mediated Ca2+ signals. Furthermore, cellular (DT40) and animal knock-out models (CRACM and STIM KO mice) will be used to assess CRACM and STIM complement and function. The proposed work will further our understanding of the molecular definition of CRAC calcium channels, greatly improving the prospects for developing therapeutic strategies involving store-operated Ca2+ influx in allergy, inflammation and autoimmune diseases. [unreadable] [unreadable] [unreadable]

Project Summary/Abstract Around 600 million people worldwide consume Areca (betel) nuts, making it the fourth-most consumed psy- choactive natural product behind alcohol, tobacco, and coffee. Betel nut consumption has significant implica- tions for public health both globally and for the U.S. Pacific Island Territory of Guam as it is a common practice among indigenous Chamorros and segments of the Micronesian communities that reside on Guam. Areca use is associated with a high prevalence of oral carcinoma, oral pre-cancerous lesions, oral submucous fibrosis, as well as periodontal and inflammatory diseases. The leading theory for the carcinogenic effects of Areca nut chewing is that some of the alkaloids found in the Areca nut undergo chemical transformations in the oral cavi- ty to produce nitrosamine derivatives, and these are implied as the principal causative agents for the resultant oral cancers. Reactive oxygen species (ROS) are proposed to be a major cause of the cell and tissue damage associated with chronic inflammatory diseases via several pathways, including stimulation of host immune cells that release a variety of inflammatory cytokines. Areca-mediated production of pro-inflammatory mediators in the oral cavity may therefore contribute to an inflammatory microenvironment that promotes dental disease, submucous fibrosis, and ultimately the development of oral cancers. Our preliminary data demonstrate that Areca nut extracts elevate intracellular calcium concentration in several inflammatory immune cells, a process that is necessary and sufficient for calcium-mediated cytokine production. The active component(s) of the ex- tract remain unknown, as are the mechanisms underlying changes in Ca2+. Therefore, we propose a multidis- ciplinary approach that combines analytical and structural chemistry with cellular assays (biochemical, phar- macological, microfluorimetric and biophysical) as well as animal carcinogenesis models to accomplish the fol- lowing specific aims: 1) extract, identify, and characterize the active chemical components of Areca nuts that mediate calcium signals in immunocytes and determine the cellular mechanisms they engage, and 2) assess the role of non-alkaloid Areca nut components in initiating or exacerbating neoplastic transformations. These studies may inform about risks of exposure and help establish guidelines for Areca use as well as instigate cessation efforts in order to reduce the incidence of cancer.

PROJECT SUMMARY/ABSTRACT Chronic pain ? often arising from musculoskeletal injury, neurological dysfunction, cancer, or autoimmune disorders ? affects ~100 million Americans. Overreliance on opioid analgesics has resulted in a national public health crisis in which opioid overdoses have claimed over 47,000 lives in 2017 and are now the leading cause of avoidable deaths in the nation. The Cannabis plant has analgesic and anti?inflammatory properties owing to its rich content of cannabinoids, terpenes, lignans, and flavonoids. However, research on the biological effects and molecular mechanisms of the numerous bioactive phytochemicals ? alone or in combination (entourage effect) ? has been limited. We have assembled a complementary and interdisciplinary team that combines expertises in molecular and cellular signaling, ion channel biology, natural products chemistry, and molecular pharmacology as well as all aspects of endocannabinoid biology. Our preliminary high? throughput screening (HTS) bioassays have identified several cannabinoids that inhibit calcium signaling in immune cells and may therefore reduce inflammation and the associated pain. Results from the work proposed here will identify the anti?inflammatory molecules contained in Cannabis sativa and characterize the mechanisms of action they engage. We hypothesize that specific phytochemicals in Cannabis suppress cellular Ca2+ signaling and subsequent release of pro? inflammatory cytokines in immunocytes that contribute to inflammatory pain. We further hypothesize that combinations of Cannabis phytochemicals synergistically inhibit certain ion channels and G protein?coupled receptors involved in immunocyte Ca2+ signaling and cytokine release, thereby ameliorating inflammatory pain. We propose to perform pharmacological profiling of individual and entourage effects of Cannabis phytochemicals on Ca2+ signaling in 5 specific pro?inflammatory human immune cells (Aim 1A). We will determine the cellular and molecular Ca2+ mobilizing mechanisms engaged by active Cannabis phytochemicals in these immune cells (Aim 1B); and profile Cannabis phytochemicals on established molecular targets of nociceptive, inflammatory and neuropathic pain (specific TRP channels and G?proteins) using heterologous expression systems, HTS bioassays and single cell electrophysiology (Aim 1C). In Aim 2 will assess analgesic properties of active cannabinoids and combinations using in vivo mouse models of inflammatory and neuropathic pain. Here, we will first determine in vitro ?Absorption, Distribution, Metabolism, and Excretion? (ADME) properties (Aim 2A) and in vivo pharmacokinetics (Aim 2B) of said cannabinoids. We will then assess the most favorable cannabinoid(s) in Complete Freund's Adjuvant (CFA)?induced inflammation and paclitaxel?mediated toxic neuropathic pain (Aim 2C). Together, these studies will create a comprehensive and mechanistic knowledge base about the efficacy, potency and suitability of Cannabis?derived phytochemicals as anti?inflammatory analgesics and may contribute to ameliorating the current opioid epidemic.