Murali Prakriya - US grants

Project Abstract Astrocytes comprise the major cell type in the brain and regulate numerous functions including neural development, clearance of neurotransmitters, and regulation of blood flow. Recent evidence indicates that astrocytes also play a direct role in regulating synaptic transmission by modulating neuronal activity through the secretion of `gliotransmitters' such as glutamate, ATP, and D-serine. Astrocytes are intimately associated with pre- and post-synaptic neuronal membranes in an anatomical unit referred to as the tripartite synapse. This close association allows astrocytes to sense neurotransmitters released by neurons, and conversely communicate with neighboring neurons through the action of gliotransmitters. Previous studies have suggested that a major mechanism mediating release of gliotransmitters is vesicular exocytosis evoked by elevations in intracellular [Ca2+]i. However, the molecules and pathways involved in generating Ca2+ elevations in astrocytes remain poorly understood. Our preliminary evidence indicates that store-operated Ca2+ release-activated Ca2+ (CRAC) channels are a major mechanism for neurotransmitter-evoked Ca2+ signals in astrocytes. We further find that ablation of CRAC channel expression or pharmacological blockade suppresses gliotransmitter release. Based on this evidence, we hypothesize that CRAC channels are essential regulators of astrocyte gliotransmitter exocytosis and the bidirectional communication between neurons and astrocytes at the tripartite synapse. We propose the three specific aims to test this hypothesis: 1) Define the molecular composition of CRAC channels in astrocytes and their contribution for the generation of complex astrocyte Ca2+ signals. 2) Determine the role of CRAC channels for secretion of gliotransmitters, and 3) Determine the role of CRAC channels for astrocyte modulation of synaptic transmission. We will approach these questions using a multidisciplinary approach that combines genetic knockouts of CRAC channel proteins with in-depth molecular and biochemical assays, whole-cell and slice electrophysiological recordings, Ca2+ imaging using wide-field and spinning disk confocal microscopy, and gliotransmission assays. Collectively, results from these studies will advance our understanding of the physiological role of CRAC channels for regulating Ca2+ homeostasis and gliotransmission in astrocytes and aid the quest for developing new therapies for pathological diseases affecting synaptic function.

DESCRIPTION (provided by applicant): Calcium (Ca2+) is a ubiquitous intracellular signal, which is responsible for controlling numerous cellular processes. This proposal focuses on understanding of Ca2+-homeostasis regulation using the power of C. elegans molecular genetics. This proposal is directed toward an investigation of functions and mechanisms of the intercellular Ca +-wave propagation and the store-operated Ca2+ (SOC) channels, which are part of the inositol 1,4,5-trisphosphate (IPS)-dependent pathway. The intercellular Ca2+-wave propagation is observed in a variety of cell types in many different species. It has been proposed that the intercellular Ca2+ waves function as synchronizing cellular activities in a particular organ. We first observed the intercellular Ca2+-wave propagation in C. elegans intestine, but its function is not known at all. Since intercellular Ca2+-wave propagation is a widely observed biological phenomena, this can be an important model system to investigate its mechanism and function using genetics. The SOC channels have an important function for Ca2+ uptake from the extracellular solution upon depletion of intracellular Ca2+ stores. It is speculated that the SOC influx has an important function for neural signaling and the pathogenesis of some neurodegenerative diseases: SOC influx may play an important role in the early development of Alzheimer's disease. Despite the biological and clinical importance of the SOC channels, their molecular identity is highly controversial. We will test the hypothesis that any transient receptor potential channels are responsible for the SOC activity in the C. elegans intestine. To study Ca2+ homeostasis, we developed a unique assay system by combining genetic, Ca2+-imaging, and electrophysiological approaches. This uniqueness of the system could contribute to revealing new aspects in the regulatory mechanisms of Ca2+ homeostasis, which were not able to be addressed well in other systems. Using this assay system, we will address the following three issues 1) investigating the function of the intercellular Ca2+-wave propagation, 2) identifying the store-operated Ca2+ channels using reverse genetics, and 3) isolation of mutations that affect Ca2+ homeostasis using forward genetics. The second and third aims will complement each other to identify important molecules for regulation of Ca2+ homeostasis. Completion of this project will provide new insights into human disorders that are caused by abnormal Ca2+ homeostasis.

? DESCRIPTION (provided by applicant): In many animal cells, stimulation of cell surface receptors coupled to G proteins or tyrosine kinases mobilizes Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. The ensuing Ca2+ entry regulates a wide variety of effector cell responses including transcription, motility, and proliferation. CRAC channels exhibit a unique biophysical fingerprint characterized by exquisite Ca2+-selectivity, store-operated gating, and distinct pore properties, and serve as fascinating ion channels for understanding the biophysical mechanisms of ion permeation and gating. Moreover, because CRAC channels sit squarely at the nexus of the cellular Ca2+ signaling network in many cells, aberrant CRAC channel function is implicated in the etiology of several diseases including chronic inflammation, muscle weakness, and a severe combined immunodeficiency syndrome. Much has been learned in the last decade of the physiological roles of CRAC channels in different tissues and the cellular choreography of the CRAC channel activation process. Still, the molecular and structural basis of how depletion of ER Ca2+ stores regulates the opening of the CRAC channel pore continues to remain poorly understood. Opening of CRAC channels is governed through direct interactions between the pore-forming Orai proteins, and the ER Ca2+ sensor, STIM1, but how STIM1 binding transduces opening of the Orai1 channel pore remains unclear. Here, we propose a multi-pronged approach that will investigate several mechanistic aspects of the CRAC channel gating process, focusing on the molecular rearrangements in the CRAC channel pore and the conformational alterations in STIM1. We will employ an interdisciplinary experimental design for these studies that combines patch-clamp electrophysiology, cysteine mutagenesis, FRET microscopy, biochemical cross-linking, and protein engineering. Using the Orai1 protein as a prototypic CRAC channel, our goals are to: (1) test the hypothesis that opening of the CRAC channel pore occurs through rotation of the TM1 helix, thereby reorienting the hydrophobic V102 side-chains to remove an energy barrier, (2) elucidate the inner pore architecture and its role in regulating ion conduction in Orai1 channels, and (3) examine the conformational rearrangements in the critically important cytoplasmic region of STIM1 during activation and binding to Orai1. These studies will significantly advance our understanding of the molecular and structural mechanisms of CRAC channel activation and open new avenues for harnessing the therapeutic potential of CRAC channels for disease intervention.

? DESCRIPTION (provided by applicant): The chemokine receptor CXCR4 and its endogenous ligand SDF-1 are expressed in the neurogenic regions of the adult brain and are critical for guiding neurogenesis and migration of neural stem and progenitor cells. Recent evidence indicates that chemokines also regulate the proliferative and restorative response of the brain following focal cell death in brain injuries and neurological diseases. Activated chemokine receptors stimulate several cellular effectors, of which the mobilization of Ca2+ is a key signaling process necessary for its biological effects. As a multifunctional second messenger, Ca2+ activates distinct genetic programs that control many processes such as the proliferation of neural progenitor cells (NPCs), NPC migration, and differentiation of NPCs into mature neurons and glia. However, how NPCs generate Ca2+ signals in response to chemokines and other regulatory factors remains unknown. Our preliminary studies indicate that store-operated Ca2+ release-activated Ca2+ channels (CRAC channels) are a major mechanism for chemokine-driven Ca2+ signals in NPCs. We further find that ablation of CRAC channel expression or pharmacological blockade suppresses chemokine-mediated Ca2+ signaling, NPC proliferation, and chemokine-mediated migration of NPCs to their terminal destinations. Based on this evidence, we hypothesize that CRAC channels are essential regulators of chemokine-mediated Ca2+ signaling and directed migration of NPCs. We propose the three specific aims to address this question: 1) Define the expression of STIM1/Orai1 proteins in the neurogenic regions of the brain. How does this correlate with the known expression of the CXCR4 receptor? 2) Elucidate the contribution of CRAC channels for the generation of complex Ca2+ signals by the chemokine, SDF1. 3) Determine the role of CRAC channels for chemokine-mediated migration of NPCs and neuroblasts. We will approach these questions using a multidisciplinary approach that combines in-depth protein expression studies using confocal microscopy, electrophysiology and Ca2+ imaging using 2-photon laser scanning microscopy, and in vitro and in vivo cell migration assays. Collectively, results from these studies will advance our understanding of the physiological role of CRAC channels for the directed migration of neural progenitors and aid the quest for developing new therapies for brain repair following injuries and in neurodegenerative diseases.

Abstract Ca2+ is a ubiquitous signaling messenger mediating many essential functions such as excitability, exocytosis and transcription. In the airway epithelial cells (AECs) lining the conducting airways of the lung, Ca2+ signals are implicated in numerous cellular functions through effects on enzymatic cascades and transcriptional factors. Ca2+ homeostasis is also closely integrated with other signaling systems including that of reactive oxygen species (ROS), which has many cellular functions of its own and is implicated in allergic lung diseases and injury. We have recently shown that store-operated Ca2+ release-activated Ca2+ (CRAC) channels serve as the main pathway for Ca2+ entry in bronchial epithelial cells and their activation leads to increases in levels of numerous proinflammatory cytokines. However, the broader significance of CRAC channels in AECs for mediating airway inflammation and potential crosstalk with ROS and other signaling pathways mediating inflammation remains unknown. We hypothesize that CRAC channels are a major mechanism for controlling the generation of inflammatory mediators from AECs in response to stimulation of cell surface G-protein coupled receptors such as protease-activated and ATP receptors, and are an essential mediator of pulmonary inflammation in diseases such as asthma. We will address this hypothesis through three specific goals: 1) examine the physiological contributions of CRAC channels for allergen- mediated release of ATP and determine how this release stimulates inflammatory cytokines from AECs, 2) investigate the functional interactions between CRAC channels and mitochondrial ROS signaling and assess the impact of this crosstalk for inflammatory cytokine generation, and 3) evaluate the contribution of CRAC channels for mediating airway inflammation in vivo in a mouse model of house dust mite-induced asthma. Together, these studies will advance our understanding of how CRAC channels regulate cellular Ca2+-dependent signaling pathways to modulate allergen-stimulated inflammatory mediators in the airways, and advance the quest for developing novel treatments for allergic airway diseases.

Ca2+ signaling mediates many essential roles in neurons including transmitter release, synaptic plasticity, and gene transcription. In dendritic spines, which constitute the sites where excitatory synaptic input is received, Ca2+ elevations drive many forms of synaptic plasticity including long-term potentiation (LTP) and spine morphogenesis. The mechanism of LTP and its physiological consequences are of intense interest in neuroscience, driven by findings showing that it likely forms the neurochemical basis of learning and information storage in the brain and is altered by numerous neurodegenerative diseases, brain injuries, and drugs of abuse. However, although the physiological phenomenon of LTP is very well characterized, its underlying molecular mechanism is less understood. One area of uncertainty is the identity of the Ca2+ entry pathways involved in generating spine Ca2+ signals and how these pathways interface with downstream signaling systems. In this work, we will investigate the contributions of a relatively poorly understood Ca2+ influx pathway formed by store-operated Orai1 channels for cognitive function, dendritic spine Ca2+ signaling, and LTP. Orai1 channels have been extensively studied in immune cells where they stimulate processes ranging from Ca2+-dependent gene expression to secretion of inflammatory mediators. Although growing evidence indicates that Orai1 is highly expressed in the brain including in many regions critical for learning and memory, the properties of these channels and their physiological roles in the brain are poorly understood. We hypothesize that Orai1 channels are a key mechanism for generating Ca2+ signals in dendritic spines and make significant contributions to synaptically-evoked Ca2+ rises in spines to regulate synaptic plasticity and cognition. Using mice lacking Orai1 or its activators, STIM1 and STIM2, we will address this hypothesis through three specific goals: 1) evaluate the contributions of Orai1 channels for cognitive processes related to learning, memory, and sensorimotor function in mouse models. 2) investigate the physiological contributions of Orai1 channels for LTP, CaMKII activation, and insertion of AMPA receptors into postsynaptic densities, and 3) examine the role of Orai1 channels for Ca2+ signaling in dendritic spines following synaptic stimulation. Together, these studies will address the role of a novel Ca2+ entry pathway for synaptic plasticity and cognitive function, and ultimately facilitate efforts to target Orai1 channels for developing novel therapeutics for cognitive dysfunctions.