REGULATION OF GLIOTRANSMISSION BY STORE-OPERATED CALCIUM CHANNELS

 描述（由申请人提供）：在中枢神经系统中，星形胶质细胞与突触前和突触后神经元末端形成三重突触，并可通过分泌“胶质递质”（如谷氨酸、ATP和D-丝氨酸）参与与神经元的双向通信，这些神经递质可影响神经元活性。以前的研究表明，介导这些分泌事件的主要机制是由细胞内钙（Ca 2+）水平升高引起的囊泡胞吐释放。然而，参与产生这些Ca 2+依赖性事件的分子和途径仍然知之甚少。虽然星形胶质细胞是电不可兴奋的细胞，但它们表现出一种形式的兴奋性， 对细胞质Ca 2+水平升高的生理和病理信号的反应。这主要是由许多G蛋白偶联细胞表面受体（GPCR）下游的内质网（ER）肌醇1，4，5三磷酸（IP 3）受体激活后从ER释放Ca 2+介导的。重要的是，ER Ca 2+储存的排空激活了被称为通过储存操作通道（SOC）的储存操作的Ca 2+进入（SOCE）的次级Ca 2+内流机制。该建议旨在研究SOCE与星形胶质细胞胶质递质胞吐的功能相关性，以获得星形胶质细胞如何调节突触生理学的机制见解。初步证据表明，在星形胶质细胞中引起Ca 2+进入的主要途径的SOC是由经典Orai 1-STIM 1蛋白产生的Ca 2+释放激活的Ca 2+（CRAC）通道。此外，初步实验表明，CRAC通道介导嘌呤能GPCR激活的星形胶质细胞中的Ca 2+信号，并且SOCE的激活刺激星形胶质细胞的胞吐和ATP分泌。因此，该项目的中心假设是CRAC通道通过在星形胶质细胞中充当重要的Ca 2+递送途径来调节胶质传递并使星形胶质细胞-神经元通信成为可能。这一假设将通过两个具体的目标，将采用多学科的方法相结合的成像，电生理和生化方法，以及缺乏CRAC通道功能的基因工程小鼠来解决。目的1将明确CRAC通道在胶质递质胞吐中的功能作用。目的2将试图确定CRAC通道对星形胶质细胞调节突触传递的意义。这些目标的发现将揭示钙离子信号通路的作用，重要的调节钙离子稳态和效应器功能的星形胶质细胞。由于星形胶质细胞具有专门的突触通路，该信号通路可能是开发用于影响突触功能的病理性疾病的新疗法的有价值的靶点。

 DESCRIPTION (provided by applicant): In the central nervous system, astrocytes form tripartite synapses with presynaptic and postsynaptic neuronal terminals and can participate in bidirectional communication with neurons through secretion of 'gliotransmitters' such as glutamate, ATP, and D-serine that can impact neuronal activity. Previous studies have suggested that a major mechanism mediating these secretion events is vesicular exocytotic release evoked by elevations in intracellular calcium (Ca2+) levels. However, the molecules and pathways involved in generating these Ca2+- dependent events remain poorly understood. Although astrocytes are electrically non-excitable cells, they exhibit a form of excitability based on elevations of cytoplasmic Ca2+ levels in response to physiological and pathological signals. This is primarily mediated by the release of Ca2+ from the endoplasmic reticulum (ER) after activation of ER inositol 1,4,5 triphosphate (IP3) receptors downstream of numerous G-protein-coupled cell surface receptors (GPCRs). Importantly, the emptying of ER Ca2+ stores activates a secondary Ca2+ influx mechanism known as store-operated Ca2+ entry (SOCE) through store-operated channels (SOCs). This proposal seeks to examine the functional relevance of SOCE to astrocyte gliotransmitter exocytosis in order to gain mechanistic insight into how astrocytes can modulate synaptic physiology. Preliminary evidence indicates that the SOCs that give rise to a major route of Ca2+ entry in astrocytes are the Ca2+ release-activated Ca2+ (CRAC) channels arising from the canonical Orai1-STIM1 proteins. Further, preliminary experiments show that CRAC channels mediate purinergic GPCR-activated Ca2+ signals in astrocytes and that activation of SOCE stimulates exocytosis and ATP secretion from astrocytes. Therefore, the central hypothesis of this project is that CRAC channels regulate gliotransmission and enable astrocyte-neuron communication by serving as a vital Ca2+ delivery pathway in astrocytes. This hypothesis will be addressed through two specific aims that will employ a multidisciplinary approach combining imaging, electrophysiological, and biochemical approaches as well as genetically engineered mice lacking CRAC channel function. Aim 1 will define the functional role of CRAC channels for gliotransmitter exocytosis. Aim 2 will seek to determine the significance of CRAC channels for astrocyte modulation of synaptic transmission. Findings from these aims will reveal the role of a Ca2+ signaling pathway important for regulating Ca2+ homeostasis and effector functions in astrocytes. Since astrocytes have specialized access to synapses, this signaling pathway may be a valuable target for the development of novel therapeutics for pathological diseases affecting synaptic function.