Depolarization-induced Ca Stores Release in CNS Axons

描述：中枢神经系统白质内的轴突连接在传递电信号方面起着至关重要的作用。常见疾病如中风、脊髓损伤和多发性硬化症等会对脑白质结构造成损害，但脑白质的病理生理机制尚不清楚。我们推测，在中枢神经系统白质中，由生理性(如活动)或病理性(如缺血、创伤)去极化触发的细胞内钙释放是由L类钙通道的门控触发的，进而激活内质网上的兰尼定受体。 具体目标包括： 1)研究生理条件下有髓轴突和神经胶质细胞内质网钙释放的机制，特别是L钙通道(DHPR)和兰尼定受体(RYRs)之间的相互作用。 2)研究病理条件(如缺氧、缺血)下有髓轴突和胶质细胞内质网钙释放的机制，重点研究DHPR和RyRs之间的相互作用。 3)研究DHPR插入轴突细胞膜及钙通道密度增加对RyRs门控和钙释放的影响。 4)研究DHPR和RyR不同亚型在轴突和胶质细胞中的分布和相互作用。有髓纤维将使用电生理学、电离钙成像、亚细胞总钙测量(这将更好地反映储存的非电离钙)来研究。初步研究将检验在生理条件下如何通过调节Cav-RyR相互作用来改变轴突钙水平。然后，我们将更详细地研究内储物过度释放如何导致轴突损伤，重点是轴突L型钙通道在激活RyRs和背柱轴突钙释放中所起的作用。我们将探讨这种机制的生理目的，并更深入地分析其对缺血损伤的贡献，目的是在缺血(以及可能的创伤和炎症发作)期间抑制这种强大的钙来源机制。 我们将探讨缺氧/缺血轴突内钙通道密度是否增加，从而促进病理性的“兴奋-收缩偶联”--如钙释放。包括电生理学、双光子共聚焦离子成像、能量色散X射线显微分析和免疫组织化学在内的技术组合，以及使用药理学和分子生物学的内部存储操作，将被应用于解决四个具体目标。

DESCRIPTION: Axonal connections within the white matter of the central nervous system play the crucial role of transmitting electrical signals. Common diseases such as stroke, spinal cord injury and multiple sclerosis cause damage to white matter structures, yet white matter pathophysiology is not well understood. We hypothesize that in CNS white matter, release of Ca from intracellular stores, triggered by physiological (eg, activity) or pathological (eg, ischemia, trauma) depolarization, is triggered by gating of L-type Ca channels which in turn activate ryanodine receptors on endoplasmic reticulum. The Specific Aims include: 1) To study the mechanisms of Ca release from endoplasmic reticulum (ER) in myelinated axons and glia under physiological conditions, with a specific focus on the interaction between L-type Ca channels (dihydropyridine receptors, DHPRs) and ryanodine receptors (RyRs). 2) To study the mechanisms of Ca release from ER in myelinated axons and glia under pathological conditions (eg, anoxia, ischemia), with a focus on the interaction between DHPRs and RyRs. 3) To study the insertion of DHPRs into axon membranes and the effect of increased Ca channel density on gating of RyRs and Ca release. 4) To study the distribution and interactions of various DHPR and RyR isoforms in axons and glia. Myelinated fibers will be studied using electrophysiology, imaging of ionized Ca, measurement of total subcellular Ca (which will better reflect stored, non-ionized Ca). Initial studies will examine how axonal Ca levels can be altered by modulation of the Cav-RyR interaction under physiological conditions. Then, we will examine in greater detail how excess release from internal stores contributes to axonal injury, with a focus on the role played by axonal L-type Ca channels that we have implicated in activating RyRs and release of Ca in dorsal column axons. The physiological purpose of such a mechanism will be explored, and the contribution to ischemic injury analyzed in greater depth, with a goal to inhibit this potent Ca sourcing machinery during ischemia (and possibly trauma and inflammatory attack as well). We will explore whether Ca channel densities are increased in anoxic/ischemic axons, thereby promoting pathological "excitation-contraction coupling"-Iike Ca release. Combinations of techniques including electrophysiology, 2-photon confocal ion imaging, energy-dispersive X-ray microanalysis and immunohistochemistry, together with manipulations of internal stores using pharmacology and molecular biology, will be applied to address the four Specific Aims.