Depolarization-induced Ca Stores Release in CNS Axons

描述：中枢神经系统白色物质内的轴突连接在传递电信号中起着至关重要的作用。常见的疾病如中风、脊髓损伤和多发性硬化会对白色物质结构造成损害，但对白色物质的病理生理学还没有很好的了解。我们假设，在CNS白色物质中，由生理性（例如，活动）或病理性（例如，缺血、创伤）去极化触发的细胞内储存的Ca释放是由L型Ca通道的门控触发的，L型Ca通道反过来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型钙通道（二氢吡啶受体，DHPRs）和兰尼碱受体（RyRs）之间的相互作用。 2)研究病理条件下（如缺氧、缺血）有髓轴突和神经胶质细胞内质网钙释放的机制，重点是DHPR和RyR之间的相互作用。 3)研究DHPRs在轴突膜上的插入以及增加钙通道密度对RyRs门控和钙释放的影响。 4)研究DHPR和RyR在神经轴突和神经胶质细胞中的分布及其相互作用。将使用电生理学、离子化Ca成像、总亚细胞Ca测量（其将更好地反映储存的非离子化Ca）研究有髓纤维。初步研究将探讨如何轴突钙水平可以通过在生理条件下的Cav-RyR相互作用的调制改变。然后，我们将更详细地研究如何从内部存储的过量释放有助于轴突损伤，重点是轴突L型钙通道，我们已经牵连在激活RyR和释放钙在背柱轴突中发挥的作用。将探索这种机制的生理目的，并更深入地分析对缺血性损伤的贡献，目标是在缺血期间抑制这种有效的Ca来源机制（以及可能的创伤和炎症发作）。 我们将探讨是否钙通道密度增加缺氧/缺血轴突，从而促进病理性的“兴奋收缩耦合”样钙释放。将应用电生理学、双光子共聚焦离子成像、能量色散X射线微量分析和免疫组织化学等技术的组合，以及使用药理学和分子生物学对内部储存的操作，以解决四个特定目标。

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.