ER Ca2+ store mediated white matter injury

ER Ca2 储存介导的白质损伤

• 批准号: 9382367
• 负责人: David Paul Stirling
• 金额: $ 38.38万
• 依托单位: UNIVERSITY OF LOUISVILLE
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-05-01 至 2022-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9382367
• 关键词: AVIL gene; Area; Atrophic; Axon; Behavioral; Binding Proteins; Calcium; Data; Degenerative Disorder; Distal; Dyes; Electron Microscopy; Elements; Event; FDA approved; Fiber; Gait; Generations; Goals; Human; ITPR1 gene; Image; Imagery; Imaging Techniques; Injury; Inositol; Intervention; Kainic Acid Receptors; Knockout Mice; Mediating; Microscopy; Modeling; Molecular; Morphology; Multiple Sclerosis; Mus; Myelin; Nature; Neurologic Deficit; Pathologic; Pathology; Patients; Pharmacology; Process; Recovery; Recovery of Function; Reproducibility; Research; Reticulum; Role; Ryanodine Receptors; Secondary to; Signal Pathway; Signal Transduction; Site; Spinal; Spinal Cord; Spinal Cord Contusions; Spinal cord injury; Stroke; Swelling; System; Technology; Testing; Therapeutic; Time; Transgenic Mice; Wallerian Degeneration; Work; axonal degeneration; carvedilol; clinically relevant; effective therapy; functional outcomes; genetic approach; improved; in vivo; injured; innovation; kinematics; lipophilicity; loss of function; nervous system disorder; neurological recovery; new therapeutic target; novel; novel therapeutics; oxidation; prevent; receptor; spinal tract; two-photon; white matter; white matter injury

Abstract: Protecting white matter following SCI is a major goal to improve neurological recovery following spinal cord injury (SCI). Whether the axoplasmic reticulum (AR), the major endomembrane system and Ca2+ store within axons, contributes to secondary “bystander” central myelinated fiber degeneration following SCI remains unknown. Furthermore, the role of the AR's major release channels, ryanodine receptors (RyR) and inositol 1,4,5-trisphophate receptors (IP3R) in white matter injury remain poorly understood; however, our preliminary data support an important role for RyR in mediating secondary degeneration of axons following a clinically-relevant contusion SCI in vivo. Our overall objective is to protect central myelinated fibers following SCI by targeting AR Ca2+ release channels. We will test the following hypothesis within this proposal. RyR and IP3R mediate intra-axonal Ca2+ store release of Ca2+ within spinal axons and cause secondary axonal degeneration following contusion SCI. Therefore, inhibiting Ca2+ store mediated Ca2+ release by targeting RyR or upstream signaling pathways that converge on RyR and IP3R will protect white matter following SCI. Our specific aims are to: 1. Determine the role of RyR and IP3R in secondary axonal degeneration following contusion SCI in real-time. 2. Determine the role of upstream signaling pathways in AR Ca2+ release in real-time. 3. Evaluate clinically relevant approaches to inhibit AR-mediated intra-axonal CICR to improve neurological recovery following contusion SCI. To accomplish this goal we will utilize two-photon microscopy combined with an ultrafast resonant scanner (capable of collecting images at up 420 frames per second) to assess axonal swelling, spheroid formation, axonal retraction, degeneration (Advillin-Cre: tdTomato transgenic mice), axonal Ca2+ wave generation and Ca2+ accumulation in axons (Advillin-Cre: tdTomato: CaMP6f and Thy1GCaMP6f transgenic mice), myelin integrity (fluorescent lipophilic dyes), and changes in AR (e.g., ER tracker dyes and 3D electron microscopy) as these dynamic events are unfolding in real-time in vivo following a contusion SCI. The technology and approach proposed may help advance the field as live imaging of axons over time allows unequivocal determination of the fate of injured axons and whether they can be rescued in real-time with treatment. Furthermore, it allows direct visualization of myelin and the fate of this vital element simultaneously with axons as these events are unfolding in the injured spinal cord. This proposal is innovative and uses advanced imaging techniques to explore overlooked areas of SCI research. The approach taken may also unveil potential novel therapeutic targets and clinically relevant treatments (e.g. FDA approved carvedilol) to promote neurological recovery after SCI. The underlying mechanisms of white matter injury may also be relevant to multiple sclerosis and other neurological diseases.

摘要：保护SCI后的白色物质是改善SCI后神经功能恢复的主要目标。 脊髓损伤（SCI）。无论是轴浆网（AR），主要内膜系统和Ca2 + 储存在轴突内，导致SCI后继发性"旁观者"中央有髓纤维变性 仍然未知。此外，AR的主要释放通道Ryanodine受体（RyR）和 白色物质损伤中的肌醇1，4，5-三磷酸受体（IP3 R）仍然知之甚少;然而，我们的研究发现， 初步数据支持RyR在介导轴突继发性变性中的重要作用， 临床相关的挫伤SCI在体内。我们的总体目标是保护中央有髓纤维， SCI通过靶向AR Ca2+释放通道。我们将在本提案中检验以下假设。RyR 和IP3R介导脊髓轴突内Ca 2+库的释放，并引起继发性轴突损伤。 脊髓损伤后的退化。因此，通过靶向RyR抑制Ca2+库介导的Ca2+释放， 或汇聚在RyR和IP3 R上的上游信号通路将保护SCI后的白色物质。我们 具体目标是：1.确定RyR和IP3R在继发性轴突变性中的作用， 实时的创伤性脊髓损伤2.确定上游信号通路在AR Ca2+释放中的作用， 实时的3.评价抑制AR介导的轴突内CICR的临床相关方法， 改善脊髓损伤后神经功能恢复。为了实现这一目标，我们将利用双光子 显微镜结合超快共振扫描仪（能够以高达420帧/秒的速度收集图像） 第二）评估轴突肿胀、球状体形成、轴突收缩、变性（Advillin-Cre：td Tomato 转基因小鼠）、轴突Ca2+波的产生和轴突中的Ca2+积累（Advillin-Cre：tdTomato： CaMP6f和Thy1GCaMP6f转基因小鼠）、髓鞘完整性（荧光亲脂性染料）和AR的变化 (e.g., ER示踪染料和3D电子显微镜），因为这些动态事件在体内实时展开 一次创伤性脊髓损伤所提出的技术和方法可能有助于推进现场成像领域 随着时间的推移，轴突的变化可以明确确定受损轴突的命运，以及它们是否可以被修复。 实时抢救治疗。此外，它允许直接可视化髓磷脂和这个重要的细胞的命运。 元件与轴突同时发生，因为这些事件在受损的脊髓中展开。这项建议是 创新和使用先进的成像技术，探索SCI研究被忽视的领域。的方法 采取的措施还可能揭示潜在的新型治疗靶点和临床相关治疗方法（例如FDA批准的 卡维地洛）促进SCI后神经功能恢复。白色物质损伤的潜在机制可能 也与多发性硬化症和其他神经系统疾病有关。