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：在SCI之后保护白质是改善神经系统恢复之后的主要目标 脊髓损伤（SCI）。是否轴突网（AR），主要的内膜系统和Ca2+ 在轴突中存储，在SCI后有助于辅助“旁观者”中央骨髓纤维变性 仍然未知。此外，AR的主要释放通道，Ryanodine受体（RYR）和 白质损伤中的肌醇1,4,5-三磷酸盐受体（IP3R）仍然了解不足；但是，我们的 初步数据支持RYR在介导轴突的次级变性中的重要作用 临床上与体内相关的挫伤SCI。我们的总体目标是保护中央髓纤维遵循 通过靶向AR CA2+释放通道的SCI。我们将在此提案中检验以下假设。里尔 IP3R培养基内Ca2+ Ca2+的存储释放在脊柱轴突中，并引起辅助轴突 挫伤科学后的变性。因此，通过靶向RYR来抑制Ca2+存储介导的Ca2+释放 或在RYR和IP3R上收敛的上游信号通路将在SCI后保护白色物质。我们的 具体目的是：1。确定RYR和IP3R在次级轴突变性中的作用 实时挫伤SCI。 2。确定上游信号通路在AR Ca2+释放中的作用 即时的。 3。评估临床相关的方法来抑制AR介导的轴内CICR至 改善挫伤后的神经恢复。为了实现这一目标，我们将利用两光子 显微镜与超快谐振扫描仪结合（能够以每帧为420帧收集图像 第二）评估轴突肿胀，球体形成，轴突回缩，变性（Advillin-Cre：TDTOMATO 转基因小鼠），轴突Ca2+波产生和Ca2+在轴突中的积累（advillin-cre：tdtomato：tdtomato： CAMP6F和THY1GCAMP6F转基因小鼠），髓磷脂完整性（荧光亲脂染料）和AR的变化 （例如，ER跟踪器染料和3D电子显微镜），因为这些动态事件正在实时展开体内 遵循挫伤科学。提出的技术和方法可能有助于作为实时成像推进该领域 随着时间的流逝，轴突可以明确地确定受伤的轴突的命运，以及它们是否可以 通过治疗实时营救。此外，它允许直接可视化髓磷脂和这种重要的命运 仅带有轴突的元素，因为这些事件正在受伤的脊髓中展开。该提议是 创新并使用先进的成像技术来探索SCI研究的被忽视领域。方法 采取的还可能揭示潜在的新型治疗靶标和临床相关治疗（例如，FDA批准 Carvedilol）促进SCI后神经恢复。白质伤害的基本机制可能 也与多发性硬化症和其他神经系统疾病有关。