ER Ca2+ store mediated white matter injury

摘要：保护脊髓损伤后的脑白质是促进脊髓损伤后神经功能恢复的主要目标。 脊髓损伤(SCI)。轴浆网(AR)、主要内膜系统和钙离子 储存在轴突内，导致脊髓损伤后继发性“旁观者”中央有髓纤维变性 仍然不为人知。此外，AR的主要释放通道Ryanodine受体(RyR)和 脑白质损伤中的肌醇1，4，5-三磷酸受体(IP3R)仍然知之甚少；然而，我们的 初步数据支持RyR在介导急性脑损伤后轴突继发性退变中的重要作用 临床相关的活体脊髓挫伤。我们的总体目标是保护中枢有髓纤维 通过靶向AR钙释放通道致脊髓损伤。我们将在这一提议中检验以下假设。RyR IP3R介导轴突内钙离子储存脊髓轴突内钙释放，引起继发性轴突 脊髓挫伤后退行性变。因此，通过靶向RyR抑制钙离子储存介导的钙释放 或者汇聚在RyR和IP3R上的上游信号通路将保护脊髓损伤后的脑白质。我们的 具体目的是：1.确定RyR和IP3R在继发性轴索变性中的作用 挫伤脊髓损伤的实时监测。2.确定上游信号通路在肾上腺皮质激素受体钙释放中的作用 实时的。3.评估抑制AR介导的轴突内CICR的临床相关方法 促进脊髓挫伤后神经功能恢复。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(能够以每帧420帧的速度采集图像 第二)评估轴突肿胀、球体形成、轴突回缩、变性(Advilin-Cre：tdTomato 转基因小鼠)，轴突钙波的产生和轴突内钙的积累(Advilin-Cre：td番茄： CaMP6f和Thy1GCaMP6f转基因小鼠)、髓鞘完整性(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)，因为这些动态事件在体内实时展开 在一次脊髓挫伤之后。所提出的技术和方法可能有助于推动实时成像领域的发展 轴突随时间的变化可以明确地确定受损轴突的命运以及它们是否可以 经治疗后实时获救。此外，它还可以直接显示髓鞘和这一至关重要的 当这些事件在受伤的脊髓中展开时，这些元素与轴突同时发生。这项建议是 创新并使用先进的成像技术来探索被忽视的脊髓损伤研究领域。该方法 Take还可能揭示潜在的新治疗靶点和临床相关治疗方法(例如，FDA批准 卡维地洛)，以促进脊髓损伤后的神经恢复。白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。

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.