The role of SOCE in microglia and secondary degeneration after SCI

翻译后摘要：脊髓损伤（SCI）后的神经功能的结果是依赖于原发性损伤的程度， 保留的白色物质（WM）和灰质的迟发性继发性变性。虽然Ca 2+超载是 被确立为继发性损伤的关键介质，迄今为止治疗靶向外部Ca 2+来源 在临床前研究和人类SCI后未能改善神经恢复。这项建议会 评估SCI后直接在脊髓神经元和小胶质细胞中钙池操纵的钙内流（SOCE）的作用 使用细胞类型特异性敲除关键SOCE介导物。尽管SOCE在维持Ca 2+浓度方面起着重要作用， 体内平衡和维持细胞内Ca 2+在免疫细胞功能的关键水平是必要的，我们 提出异常和过量的SOCE导致WM的继发性变性。SOCE在WM中的作用 损伤、小胶质细胞活化和功能仍然知之甚少。此外， 必需的SOCE组分，包括基质相互作用分子（stim 1和stim-2）和奥赖1-3（形成 Ca 2+通道孔）在小胶质细胞功能和神经元损伤中的作用尚不清楚。我们的初步数据支持 SOCE在介导继发性变性、小胶质细胞活化和促炎中重要作用 细胞因子释放和神经恢复恶化。我们假设SOCE的失调介导了 SCI后“旁观者”继发性轴突变性通过增加通过Stim的Ca 2+通透性和/或 奥赖通道导致轴突中的Ca 2+过载。此外，我们假设SOCE调节小胶质细胞， 激活和释放促炎因子，对SCI后的神经恢复产生负面影响。我们 具体目标是，1。确定SOCE在继发性变性和神经恢复中的作用 SCI之后。2.确定小胶质细胞SOCE在继发性变性、伤口愈合和 脊髓损伤后神经功能恢复为了实现这些目标，我们将使用双光子激发活体成像 真实的观察脊髓损伤后脊髓轴突和小胶质细胞的动态反应。我们将使用两者 药理学SOCE抑制剂和细胞类型特异性敲除关键SOCE介质直接在脊髓 神经元和小胶质细胞。最有效的方法将使用行为测试进行评估， 保留脑白质和灰质以及伤口愈合这项技术和方法可能有助于推动该领域的发展 因为轴突随时间的实时成像可以明确确定受损轴突的命运， 他们可以通过治疗实时获救。此外，它允许直接可视化小胶质细胞 与轴突及其相互作用同时发生，因为这些事件在受损的脊髓中展开。这 该提案具有创新性，并使用先进的成像技术来探索SCI研究中被忽视的领域。 所采取的方法也可能揭示新的药物靶点，并重新使用FDA批准的药物， SOCE（例如，特立氟胺）。WM损伤的潜在机制也可能与其他相关因素有关。 神经系统的神经疾病，如多发性硬化症、中风和TBI。

Abstract: Neurological outcome after spinal cord injury (SCI) is dependent on the extent of primary injury and the delayed secondary degeneration of spared white matter (WM) and grey matter. Although Ca2+ overload is established as a key mediator of secondary injury, therapeutically targeting external sources of Ca2+ to date have failed to improve neurological recovery in pre-clinical studies and following human SCI. This proposal will assess the role of store-operated Ca2+ entry (SOCE) directly in spinal cord neurons and microglia after SCI using cell type specific knockout of key SOCE mediators. Although, SOCE plays a vital role in maintaining Ca2+ homeostasis and is necessary to sustain intracellular Ca2+ at critical levels for immune cell function, we propose that aberrant and excessive SOCE causes secondary degeneration of WM. The role of SOCE in WM injury, microglial activation, and function remains poorly understood. Furthermore, the precise role of the essential SOCE components including stromal interaction molecule (stim 1 and -2), and Orai 1-3 (form the Ca2+ channel pore) in microglial function and neuronal injury remains unclear. Our preliminary data support an important role for SOCE in mediating secondary degeneration, microglial activation and proinflammatory cytokine release, and worsening neurological recovery. We hypothesize that dysregulation of SOCE mediates “bystander” secondary axonal degeneration following SCI by increasing Ca2+ permeability through Stim and/or Orai channels causing Ca2+ overload in axons. In addition, we hypothesize that SOCE regulates microglial activation and release of proinflammatory factors that negatively impacts neurological recovery after SCI. Our specific Aims are to, 1. Determine the role of SOCE in secondary degeneration and neurological recovery following SCI. 2. Determine the role of microglial SOCE in secondary degeneration, wound healing, and neurological recovery after SCI. To accomplish these aims, we will use two photon excitation intravital imaging to visualize the dynamic response of spinal cord axons and microglia after SCI in real time. We will use both pharmacological SOCE inhibitors and cell type specific knockout of key SOCE mediators directly in spinal neurons and microglia. The most efficacious approach will then be assessed using behavioral testing, amount of spared WM and grey matter, and wound healing. The technology and approach 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 microglia simultaneously with axons and their interactions 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 novel drug targets and repurpose FDA approved drugs found to inhibit SOCE (e.g., teriflunomide). The underlying mechanisms of WM injury may also be relevant to other neurological diseases of the nervous system such as multiple sclerosis, stroke, and TBI.