Sodium Channels and Neuroprotection in Neuroinflammatory Disorders

描述(由申请人提供)： 我的实验室对神经炎性疾病的神经保护方法感兴趣，包括多发性硬化症和脊髓损伤，在这些疾病中，炎症已经被证明发生在受伤的脊髓内。除了针对MS等疾病的轴突离子通道进行轴突神经保护的研究外，我们感兴趣的是神经胶质细胞和免疫细胞内的电压门控Na通道在神经炎症性疾病中对其功能的贡献，以及Na通道阻断在这些细胞中的作用。在这项建议中，我们重点关注以下具体目标：1.钠通道阻滞剂与神经炎性疾病的神经保护我们已经证明，钠通道阻滞剂对EAE小鼠具有抗炎和神经保护作用，但我们观察到随着钠通道阻滞剂的停用，情况急剧恶化。钠通道阻滞剂治疗多发性硬化症的几项临床研究正在进行中。我们现在将确定EAE的恶化是否是C57/BL6小鼠独有的，以及其他钠通道阻滞剂的停用是否会加剧EAE。II.钠通道NaV1.5与星形胶质细胞在神经炎症中的功能我们已经证明，人类星形胶质细胞表达Na通道，反应性星形胶质细胞在MS病变的边缘和内部表现出强烈的NaV1.5上调。我们现在将使用包括shRNA敲除和Cre/lox敲除的方法来确定钠通道在神经炎性疾病中对星形胶质细胞的效应作用的贡献。小胶质细胞的钠通道和功能我们已经证明，小胶质细胞/巨噬细胞内存在并发挥功能的NaV1.5和Nav1.6，钠通道阻滞剂减弱了它们的迁移和吞噬作用。然而，钠通道阻断对其他小胶质细胞/巨噬细胞功能的影响还不是很清楚，有必要了解控制小胶质细胞/巨噬细胞功能的信号通路。我们已经证明ERK、p38和JNK调节小胶质细胞的迁移，并且Na通道阻断后小胶质细胞中磷酸化的ERK水平减弱。最近，有研究表明，丝裂原激活的蛋白激酶磷酸酶(MPK)的诱导，使ERK去磷酸化，减少了小胶质细胞的迁移。我们现在将研究钠通道对激活的小胶质细胞的多种功能和信号通路的贡献。中枢神经损伤的轴突可以从轴突切断的初始位置发生实质性的回缩(回缩)。这可能会干扰沿更近端的、最初未受损伤的轴突分支的功能传递；或者可能导致持续侧支的退化和神经元的退化。我们和其他人已经证明了小胶质细胞/巨噬细胞可以吞噬横断的轴突卵圆形，但关于小胶质细胞在轴突变性中所起作用的细节还不清楚。因此，我们将研究小胶质细胞/巨噬细胞在轴突变性中的作用。IV.人类多发性硬化症组织的研究在我们先前对人类多发性硬化症组织的研究的基础上，我们将从我们的体外研究和对EAE的研究中推断，在人类多发性硬化症病变中，假设MS病变活跃边缘的星形胶质细胞在Na通道表达方面与这些病变中心部分的星形胶质细胞不同；MS病变活跃边缘的小胶质细胞/巨噬细胞在这些病变中心部分的Na通道表达方面与这些病变中心部分的小胶质细胞/巨噬细胞相比显示不同的特性；以及小胶质细胞/巨噬细胞介导MS中的轴突收缩。

DESCRIPTION (provided by applicant): My laboratory is interested in neuroprotective approaches in neuroinflammatory disorders, including MS and SCI, where inflammation has been shown to occur within the injured spinal cord. In addition to studies that target axonal ion channels for axonal neuroprotection in disorders such as MS, we are interested in the contribution of voltage-gated Na channels within glial and immune cells to their functions in neuroinflammatory diseases, and in the effects of Na channel block in these cells. In this proposal, we focus on the following Specific Aims: I. Sodium Channel Blockers and Neuroprotection in Neuroinflammatory Disorders We have demonstrated that Na channel blockers have anti-inflammatory and neuroprotective effects in mice with EAE, but we observed acute worsening following Na channel blocker withdrawal. Several clinical studies of Na channel blockers in MS are ongoing. We will now determine whether exacerbation of EAE is unique to C57/BL6 mice and whether withdrawal of other Na channel blockers exacerbates EAE. II. Sodium Channel Nav1.5 and Functions of Astrocytes in Neuroinflammation We have demonstrated that human astrocytes express Na channels and that reactive astrocytes exhibit robust upregulation of Nav1.5 at the borders of, and within, MS lesions. We will now determine the contribution of Na channels to effector roles of astrocytes in neuroinflammatory disorders, using methods that include shRNA knock-down and Cre/lox knockouts. III. Sodium Channels and Functions of Microglia We have shown that Nav1.5 and Nav1.6 are present and functional within microglia / macrophages, and that Na channel blockade attenuates their migration and phagocytosis. However, the effects of Na channel blockade on other microglial/macrophage functions is less well understood, and there is a need to understand signaling pathways that control microglial/macrophage function. We have shown that ERK, p38 and JNK regulate migration of microglia, and that levels of phosphorylated ERK are attenuated in microglia following Na channel blockade. Recently, it was shown that induction of mitogen-activated protein kinase phosphatase (MPK), which dephosphorylates ERK, reduces migration of microglia. We will now examine the contribution of Na channels to multiple functions and signaling pathways of activated microglia. Injured axons in the CNS can undergo substantial retraction (die-back) from the initial site of axotomy. This can interfere with functional transmission along more proximal, initially uninjured axonal branches; or may lead to degeneration of sustaining collaterals and neuronal degeneration. We and others have demonstrated that microglia/macrophages can engulf transected axonal ovoids, but details of the role of microglia in axonal degeneration are not yet understood. We will therefore study the role of microglia/macrophages in axonal degeneration. IV. Studies in Human MS Tissue Building upon our prior studies on human MS tissue, we will extrapolate from our in vitro studies and studies with EAE to test the hypotheses in human MS lesions that astrocytes at the active edge of MS lesions show different properties in terms of Na channel expression compared to astrocytes in central parts of these lesions; that microglia/macrophages at the active border of MS lesions show different properties in terms of Na channel expression compared to microglia/macrophages in central parts of these lesions; and that microglia/ macrophages mediate axonal die-back in MS.