Effects of Semaphorin 3A on Stroke Recovery

描述(由申请者提供)：中风发生后，大脑上调几条修复途径，试图自我修复。这种修复过程效果有限的原因之一是大脑受损区域存在生长抑制因子。在这里，我们建议研究通过轴突引导分子信号素3A(Sema3A)发出的信号对实验性卒中后恢复过程的影响。我们的假说表明，与其在发育中的大脑中的功能类似，Sema3A在成人神经发生和血管重建过程中对生长的轴突和内皮细胞起到排斥作用，这种活动将对恢复过程不利。我们的初步数据表明，i)Sema3A及其受体Neuropilin-1(NRP-1)在缺血脑中上调；ii)重组Sema3A导致培养的皮质神经元轴突回缩，并抑制脑内皮细胞系中管状细胞的形成；iii)Sema3A信号可被抑制12/15-LOX干扰。为了扩大这些发现，我们提出了以下具体目标。在目标1中，我们使用培养的原代神经元和脑微血管内皮细胞系来研究Sema3A作用的信号通路。实验性卒中后神经元中轴突的寻路和新血管的形成可能是卒中康复的关键，两者都受到Sema3A的影响。我们将集中于通过MAP激酶蛋白和12/15-脂氧合酶的代谢产物来传递信号，这些已经被证明介导了Sema3A的排斥作用。目的2研究Sema3A及其受体Neuropilin 1(Nrp1)在小鼠大脑中动脉闭塞(MCAO)模型中的表达。我们将研究SEMA表达对神经发生相关轴突连接和脑微血管形成的不同影响，以及Sema3A水平与功能恢复的相关性。在目标3中，我们通过在大脑MCAO的同侧脑内注射可溶性信号素来抑制新生神经元的轴突寻径。或者，我们通过注射抑制性类肽和抑制下游的12-脂氧合酶来干扰信号素信号传递。中风后激活的内源性修复过程可能会恢复受损大脑的功能。操纵信号素通路能否导致更有效的神经元整合和改善的血管重建？本申请与PA-08-099《卒中后功能恢复机制》相关。 公共卫生相关性：中风的康复可能会从内源性修复过程中受益，通过内源性修复过程，受伤的大脑试图自我愈合。不幸的是，仍然存在许多挑战，因为包括信号素3A(Sema3A)在内的抑制分子不利于成功的重建过程。通过研究Sema3A抑制轴突延伸和新血管形成的分子途径，我们希望找到促进中风后恢复过程的治疗方案。

DESCRIPTION (provided by applicant): After a stroke occurs, the brain up-regulates several repair pathways in an attempt to heal itself. One of the reasons for limited efficacy of this repair process lies in the presence of growth inhibitory factors in the injured region of the brain. Here, we propose to investigate the influence of signaling via the axon guidance molecule semaphorin 3A (Sema3A) on the recovery process after experimental stroke. Our hypothesis states that, analogous to its function in the developing brain, Sema3A acts as a repellent for growing axons and endothelial cells during adult neurogenesis and revascularization, and that this activity will be detrimental for the recovery process. Our pilot data suggest that i) Sema3A and its receptor neuropilin-1 (NRP-1), are up- regulated in the ischemic brain; ii) recombinant Sema3A leads to axon retraction in cultured cortical neurons, and inhibits tube formation in a brain endothelial cell line; and iii) Sema3A signaling can be disrupted by inhibition of 12/15-LOX. To expand these findings, we propose the following specific aims. In Aim 1, we use cultured primary neurons and a brain microvascular endothelial cell line to study the signaling pathways through which Sema3A operates. Both axonal pathfinding in neurons and development of new blood vessels following experimental stroke may be crucial for stroke recovery, and both are impacted by Sema3A. We will focus on signaling through the MAP kinase proteins, and on metabolites of 12/15-lipoxygenase, which have been shown to mediate the repulsive actions of Sema3A. In Aim 2, we study the expression of Sema3A and its receptor Neuropilin 1 (NRP1), in a mouse model of middle cerebral artery occlusion (MCAO). We will investigate differential effects of sema expression on neurogenesis-related axonal connectivity and the formation of brain microvessels, and correlate levels of Sema3A with functional recovery. In Aim 3, we inhibit axonal pathfinding of newly born neurons by injecting soluble semaphorin on the ipsilateral side of the brain subjected to MCAO. Alternatively, we disrupt semaphorin signaling by injecting an inhibitory peptoid, and by inhibiting the downstream mediator 12-lipoxygenase. Endogenous repair processes activated after stroke can potentially restore functionality to the damaged brain. Can manipulating the semaphorin pathway lead to more efficient integration of neurons and an improved revascularization? This application is relevant to PA-08-099 "Mechanisms of functional recovery after stroke". PUBLIC HEALTH RELEVANCE: Recovery from stroke could potentially benefit from endogenous repair processes, by which the injured brain tries to heal itself. Unfortunately, many challenges remain, because inhibitory molecules including semaphorin 3A (Sema3A) are detrimental to a successful rebuilding process. By investigating the molecular pathways by which Sema3A inhibits axon extension and the formation of new blood vessels, we hope to find treatment options to enhance the recovery process following stroke.