Effects of Semaphorin 3A on Stroke Recovery

描述（由申请人提供）：中风发生后，大脑上调几个修复途径，试图自我愈合。这种修复过程的功效有限的原因之一在于大脑受损区域中存在生长抑制因子。在这里，我们建议通过轴突导向分子semaphorin 3A（Sema 3A）对实验性中风后恢复过程中的信号传导的影响进行调查。我们的假设指出，类似于其在发育中的大脑中的功能，Sema 3A在成人神经发生和血管再生期间充当生长轴突和内皮细胞的排斥剂，并且这种活性将不利于恢复过程。我们的试验数据表明i）Sema 3A及其受体神经纤毛蛋白-1（NRP-1）在缺血性脑中上调; ii）重组Sema 3A导致培养的皮层神经元中的轴突回缩，并抑制脑内皮细胞系中的管形成;和iii）Sema 3A信号传导可通过抑制12/15-LOX而被破坏。为了扩大这些发现，我们提出了以下具体目标。在目的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Sema 3A通过其运作的信号通路。实验性中风后神经元中的轴突寻路和新血管的发育可能对中风恢复至关重要，两者都受到Sema 3A的影响。我们将专注于通过MAP激酶蛋白质的信号传导，以及12/15-脂氧合酶的代谢物，这些代谢物已被证明介导Sema 3A的排斥作用。目的二：研究Sema 3A及其受体神经纤毛蛋白1（Neuropilin 1，NRP 1）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，并将Sema 3A水平与功能恢复相关联。在目的3中，我们通过在MCAO的大脑同侧注射可溶性semaphorin来抑制新生神经元的轴突寻路。或者，我们通过注射抑制性类肽和抑制下游介体12-脂氧合酶来破坏脑信号蛋白信号传导。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。操纵脑信号蛋白通路能导致更有效的神经元整合和改善血管重建吗？本申请与PA-08-099“卒中后功能恢复机制”相关。

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".