Effects of Semaphorin 3A on Stroke Recovery

描述（由申请人提供）：中风发生后，大脑上调几种修复途径，试图自愈。这种修复过程效果有限的原因之一在于大脑受损区域存在生长抑制因子。本研究拟探讨轴突引导分子信号蛋白3A （Sema3A）对实验性脑卒中后恢复过程的影响。我们的假设表明，Sema3A类似于其在大脑发育中的功能，在成人神经发生和血运重建过程中，Sema3A作为生长轴突和内皮细胞的排斥剂，这种活性将对恢复过程有害。我们的前期数据表明，i) Sema3A及其受体神经肽-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收缩，并抑制脑内皮细胞系的管状形成；iii)抑制12/15-LOX可破坏Sema3A信号通路。为了扩展这些发现，我们提出以下具体目标。在Aim 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的信号通路。实验性脑卒中后神经元的轴突寻路和新血管的发育可能对脑卒中恢复至关重要，而这两者都受到Sema3A的影响。我们将专注于通过MAP激酶蛋白和12/15脂氧合酶的代谢物进行信号传导，这些代谢物已被证明可以介导Sema3A的排斥作用。在Aim 2中，我们研究了Sema3A及其受体Neuropilin 1 （NRP1）在大脑中动脉闭塞（MCAO）小鼠模型中的表达。我们将研究Sema3A表达对神经发生相关轴突连通性和脑微血管形成的差异影响，以及Sema3A水平与功能恢复的相关性。在Aim 3中，我们通过在MCAO的大脑同侧注射可溶性信号蛋白来抑制新生神经元的轴突寻路。或者，我们通过注射抑制性肽和抑制下游介质12-脂氧合酶来破坏信号素信号。中风后激活的内源性修复过程可以潜在地恢复受损大脑的功能。操纵信号素通路能导致更有效的神经元整合和改善血运重建吗？该应用与PA-08-099“中风后功能恢复机制”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".