Ephrins in Myelin-based inhibition of Regeneration

描述（由申请人提供）：本研究的目的是了解髓磷脂抑制轴突再生作用的分子基础。要验证的具体假设是，在发育过程中已知的轴突排斥剂ephrinB3是否是一种基于髓鞘的抑制剂，可以抑制表达EphA4受体的受损皮质脊髓束（CST）轴突的再生。Aim 1的实验将采用体外实验来确定ephrinB3是否实际上抑制出生后皮质神经元的神经突生长，与其他已知的髓磷脂抑制剂相比，它作为抑制剂的效力以及它对髓磷脂总体抑制活性的贡献。目的2将在神经细胞培养系统中使用EphA4受体酪氨酸激酶（CST发育中ephrinB3的受体）的位点定向突变体来确定分子胞内部分的哪些氨基酸是ephrin诱导的排斥/抑制所必需的。这些实验的结果将有望导致识别关键信号分子参与抑制ephrin轴突再生。Aim 3的研究将通过检查慢性脊髓损伤小鼠模型，结合Eph/ephrin功能的遗传和药理学阻断，寻求扩展Aim 1的实验结果。

DESCRIPTION (provided by applicant): The goal of this study is to understand the molecular basis of myelin's inhibitory effect on axonal regeneration. The specific hypothesis to be tested is whether ephrinB3, a known axon repellant during development, is a myelin-based inhibitor with respect to the regeneration of injured corticospinal tract (CST) axons that express the EphA4 receptor. Experiments in Aim 1 will employ an in vitro assay deteremine if ephrinB3 is, in fact, inhibitory to neurite outgrowth from postnatal cortical neurons, its potency as an inhibitor compared to other known myelin-based inhibitors and the contribution it makes to the overall inhibitory activity of myelin. Aim 2 will use site directed mutants of the EphA4 receptor tyrosine kinase (the receptor for ephrinB3 in CST development) in a neuronal cell culture system to determine which amino acids in the intracellular portion of the molecule are required for ephrin-induced repulsion/inhibition. Results from these experiments will, hopefully, lead to the identification of key signalling molecules involved in inhibition by ephrins of axonal regeneration. Studies in Aim 3 will seek to extend the findings of experiments in Aim 1 by examining a mouse model of chronic spinal cord injury coupled with genetic and pharmicological blockage of Eph/ephrin function.