Ephrins in Myelin-based inhibition of Regeneration

描述(申请人提供)：本研究的目的是了解髓鞘抑制轴突再生的分子基础。需要检验的具体假设是，ewitinB3，一种已知的发育中的轴突排斥剂，是否是一种基于髓鞘的抑制物，与表达EphA4受体的受损皮质脊髓束(CST)轴突的再生有关。Aim 1中的实验将使用体外测试方法来确定ewitinB3实际上是否抑制出生后皮质神经元的轴突生长，与其他已知的基于髓鞘的抑制剂相比，它作为一种抑制剂的效力，以及它对髓鞘整体抑制活性的贡献。目的2将在神经细胞培养系统中使用EphA4受体酪氨酸激酶(在CST发育过程中EphA4受体酪氨酸激酶的受体)的定点突变体，以确定该分子细胞内部分的哪些氨基酸是eaffin诱导的排斥/抑制所必需的。这些实验的结果将有望导致关键信号分子的识别，这些分子参与了肾上腺素对轴突再生的抑制作用。目标3中的研究将通过检测慢性脊髓损伤的小鼠模型以及Eph/Ephin功能的遗传和药物阻断来寻求扩展目标1中的实验结果。

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.