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鞘对轴突再生的抑制作用的分子基础。待检验的具体假设是，ephrinB 3（发育期间已知的轴突排斥剂）是否是关于表达EphA 4受体的受损皮质脊髓束（CST）轴突的再生的基于髓鞘的抑制剂。目的1中的实验将采用体外测定法确定埃甫蛋白B3是否实际上抑制来自出生后皮质神经元的神经突生长，其作为抑制剂与其它已知的基于髓鞘的抑制剂相比的效力以及其对髓鞘的总体抑制活性的贡献。目的2将在神经元细胞培养系统中使用EphA 4受体酪氨酸激酶（CST发育中ephrinB 3的受体）的定点突变体，以确定该分子的细胞内部分中的哪些氨基酸是ephrin诱导的排斥/抑制所需的。从这些实验的结果，希望，导致识别参与抑制轴突再生的ephrins的关键信号分子。目标3中的研究将通过检查慢性脊髓损伤与Eph/ephrin功能的遗传和药物阻断相结合的小鼠模型来寻求扩展目标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.