Novel Compounds That Overcome Glial Inhibition of Axonal Regeneration

项目摘要 CNS轴突再生的一个主要障碍是存在与轴突再生相关的生长抑制蛋白。 髓鞘碎片和神经胶质疤痕。中枢神经系统损伤后的功能恢复需要这种抑制作用， 克服最近的研究表明，cAMP的变化，沿着PKC，EGFR和RhoA的增加， 活动，是抑制信号的重要方面。然而，我们仍然缺乏知识， 与损伤部位抑制相关的抑制蛋白的数量/身份，详细的信号传导 抑制性受体所采用的机制，以及受损轴突的细胞类型特异性反应。 此外，存在与当前药理学策略相关的问题，包括缺乏特异性， 不确定的毒性，以及具有多效性功能的靶向途径。克服这些 困难，我们已经启动了一个基于表型的无偏筛选一个新的化合物库 选择其有利的化学性质，而不是已知的生物功能。屏幕是基于 化合物增加来自用抑制性髓鞘激发的CNS神经元的神经突生长的能力 印刷受体.初步结果已经产生了4种能够强烈增加神经突生长的“命中化合物”。 随后的研究表明，命中化合物a）作用于不同的神经元类型，B）选择性地 克服抑制而不是促进生长，c）是非常有效的，d）克服抑制， 与损伤相关的测定，e）不影响cAMP水平、PKC活性或EGFR活化，f）改变 微管动力学，和g）促进体内再生。因为这些化合物很有效， 选择性，并可能通过新的机制，他们是令人兴奋的候选人治疗 开发和再生抑制的机理研究。建议是1）调查 4种命中化合物的信号传导机制和蛋白靶点，2）检查1种命中化合物的能力， 化合物以促进体内脊髓损伤或视神经损伤后的再生，以及3）筛选 在新的抑制性（蛋白聚糖）底物上的完整的4000个化合物文库。这些实验可以提供 再生抑制的关键见解，并为CNS损伤的新方法铺平了道路。

Project Summary A major barrier to regeneration of CNS axons is the presence of growth-inhibitory proteins associated with myelin debris and the glial scar. Functional recovery after CNS injury requires that this inhibition be overcome. Recent studies suggest that changes in cAMP, along with increases in PKC, EGFR, and RhoA activities, are important aspects of inhibitory signaling. However, we still lack knowledge about the number/identity of inhibitory proteins associated with inhibition at injury sites, the detailed signaling mechanisms employed by inhibitory receptors, and the cell type-specific responses of damaged axons. Further, there are problems associated with current pharmacological strategies, including lack of specificity, uncertain toxicities, and the targeting of pathways with pleiotrophic functions. To overcome these difficulties, we have initiated a phenotype-based unbiased screen of a novel chemical compound library chosen for its favorable chemical properties rather than known biological function. The screen is based on the ability of compounds to increase neurite outgrowth from CNS neurons challenged with inhibitory myelin substrates. Initial results have produced 4 "hit compounds" capable of strongly increasing neurite growth. Subsequent investigations indicate that the hit compounds a) act on different neuronal types, b) selectively overcome inhibition rather than promote growth, c) are highly potent, d) overcome inhibition in distinct assays relevant to injury, e) do not affect cAMP levels, PKC activity, or EGFR activation, f) alter microtubule dynamics, and g) promote regeneration in vivo. Because the compounds are potent and selective, and may act through novel mechanisms, they are exciting candidates for therapeutic development and for mechanistic studies of regeneration inhibition. The proposal is to 1) investigate the signaling mechanisms and protein targets of the 4 hit compounds, 2) examine the ability of 1 hit compound to promote regeneration after spinal cord injury or optic nerve crush in vivo, and 3) screen the full 4000 compound library on a novel inhibitory (proteoglycan) substrate. These experiments could provide key insights into regeneration inhibition, and pave the way for a novel approach to CNS injury.