Novel Compounds That Overcome Glial Inhibition of Axonal Regeneration

项目概要 中枢神经系统轴突再生的一个主要障碍是与以下因素相关的生长抑制蛋白的存在 髓磷脂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 克服。最近的研究表明，cAMP 的变化以及 PKC、EGFR 和 RhoA 的增加 活性，是抑制信号传导的重要方面。然而，我们仍然缺乏这方面的知识 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 抑制性受体采用的机制，以及受损轴突的细胞类型特异性反应。 此外，目前的药理学策略还存在一些问题，包括缺乏特异性、 不确定的毒性，以及具有多效功能的靶向途径。为了克服这些 克服困难，我们启动了基于表型的新型化合物库的无偏筛选 选择它是因为其有利的化学特性而不是已知的生物功能。画面是基于 化合物增加受到抑制性髓磷脂攻击的中枢神经系统神经元的神经突生长的能力 基材。初步结果产生了 4 种能够强烈促进神经突生长的“热门化合物”。 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) 非常有效，d) 以不同的方式克服抑制 与损伤相关的测定，e) 不影响 cAMP 水平、PKC 活性或 EGFR 激活，f) 改变 微管动力学，g) 促进体内再生。因为这些化合物非常有效并且 有选择性，并且可能通过新颖的机制发挥作用，它们是令人兴奋的治疗候选者 开发和再生抑制的机制研究。建议 1) 调查 4 个命中化合物的信号传导机制和蛋白质靶标，2) 检查 1 个命中化合物的能力 促进体内脊髓损伤或视神经挤压后再生的化合物，以及3)筛选 新型抑制性（蛋白聚糖）底物上的完整 4000 种化合物库。这些实验可以提供 再生抑制的关键见解，并为中枢神经系统损伤的新方法铺平道路。

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.