Novel Compounds That Overcome Glial Inhibition of Axonal Regeneration

项目摘要 中枢神经系统轴突再生的一个主要障碍是与以下相关的生长抑制蛋白的存在 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 克服困难。最近的研究表明，cAMP的变化以及PKC、EGFR和RhoA的增加 活性，是抑制信号的重要方面。然而，我们仍然缺乏关于 与损伤部位抑制相关的抑制蛋白的数量/特性，详细的信号转导 抑制性受体使用的机制，以及受损轴突的细胞类型特异性反应。 此外，还存在与当前药理策略相关的问题，包括缺乏特异性， 不确定的毒性，以及具有多营养功能的通路的靶向。要克服这些障碍 困难，我们已经启动了一种基于表型的新化合物文库的无偏筛选 因其良好的化学性质而不是已知的生物功能而被选择。该屏幕基于 化合物促进抑制髓鞘刺激的中枢神经系统神经元突起生长的能力 底物。初步结果已经产生了4种能够强烈促进神经突起生长的“命中化合物”。 随后的研究表明，HIT化合物a)作用于不同类型的神经元，b)选择性地作用于不同类型的神经元 克服抑制而不是促进生长，c)是高度有效的，d)在不同的 与损伤相关的检测，e)不影响cAMP水平、PKC活性或EGFR激活，f)改变 微管动力学，和g)促进体内再生。因为这些化合物是强效的 选择性的，并可能通过新的机制发挥作用，它们是令人兴奋的治疗候选 发展和再生抑制的机理研究。建议是1)调查 4个HIT化合物的信号转导机制和蛋白质靶标；2)检测1个HIT的能力 体内脊髓损伤或视神经挤压后促进再生的化合物，以及3)筛选 在一种新的抑制性(蛋白多糖)底物上构建了完整的4000个化合物文库。这些实验可以提供 对再生抑制的关键见解，并为治疗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.