Novel Compounds That Overcome Glial Inhibition of Axonal Regeneration

描述(申请人提供)：中枢神经系统轴突再生的一个主要障碍是与髓鞘碎片和胶质瘢痕相关的生长抑制蛋白的存在。中枢神经系统损伤后的功能恢复需要克服这种抑制。最近的研究表明，cAMP的变化以及PKC、EGFR和RhoA活性的增加是抑制信号转导的重要方面。然而，我们仍然缺乏与损伤部位抑制相关的抑制蛋白的数量/特性，抑制受体所使用的详细信号机制，以及受损轴突的细胞类型特异性反应。此外，目前的药理策略还存在一些问题，包括缺乏特异性，毒性不确定，以及靶向具有多种营养功能的途径。为了克服这些困难，我们启动了基于表型的无偏筛选，选择了一个新的化合物文库，因为它具有良好的化学性质，而不是已知的生物学功能。筛选是基于化合物增加用抑制性髓鞘底物挑战的中枢神经系统神经元突起的能力。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)选择性地克服抑制而不是促进生长，c)高度有效，d)在与损伤相关的不同检测中克服抑制，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。由于这些化合物是有效的和选择性的，并可能通过新的机制发挥作用，它们是治疗开发和再生抑制机制研究的令人兴奋的候选者。建议1)研究4个HIT化合物的信号机制和蛋白靶点，2)检测1个HIT化合物在体内脊髓损伤或视神经挤压后促进再生的能力，3)在一种新的抑制性(蛋白多糖)底物上筛选完整的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。与公共健康相关：拟议的实验将研究促进再生的新化合物的作用机制，并阐明它们在中枢神经系统损伤后增加轴突再生的能力。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: The proposed experiments will investigate the mechanisms of action of novel compounds promoting regeneration, and elucidate their ability to increase axonal regrowth after CNS injury.