Phenotypic Assays to Identify Agents That Induce Reactive Gliosis: A Counter-Screen to Prioritize Compounds for Preclinical Animal Studies
Phenotypic Assays to Identify Agents That Induce Reactive Gliosis: A Counter-Screen to Prioritize Compounds for Preclinical Animal Studies
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DOI：
10.1089/adt.2015.654
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发表时间：
2015-09-01
影响因子：
1.8
通讯作者：
Lemmon, Vance P.
中科院分区：
文献类型：
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作者：
Beckerman, Samuel R.;Jimenez, Joaquin E.;Lemmon, Vance P.
创伤性中枢神经系统 (CNS) 损伤后，星形胶质细胞表型在称为反应性神经胶质增生的过程中发生变化。星形胶质细胞增生的特征是胶质纤维酸性蛋白（GFAP）细胞骨架的扩张、星状形态的采用以及一些细胞外基质分子的差异表达。损伤后立即发生的星形胶质细胞反应是有益的，但在慢性损伤阶段，反应性星形胶质细胞会产生抑制因子（即硫酸软骨素蛋白聚糖[CSPG]），限制受损轴突的再生。目前还没有药物可以促进人类中枢神经系统创伤后轴突再生或功能恢复。为了开发针对受损中枢神经系统的新疗法，我们在表型测定中筛选了各种文库，以鉴定促进神经突生长的化合物。然而，这些化合物对星形胶质细胞的影响尚不清楚。具体来说，我们感兴趣的是化合物是否可以以模仿神经胶质细胞对损伤的反应的方式改变星形胶质细胞。为了检验这一假设，我们开发了基于细胞的表型生物测定法来测量原代星形胶质细胞培养物中 (1) GFAP 形态/定位和 (2) CSPG 表达/免疫反应性的变化。这些测定针对 96 孔板中的六点剂量反应实验进行了优化。 GFAP 形态学测定适用于 Z 因子为 0.44 +/- 0.03（平均值 +/- 平均值的标准误差；N=3 生物重复）的反筛选。 CSPG 测定具有可重复性和信息丰富性，但不满足“可筛选”测定的常见指标。作为原理证明，我们测试了来自神经突生长生物测定的一小组命中化合物，并确定了一种可以增强轴突生长而不加剧反应性神经胶质增生的有害特征的化合物。
Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors (i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44 +/- 0.03 (mean +/- standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a "screenable" assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis.