Evaluation of Novel Targets for Retinal Ganglion Cell Axon Regeneration

摘要 国家眼科研究所已将恢复视力确定为一个“大胆的目标”，要求 从功能视网膜到大脑的神经连接的重建。不幸的是，人类的再生 成年哺乳动物的中枢神经系统轴突极为有限或为零。视网膜神经节细胞与外侧神经节细胞的再连接 支持视力恢复的膝状神经元需要克服轴突的内源性限制 再生。一小群限制再生的基因，特别是视网膜神经节细胞亚型 已经被确认，但只有很小一部分基因组被测试了在限制 轴突再生。我们假设存在额外的再生调节因素，而且守恒 跨细胞类型、物种和生长模式的功能研究将促进它们的发现。 我们已经完成了一项基于shRNA的全基因组筛选，寻找限制轴突修复的内源性基因 利用培养的小鼠大脑皮质神经元获得超过135,000个分离再生的哺乳动物中枢神经系统 实验。在17,000个基因中，这种功能丧失的筛查产生了500个再生基因。在这里，我们的 主要目标是测试这个选定的清单在体内促进视网膜神经节细胞轴突再生的能力。 我们预计，具有最强再生限制功能的基因将具有保守功能 皮质神经元到视网膜神经节细胞。在皮质轴突再生基因列表中，我们将优先 使用另外两个数据集进行视神经再生实验的基因。在一个实验中，我们 测定了发芽和未发芽的小鼠CST神经元在 中枢神经系统损伤，假设最相关的基因将参与萌发和再生 上述再生系数的情况也是如此。我们还进行了实验，以确定 通过筛选调节运动轴突再生的基因来保护物种的再生 线虫线虫。多个实验系统共有的因素有望确定基本 再生机制可能会影响人类视觉系统中的等同过程。 对于那些新发现的功能丧失支持视神经再生的基因，二次研究 将评估阳性基因、视网膜神经节细胞类型特异性作用和中央轴突之间的协同作用 寻路功能。该项目建立在成熟的小鼠中枢神经系统和C. Elgans将分析促进哺乳动物视网膜神经节细胞后轴突再生的新机制 轴突切断术。

SUMMARY The National Eye Institute has identified the restoration of vision as an “audacious goal” requiring the reestablishment of neural connections from a functional retina with the brain. Unfortunately, the regeneration of adult mammalian CNS axons is extremely limited or nil. The reconnection of retinal ganglion cells with lateral geniculate neurons to support vision recovery requires overcoming endogenous limitations on axonal regeneration. A small group of genes limiting regeneration in particular retinal ganglion cell subtypes have been identified, but only a very small fraction of the genome has been tested with regard to a role in limiting axon regeneration. We hypothesize that additional regeneration-regulating factors exist, and that conservation of function across cell types, species and modes of growth will facilitate their discovery. We have completed a genome-wide shRNA-based screen for endogenous genes limiting the repair of axons in the mammalian CNS using cultured mouse cerebral cortex neurons with over 135,000 separate regeneration experiments. Across 17,000 genes, this loss of function screen yielded 500 regeneration genes. Here, our primary goal is to test this selected list for the ability to promote retinal ganglion cell axon regeneration in vivo. We expect that genes with the most robust regeneration-limiting function will have conserved function from cortical neurons to retinal ganglion cells. Amongst the cortical axon regeneration gene list, we will prioritize genes for optic nerve regeneration experiments using two additional datasets. In one experiment, we determined the distinct transcriptional profiles of sprouting versus non-sprouting mouse CST neurons after CNS injury, hypothesizing that the most relevant genes will participate in both sprouting and regeneration, as is the case for previously described regeneration factors. We have also conducted experiments to identify species conservation of regeneration by screening for genes that regulate motor axon regeneration in the nematode C. elegans. Factors common to multiple experimental systems are expected to identify fundamental mechanisms in regeneration that are likely to affect the equivalent process in human visual system. For those newly discovered genes whose loss of function supports optic nerve regeneration, secondary studies will assess synergy amongst positive genes, retinal ganglion cell type specificity of action, and central axonal pathfinding function. This project builds on genetic screens in the mature mouse central nervous system and C. elegans to analyze novel mechanisms that promote axon regeneration after mammalian retinal ganglion cell axotomy.