Reprogramming retinal ganglion cells for optic nerve regeneration and guidance

成功的视神经再生和功能恢复有三个主要目标：1)识别 更多可以通过不同机制操纵以促进视神经再生的基因，2)识别 能促进不同子集再生足够轴突的最佳组合方法 视网膜神经节细胞(RGC)穿过视交叉到达大脑，以及3)精确地引导再生 视神经轴突从不同类型的视网膜节细胞到其原始的大脑靶点。目标3是最难的一个 鉴于目标1和目标2是实现目标3的先决条件。本研究的总体目标主要是 解决前两个目标。诱导多能干细胞的两种因子KLF4和c-Myc 重编程被证明是视神经再生的重要调节因素。初步研究 结果显示，重编程因子Lin28在小鼠视网膜神经节细胞中的过表达也显著促进了视神经 再生。最近完成但未发表的一项研究表明，H3K27甲基化是必要的，并且 通过抑制KLF4，足以在体内再生感觉轴突。敲除脱甲基酶UTX 视网膜节细胞显著促进视神经再生。因为H3K27甲基化和相关的组蛋白 甲基转移酶和去甲基酶已被证明在IPSC过程中与重编程因子一起工作 通过改变染色质结构，我们假设成熟的小鼠视网膜神经节细胞可以被重新编程 通过重新编程因子或染色质重塑其表观遗传景观而进入再生状态 调制器。作为支持，CHIP-SEQ分析H3K27me3在再生神经元中发现了MAGI3，一种膜 相关鸟苷酸激酶，作为一个被H3K27me3抑制的基因。感觉神经元或视网膜节细胞中MAGI3的缺失 分别导致明显的感觉神经轴突和视神经再生。除了MAGI3，许多单元重新编程 Oct4、FoxA1/2、GATA3/4、PAX6等因子是H3K27me3基因调控的首选候选基因 在再生神经元方面。因此，在目标1中，本研究将探讨 LIN28和MAGI3调控视神经再生。初步研究表明，删除肌球蛋白IIA/B 视网膜节细胞本身可促进视神经再生，消除再生轴突的后退 当与Pten缺失结合使用时。增强的RGC神经活动，当与mTOR激活相结合时，可能 诱导远距离视神经再生。因此，在目标2中，这项研究将确定基因组合是否 用两种机械上不同的方法重新编程，细胞骨架调节或神经活动，可能会导致 更有效地将视神经再生到大脑中。在目标3中，该研究将调查潜在的作用 以上提到的调节视神经再生的新的细胞重编程基因。建议数 这项研究是基于非常强大的初步数据。这一结果将为识别新基因开辟新的方向 促进视神经再生，为今后视力功能恢复奠定坚实基础。

There are 3 major goals for successful optic nerve regeneration and functional recovery: 1) identify more genes that can be manipulated to promote optic nerve regeneration via different mechanisms, 2) identify the optimal combinatory approaches that can promote sufficient regenerating axons from different subsets of retinal ganglion cells (RGCs) to cross the optic chiasm and reach the brain, and 3) precisely guiding regenerating optic nerve axons from different types of RGCs to their original brain targets. The goal 3 is the most difficult one whereas the goal 1 and 2 are the prerequisites to achieve goal 3. The overall goal of this study is to mainly address the first 2 goals. KLF4 and c-Myc, two factors used for the induced pluripotent stem cells (iPSCs) reprogramming, were shown to be important regulators of optic nerve regeneration. The preliminary study showed that overexpression of reprogramming factor Lin28 in mouse RGCs also drastically promoted optic nerve regeneration. A recently completed but unpublished study showed that H3K27 methylation is necessary and sufficient for sensory axon regeneration in vivo by suppressing KLF4. Knocking out the demethylase UTX in RGCs dramatically enhanced optic nerve regeneration. Because H3K27 methylation and associated histone methyltransferase and demethylases have been shown to work together with reprogramming factors during iPSC process by modifying chromatin structure, we hypothesize that mature mouse RGCs can be reprogrammed into a regenerating state via remodeling their epigenetic landscape through reprogramming factors or chromatin modulators. In support, ChIP-seq analysis of H3K27me3 in regenerating neurons identified Magi3, a membrane associated guanylate kinase, as a gene suppressed by H3K27me3. Deleting Magi3 in sensory neurons or RGCs led to marked sensory axon and optic nerve regeneration, respectively. Besides Magi3, many cell reprogramming factors, such as Oct4, FoxA1/2, GATA3/4, PAX6, were identified as top candidate genes regulated by H3K27me3 in regenerating neurons. Therefore, in Aim 1, the study will investigate the roles and mechanisms by which Lin28 and Magi3 regulate optic nerve regeneration. Preliminary study demonstrated that deleting myosin IIA/B in RGCs by itself could promote optic nerve regeneration and abolish backward turning of regenerating axons when combined with Pten deletion. Enhanced RGC neural activity, when combined with mTOR activation, could induce long distance optic nerve regeneration. Thus, in Aim 2, the study will determine if combination of genetic reprogramming with 2 mechanistically different approaches, cytoskeletal modulation or neural activity, can lead to more efficient optic nerve regeneration into the brain. In Aim 3, the study will investigate the potential roles of novel cell reprogramming genes mentioned above in regulation of optic nerve regeneration. The proposed study is based on very strong preliminary data. The results will open a new direction to identify novel genes promoting optic nerve regeneration and build a solid foundation for future functional recovery of vision.