Reprogramming retinal ganglion cells for optic nerve regeneration and guidance

成功的视神经再生和功能恢复有3个主要目标：1）识别 更多的基因可以通过不同的机制来促进视神经再生，2）识别 最佳的组合方法，可以促进足够的再生轴突从不同的子集， 视网膜神经节细胞（RGC）穿过视交叉到达大脑，以及3）精确引导再生 视神经轴突从不同类型的RGC到它们最初的大脑目标。目标3是最难的一个 而目标1和2是实现目标3的先决条件。本研究的总体目标主要是 实现前两个目标。KLF 4和c-Myc，用于诱导多能干细胞（iPSC）的两种因子 重编程，被证明是视神经再生的重要调节因子。初步研究 显示小鼠RGCs中重编程因子Lin 28的过表达也显著促进视神经 再生一项最近完成但未发表的研究表明，H3 K27甲基化是必要的， 足以通过抑制KLF 4在体内再生感觉轴突。敲除去甲基酶UTX RGC显著增强视神经再生。因为H3 K27甲基化和相关的组蛋白 甲基转移酶和脱甲基酶已经显示在iPSC期间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RGCs可以被重编程， 通过重编程因子或染色质重塑其表观遗传景观， 调制器。作为支持，再生神经元中H3 K27 me 3的ChIP-seq分析鉴定了Magi 3，一种膜蛋白， 相关的鸟苷酸激酶，作为被H3 K27 me 3抑制的基因。在感觉神经元或RGC中删除Magi 3 分别导致明显的感觉轴突和视神经再生。除了Magi 3，许多细胞重编程 H3 K27 me 3调控的主要候选基因有Oct 4、FoxA 1/2、GATA 3/4、PAX 6等 在再生神经元中。因此，在目标1中，本研究将研究 Lin 28和Magi 3调节视神经再生。初步研究表明，去除肌球蛋白IIA/B RGC本身能促进视神经再生，并能消除再生轴突的后转 当与Pten缺失结合时。增强的RGC神经活动，当与mTOR激活结合时， 诱导长距离视神经再生。因此，在目标2中，该研究将确定基因的组合是否 用2种不同的机制方法，细胞骨架调节或神经活动，重新编程可以导致 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 上述新的细胞重编程基因在视神经再生调控中的作用。拟议 这项研究是基于非常有力的初步数据。这一结果将为识别新基因开辟新的方向 促进视神经再生，为将来视力功能恢复打下坚实基础。

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 A recently completed but unpublished study showed that H3K27 methylation is necessary and sufficient for sensory axon regeneration in vivo by suppressing KLF4.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.