Targeting aging genes and pathways to promote optic nerve regeneration

总结 迄今为止，靶向调节内在轴突生长能力的基因已经产生了迄今为止最有希望的 导致视神经再生最近的研究，包括我们的研究，提供了强有力的证据， 衰老可能是中枢神经系统神经元内在轴突生长能力丧失的关键收敛过程。 事实上，许多调节轴突再生的基因也是衰老的标志基因（基因组不稳定性， 端粒磨损、表观遗传改变和营养感测等）。首先，最近的研究和我们的初步 结果表明，c-Myc和p53，两个众所周知的基因参与DNA修复和基因组不稳定性， 老化，支持视神经再生。其次，我们的初步研究表明，端粒酶逆转录酶 转录酶（TERT）是感觉轴突再生所必需的。第三，衰老往往与 组蛋白3在赖氨酸27（H3 K27）处的甲基化降低和H3 K4的甲基化增加，导致组蛋白3在赖氨酸27（H3 K27）处的甲基化降低和H3 K4的甲基化增加， 异染色质的量。作为支持，H3 K27脱甲基酶UTX的水平在老化过程中增加， 在c中敲除UTX。秀丽隐杆草能促进长寿。我们未发表的研究表明，敲除UTX和 其靶基因Magi 3在RGCs中显著促进视神经再生。第四，胰岛素和IGF-1 信号传导（IIS）途径是营养感受的关键调节因子，是最保守的衰老控制途径 进化的道路。IGF-1和许多IIS下游靶点，如Pten/PI 3 K，Akt和mTor，都是 视神经再生的重要调节因子。我们发表的研究和最近的一项研究表明，Sirt 1 和LKB 1，两种重要的营养感受器，具有调节感觉轴突和脊髓再生的功能， 分别Foxo 3是Akt信号传导的另一个关键靶点，最近显示其促进血管细胞增殖， 通过Sirt 1再生。最后，最近的研究结果表明，细胞重编程过程可以逆转 老化并使细胞恢复活力。重要的是，操纵几种重编程因子，如KLF 4和 Lin 28已被证明可以促进视神经再生。因此，我们假设老化调节 基因/通路可以通过成熟CNS的再生来促进视神经再生 神经元在目的1中，我们将确定在RGC中操纵miR-138/Sirt 1、TERT和Foxo 3是否可以促进 视神经再生在目标2中，我们将首先确定这些衰老基因与肌球蛋白II的结合是否 敲除或增强的神经活性将对再生具有协同效应。我们将使用RNA-seq 和纯化RGC的ATAC-seq，以探索这些衰老基因如何调节视神经再生。在目标3中， 对不同发育、成熟和衰老阶段的纯化RGC进行RNA-seq和ATAC-seq， 将首先使用先进的综合生物信息学分析，以确定顶级候选衰老基因及其 相关的转录因子，这两种作用协调RGCs老化。然后我们将执行功能 筛选实验以确定它们在轴突生长和视神经再生调节中的作用。

Summary To date, targeting the genes regulating intrinsic axon growth ability have produced by far the most promising results in optic nerve regeneration. Recent studies, including ours, have provided strong evidence that neuronal aging might be a key converging process underlying the loss of intrinsic axon growth ability of CNS neurons. Indeed, many genes that act to regulate axon regeneration are also hallmark genes of aging (genomic instability, telomere attrition, epigenetic alteration, and nutrient sensing, etc.). First, recent studies and our preliminary results showed that c-Myc and p53, two well-known genes involved in DNA repair and genomic instability during aging, act to support optic nerve regeneration. Second, our preliminary study showed that telomerase reverse transcriptase (TERT) was necessary for sensory axon regeneration in vivo. Third, aging is often associated with decreased methylation of histone 3 at lysine 27 (H3K27) and increased methylation of H3K4, resulting in reduced amount of heterochromatin. In support, the level of H3K27 demethylase UTX increases during aging and knocking out UTX in c. elegans promotes longevity. Our unpublished study showed that knocking out UTX and its targeted gene, Magi3, in RGCs drastically promoted optic nerve regeneration. Fourth, the insulin and IGF-1 signaling (IIS) pathways, the key regulators of nutrient sensing, are the most conserved aging controlling pathway in evolution. IGF-1 and many IIS downstream targets, such as Pten/PI3K, Akt, and mTor, are all important regulators of optic nerve regeneration. Our published study and a recent study have shown that Sirt1 and LKB1, two important nutrient sensors, function to regulate sensory axon and spinal cord regeneration, respectively. Foxo3, another key target of Akt signaling, has recently been shown to promote vascular cell regeneration through Sirt1. Lastly, recent findings indicated that cellular reprogramming process can reverse aging and rejuvenate the cells. Importantly, manipulations of several reprogramming factors, such as KLF4 and Lin28, have been shown to promote optic nerve regeneration. Therefore, we hypothesize that aging regulatory genes/pathways can be manipulated to promote optic nerve regeneration through rejuvenation of mature CNS neurons. In Aim 1, we will determine if manipulation of miR-138/Sirt1, TERT, and Foxo3 in RGCs can promote optic nerve regeneration. In Aim 2, we will first determine if combination of these aging genes with myosin II knockout or enhanced neural activity would have synergistic effects on regeneration. We will then use RNA-seq and ATAC-seq of purified RGCs to explore how these aging genes regulate optic nerve regeneration. In Aim 3, by performing RNA-seq and ATAC-seq of purified RGCs at different developing, maturation, and aging stages, we will first use advanced integrative bioinformatics analyses to identify top candidate aging genes and their associated transcription factors, both of which act to orchestrate RGCs aging. We will then perform functional screening experiments to determine their roles in regulation of axon growth and optic nerve regeneration.