Targeting aging genes and pathways to promote optic nerve regeneration

摘要 到目前为止，靶向调节内在轴突生长能力的基因已经产生了到目前为止最有希望的 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表明，神经元 衰老可能是中枢神经系统神经元内源性轴突生长能力丧失的关键汇聚过程。 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， 端粒磨损、表观遗传学改变和营养感知等)。第一，近期的研究和我们的初步研究 结果表明，c-Myc和P53这两个参与DNA修复和基因组不稳定的基因在 老化，支持视神经再生。第二，我们的初步研究表明，端粒酶逆转 转录酶(TERT)是体内感觉神经轴突再生所必需的。第三，衰老往往与 组蛋白3赖氨酸27(H3K27)的甲基化减少，H3K4的甲基化增加，导致 异染色质的量。在支持方面，H3K27脱甲基酶UTX的水平在衰老和 剔除线虫中的UTX可以延长寿命。我们未发表的研究表明，敲除UTX和 其靶向基因MAGI3在视网膜节细胞中极大地促进了视神经再生。第四，胰岛素和IGF-1 信号通路(IIS)是营养感知的关键调节器，是最保守的衰老控制途径 进化中的路径。IGF-1和许多IIS下游目标，如Pten/PI3K、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，Sirt1 和LKB1，两个重要的营养感受器，调节感觉神经轴突和脊髓再生， 分别进行了分析。FOXO_3，Akt信号的另一个关键靶点，最近被证明促进血管细胞 通过Sirt1再生。最后，最近的发现表明，细胞重新编程过程可以逆转 使细胞老化和恢复活力。重要的是，几个重新编程因素的操作，如KLF4和 LIN28，已被证明可以促进视神经再生。因此，我们假设老龄化的监管机构 基因/通路可以通过成熟中枢神经系统的再生来促进视神经再生 神经元。在目标1中，我们将确定在RGC中操纵miR-138/Sirt1、TERT和Foxo3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是否与肌球蛋白II结合 基因敲除或增强的神经活性将对再生产生协同效应。然后我们将使用RNA-seq 并对纯化的视网膜节细胞进行ATAC-SEQ，以探索这些老化基因如何调控视神经再生。在目标3中，由 对不同发育、成熟和老化阶段的视网膜神经节细胞进行rna-seq和atac-seq检测。 将首先使用先进的综合生物信息学分析来确定最佳候选衰老基因及其 相关的转录因子，两者都起到协调视网膜节细胞衰老的作用。然后我们将执行功能 筛选实验以确定它们在调节轴突生长和视神经再生中的作用。

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 Our unpublished study showed that knocking out UTX and its targeted gene, Magi3, in RGCs drastically promoted optic nerve regeneration.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.