Investigating the roles of a Topoisomerase complex in autophagy and lifespan regulation

这项新的提议是研究RNA/DNA双活性拓扑异构酶如何调节自噬并影响动物模型的寿命。自噬是一种进化上保守的自食过程，当细胞和生物体处于饥饿状态时会被激活。最佳的自噬活性通过去除受损的细胞器和回收必要的蛋白质和营养物质对应激生存至关重要。值得注意的是，受损的自噬是衰老的标志（Aman等人，Nature Aging，2021），并已在多种年龄相关的神经退行性疾病和自身免疫性疾病中观察到4，5。在模式生物中，减少的自噬与缩短的寿命相关，而增加的自噬与延迟的衰老相关（Aman等人，Nature Aging，2021）。自噬是一种治疗靶点，因为增加自噬的化合物可以提高模型生物的寿命并减少与年龄相关的疾病。这些化合物中的一些正在临床试验中进行检查（Aman等人，Nature Aging，2021）。 我们的小组在真核生物中发现了第一个DNA/RNA双活性拓扑异构酶复合物，拓扑异构酶3 β（Top 3b）-TDRD 3。这种复合物可以解决DNA和RNA的拓扑复杂性。Top 3b-Tdrd 3复合物可调节神经元活性激活的转录6、mRNA翻译和周转7，8。值得注意的是，Top 3b突变与多种精神疾病有关，包括精神分裂症，自闭症和智力残疾6，9，10。此外，携带Top 3b突变的小鼠表现出较短的寿命11。然而，Top 3b失活如何导致寿命缩短和神经系统疾病的机制仍不清楚。一种可能的机制是Top 3b-Tdrd 3可以通过沉默转座子12，13来防止早期衰老，转座子12，13是可以成为衰老和衰老相关疾病的驱动因素的跳跃遗传元件。在这里，我们提出了另一种可能的机制--TOP 3B-TDRD 3可能通过促进自噬来预防过早衰老和与年龄相关的疾病。我们将通过使用三个小组的综合专业知识来研究这个建议，Wang（Top 3b，果蝇，基因组学），DeCabo（饥饿，小鼠）和Cai（神经疾病，小鼠）。 已知响应于饥饿，细胞转录和翻译都被重编程（Feng et al.，细胞生物学趋势，2015）（Fullgrabe等人，NRMCB 2014）;并且许多关键的自噬相关基因（ATG）的表达被激活（Peeters等人，14.第十四章自噬基因激活的破坏可导致寿命缩短。 由于Top 3b-TDRD 3可以调节转录和翻译，我们假设这种复合物也可能促进饥饿诱导的自噬基因在转录或翻译步骤的表达。我们计划研究TOP 3B-TDRD 3失活的细胞或动物模型是否表现出自噬基因表达缺陷，以及三个特定目标中的自噬减少。 Top 3b促进饥饿激活和抑制的转录（Aim 1） 我们已经建立了TOP 3B-KO和TDRD 3-KO HCT 116细胞培养系统，用于研究饥饿诱导的基因表达和自噬（Su et al.，修订稿）。我们的RNA-seq显示，许多基因在饥饿时上调或下调，我们分别将它们命名为SAG（饥饿激活基因）和SRG（饥饿抑制基因）。值得注意的是，我们使用RNAPII-ser 2 p抗体（一种转录延伸标记物）的ChIP-seq揭示了许多这些SAG和SRG显示出改变的转录，这表明Top 3b对于饥饿诱导的转录激活和抑制至关重要。 TOP 3B-KO改变自噬相关基因的转录（Aim 1） 我们的RNA-seq和RNAP 2-ser 2 p ChIP-seq揭示了几个自噬相关基因在饥饿时在Top 3b-KO HCT 116细胞中显示出转录降低。例如，对于关键的活性自噬标记物，MAP 1 LC 3B转录在RNA-seq中通过饥饿增加，而在WT中通过RNAPII-ser 2 p ChIP-seq增加，但这种增加在Top 3b-KO中减少。Western印迹证实了这种蛋白质的一种亚型的减少。我们还进行了lysotracker染色，一种检测自噬的常用方法;并在饥饿后6小时观察到T0 P3 B-KO细胞中染色减少。 果蝇表现出减少的自噬和较短的寿命响应饥饿（目的2，3） 我们测试了在TOP 3b-KO果蝇中响应于饥饿的自噬激活是否有缺陷。6小时饥饿诱导WT果蝇3龄幼虫脂肪体中LysoTracker信号显著增加，然而，这种激活在Top 3b-/-中几乎完全不存在，这意味着Top 3b对于饥饿诱导的自噬形成是必需的。值得注意的是，我们发现Top 3b-/-果蝇在饥饿时比WT果蝇显示出显著更短的寿命。这些数据支持我们的假设，即Top 3b通过促进自噬促进正常寿命。 我们已经发表了一篇论文，显示Top 3b-TDRD 3复合物促进人类癌细胞中饥饿诱导基因的转录和自噬本身。我们还证明Top 3b-KO果蝇显示出减少的自噬（Su et al.，NAR 2023）。

This new proposal is to investigate how a RNA/DNA dual-activity topoisomerase regulates autophagy and affects lifespan in animal models. Autophagy is an evolutionarily conserved self eating process that is activated when cells and organisms are under starvation3. The optimal autophagic activity is critical to stress survival by removing damaged organelles and recycling necessary proteins and nutrients. Notably, compromised autophagy is a hallmark of aging (Aman et al., Nature Aging, 2021), and has been observed in multiple age related neurodegenerative and autoimmune diseases4,5. In model organisms, reduced autophagy is associated with shortened lifespan, whereas increased autophagy is associated with delayed aging (Aman et al., Nature Aging, 2021). Autophagy is a therapeutic target, as compounds that increase autophagy can improve lifespan and reduce age-associated disease conditions in model organisms. Some of these compounds are being examined in clinical trials (Aman et al., Nature Aging, 2021). Our group has discovered the first DNA/RNA dual activity topoisomerase complex in eucaryotes, Topoisomerase 3 beta (Top3b)-TDRD3. This complex can resolve both DNA and RNA topological complexity. Top3b-Tdrd3 complex can regulate neuronal activity-activated transcription6, mRNA translation and turnover7,8. Notably, the Top3b mutation has been linked to multiple mental disorders including schizophrenia, autism, and intellectual disability6,9,10. Moreover, mouse carrying Top3b mutations exhibits shorter lifespans11. However, the mechanisms underlying how Top3b inactivation can lead to reduced lifespan and neurological disorders remain unclear. One possible mechanism is that Top3b-Tdrd3 may prevent early aging by silencing transposons12,13,the jumping genetic elements that can be a driver of aging and aging associated diseases. Here we propose another possible mechanism-- TOP3B-TDRD3 may prevent premature aging and age associated conditions by promoting autophagy. We will investigate this proposal by using combined expertise of three groups, Wang (Top3b, Drosophila, genomics), DeCabo (starvation, mouse), and Cai (neurological diseases, mouse). It is known that in response to starvation, cellular transcription and translation are both reprogramed (Feng et al., Trends Cell Biol., 2015)(Fullgrabe et al., NRMCB 2014); and expression of many critical autophagy-related genes (ATGs) are activated (Peeters, et al., Autophagy 2019)14. Disruption of activation of autophagy genes can lead to shortened lifespan. Because Top3b-TDRD3 can regulate both transcription and translation, we hypothesize that this complex may also facilitate starvation-induced expression of autophagy genes at either transcription or translation steps. We plan to investigate whether cells or animal models inactivated of TOP3B-TDRD3 exhibit defective expression of autophagy genes, as well as reduced autophagy in three specific aims. Top3b promotes starvation-activated and repressed transcription (Aim 1) We have established the TOP3B-KO and TDRD3-KO HCT116 cell culture system for studies of starvation-induced gene expression and autophagy (Su et al., manuscript in revision). Our RNA-seq shows that many genes are up- or down-regulated in response to starvation, and we named them SAGs (starvation activated genes) and SRGs (starvation repressed genes), respectively. Notably, our ChIP-seq using RNAPII-ser2p antibodies, a transcription elongation marker, revealed that many of those SAGs and SRGs display altered transcription, suggesting that Top3b is critical for starvation induced transcriptional activation and repression. TOP3B-KO alters the transcription of autophagy-associated genes (Aim 1) Our RNA-seq and RNAP2-ser2p ChIP-seq revealed that several autophagy-associated genes show reduced transcription in Top3b-KO HCT116 cells upon starvation. For instance, with a key active autophagy marker, MAP1LC3B transcription is increased by starvation in RNA-seq and RNAPII-ser2p ChIP-seq in WT, but this increase is diminished in Top3b-KO. Western blotting confirmed the reduction of one isoform of this protein. We have also performed lysotracker staining, a common method to detect autophagy; and observed reduced staining in TOP3B-KO cells at 6 hours post starvation. Drosophila exhibits reduced autophagy and shorter lifespan responding to starvation (Aim 2,3) We tested whether autophagy activation in response to starvation is defective in TOP3b-KO Drosophila. Six hours of starvation induced a significant increase of LysoTracker signals in the fat bodies of WT Drosophila 3rd instar larva, however, this activation is nearly completely absent in Top3b-/-, implying that Top3b is necessary for starvation-induced autophagy formation. Notably, we found that the Top3b-/- flies show a significantly shorter lifespan upon starvation vs. WT flies. The data support our hypothesis that Top3b facilitates normal lifespan by promoting autophagy. We have published one paper showing that Top3b-TDRD3 complex promotes transcription starvation-induced genes and autophagy per se in human cancer cells. We also demonstrate that Top3b-KO flies show reduced autophagy (Su et al., NAR 2023).