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

这项新的提议是为了研究RNA/DNA双活性拓扑异构酶如何在动物模型中调节自噬和影响寿命。自噬是一种进化上保守的自噬过程，当细胞和生物体处于饥饿状态时会被激活。最佳的自噬活性对于通过移除受损的细胞器和循环必要的蛋白质和营养物质而应激生存至关重要。值得注意的是，自噬受损是衰老的一个标志(Aman等人，自然衰老，2021年)，并已在多种与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，自噬减少与寿命缩短有关，而自噬增加与延迟衰老有关(Aman等人，自然衰老，2021年)。自噬是一种治疗目标，因为在模式生物中，增加自噬的化合物可以延长寿命，减少与年龄相关的疾病状况。其中一些化合物正在进行临床试验(Aman等人，自然老龄化，2021年)。 本课题组首次在真核生物中发现了DNA/RNA双活性拓扑异构酶复合体TOP3B-TDRD3。这种复合体可以同时解决DNA和RNA的拓扑复杂性。TOP3B-Tdrd3复合体可以调节神经元活性激活的转录6、mRNA翻译和翻转7、8。值得注意的是，TOP3B突变与多种精神障碍有关，包括精神分裂症、自闭症和智力残疾6、9、10。此外，携带TOP3B突变的小鼠的寿命较短11。然而，TOP3B失活如何导致寿命缩短和神经疾病的潜在机制仍不清楚。一种可能的机制是，TOP3B-Tdrd3可能通过沉默转座子12，13来防止早期衰老，转座子12，13是导致衰老和衰老相关疾病的跳跃遗传元件。在这里，我们提出了另一种可能的机制--TOP3B-TDRD3可能通过促进自噬来防止过早衰老和年龄相关的疾病。我们将利用三个小组的联合专业知识来调查这一建议，王(TOP3B，果蝇，基因组学)，DeCabo(饥饿，小鼠)，和CAI(神经疾病，小鼠)。 众所周知，为了响应饥饿，细胞转录和翻译都被重新编程(冯等人，Trends Cell Biol.，2015)(Fullgrabe等人，NRMCB 2014)；许多关键的自噬相关基因(ATGs)的表达被激活(Peters等人，自噬2019)14.自噬基因激活的中断可能导致寿命缩短。由于TOP3B-TDRD3可以同时调节转录和翻译，我们假设该复合体也可能在转录或翻译步骤促进饥饿诱导的自噬基因的表达。我们计划研究TOP3B-TDRD3失活的细胞或动物模型是否表现出自噬基因的缺陷表达，以及在三个特定目的中自噬减少。 TOP3B促进饥饿激活和抑制转录(目标1) 我们已经建立了TOP3B-KO和TDRD3-KO HCT116细胞培养系统，用于研究饥饿诱导的基因表达和自噬(Su等人，手稿正在修订中)。我们的RNA-SEQ显示，许多基因对饥饿的反应是上调或下调，我们分别将它们命名为SAGs(饥饿激活基因)和SRGs(饥饿抑制基因)。值得注意的是，我们使用RNAPII-ser2p抗体(一种转录延伸标记)的CHIP-SEQ显示，许多SAG和SRG显示出转录变化，这表明TOP3B在饥饿诱导的转录激活和抑制中起关键作用。 TOP3B-KO改变自噬相关基因的转录(目的1) 我们的RNA-SEQ和RNAP2-SER2P芯片-SEQ显示，在TOP3B-KO HCT116细胞中，几个自噬相关基因在饥饿时转录减少。例如，在一个关键的活性自噬标记的作用下，在WT的RNA-seq和RNAPII-ser2p Chip-seq中，MAP1LC3B的转录因饥饿而增加，但在TOP3B-KO中这种增加被减弱。Western blotting证实该蛋白的一种异构体减少。我们还进行了溶菌素追踪器染色，这是一种检测自噬的常用方法；在饥饿后6小时，我们观察到TOP3B-KO细胞染色减少。 果蝇表现出自噬减少和对饥饿的反应寿命缩短(目标2，3) 我们测试了TOP3B-KO果蝇对饥饿反应的自噬激活是否存在缺陷。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，而TOP3B-/-几乎完全不存在这种激活，这表明TOP3B是饥饿诱导自噬形成所必需的。值得注意的是，我们发现TOP3B-/-果蝇在饥饿时的寿命比WT果蝇要短得多。这些数据支持我们的假设，即TOP3B通过促进自噬来促进正常寿命。 我们发表的一篇论文表明，TOP3B-TDRD3复合体促进了转录饥饿诱导的基因和人类癌细胞本身的自噬。我们还证明了TOP3B-KO苍蝇显示出减少的自噬(Su等人，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).