Investigating the roles of Topoisomerase 3b-TDRD3 complex in neurodegeneration and Alzheimer's disease

研究拓扑异构酶 3b-TDRD3 复合物在神经退行性疾病和阿尔茨海默病中的作用

• 批准号: 10469229
• 负责人: Weidong Wang
• 金额: $ 21.36万
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10469229
• 关键词: 2019-nCoV; Affect; Age; Aging; Alzheimer' s Disease; Alzheimer' s disease patient; Animal Model; Animals; Aubergine; Behavior; Binding; Biochemical; Biogenesis; Biological Assay; Brain; COVID-19; Cell Death; Cell Line; Cells; Cognition Disorders; Collaborations; Complex; Cytoplasmic Granules; DNA; DNA Transposable Elements; Data; Defect; Dissociation; Drosophila genus; Drug Targeting; Eukaryota; Exhibits; Eye; Eye Development; FMR1; FMRP; Functional disorder; Genes; Genetic; Genetic Transcription; Genetic Translation; Goals; Hela Cells; Heterochromatin; Human; Inflammation; Knockout Mice; Link; Longevity; Mammalian Cell; Mental disorders; Messenger RNA; Modeling; Modification; Mus; Mutation; Nerve Degeneration; Neurologic; Optic Nerve; Ovary; Pathogenicity; Pathologic; Pathway interactions; Phenotype; Phosphotransferases; Play; Polyribosomes; Proteins; RNA; RNA Viruses; RNA-Binding Proteins; RNA-Induced Silencing Complex; Reporter; Role; Science; Secondary to; Seeds; Stress; Tauopathies; Topoisomerase; Topoisomerase Inhibitors; Toxic effect; Transcriptional Silencer Elements; Update; Variant; Wing; Work; base; biological adaptation to stress; druggable target; fly; frontotemporal lobar dementia-amyotrophic lateral sclerosis; inhibitor/antagonist; mRNA Transcript Degradation; misfolded protein; mouse model; mutant; nervous system disorder; neurotoxicity; pandemic disease; piRNA; protein TDP-43; stress granule; stress reduction; synaptogenesis; tau Proteins; tau mutation; therapeutic target; transcriptome sequencing

Our group has discovered Top3b as the first dual-activity topoisomerase in eukaryotes(Xu et al., Nat. Neurosci., 2013). This discovery has greatly expanded the topoisomerase field, because it suggests that not only DNA, but also RNA, may have topological problems that require topoisomerases to solve. We have since discovered that Top3b is an RBP and the major mRNA-binding topoisomerase in mammalian cells, which forms a conserved complex with TDRD3. Top3b-TDRD3 interacts with an RBP, FMRP (Fragile-X Mental Retardation Protein); and they co-localize in SGs, co-fractionate in polyribosomes, and facilitate mRNA translation and synapse formation. Like other RBPs and SG components, Top3b mutations are associated with neurological disorders; and Top3b-KO mice display behavior and neurological defects observed in psychiatric and cognitive disorders (Joo et al., Nat. Comm. 2020). Furthermore, Top3b-TDRD3 interacts with RNA-induced silencing complex to promote heterochromatin formation and silencing of transposons in Drosophila (Lee et al., Nat. Comm., 2018). Recently, Top3b-TDRD3 has been shown to be required for efficient replication of positive-strand RNA virus, including SARS-CoV-2, suggesting that Top3b could be a drug target for COVID-19 and other pandemics caused by RNA virus. There are three main reasons that prompt us to investigate the roles of Top3b in AD and tauopathy. First, Top3b-TDRD3 is a component of SGs, which can act as pathological seeds where misfolded proteins can find low complexity proteins to aggregate, leading to AD and tauopathy. Neuronal toxicity induced by Tau, TDP-43 and C90orf72 can be alleviated by genetic reduction of SG components; or by inhibition of the kinases that initiate SG assembly. Thus, SGs have been suggested as a drug target for neurodegeneration. Importantly, Top3b-TDRD3 KO human cells and Drosophila display accelerated dissociation of SGs. Moreover, genetic reduction of Top3b suppresses Tau-induced neurodegeneration in Drosophila. The findings support the notion that Top3b could be a target for tauopathy like other SG components. Second, we discovered that several genes that show defective transcription in Top3b-KO mouse brains are critical for AD, such as APP and Tau (11). This suggests that genetic reduction of Top3b may decrease the levels of these pathogenic proteins, leading to suppression of neurodegeneration. Third, we found that Top3b biochemically and genetically interacts with piRNA machinery to promote silencing of transposable elements (TEs) in flies; and this biochemical interaction is conserved in mice. TE dysregulation has been observed in AD patients and animal models of tauopathy. Moreover, they have been suggested as drivers in aging, age-associated inflammation and neurodegeneration. We plan to study whether Top3b and Tau may work together in piRNA and/or other pathways to promote TE silencing. Specific Aims Aim 1. Use cell lines to study if Top3b-TDRD3 regulates normal and tau-induced SG dynamics and cellular toxicity Sub-Aim 1-1. Study how Top3b-TDRD3 functions in normal SGs We have obtained Top3b and TDRD3-KO HeLa cells; and found that SG assembly is normal, but SG disassembly is accelerated in both KO cells. We have identified a new interacting partner, PRRC2A/C, which is a known SG component; and found that this protein genetically interacts with TDRD3 in Drosophila animal survival and eye development. Sub-Aim 1-2. Study if and how Top3b-TDRD3 affects tau-associated SGs and toxicity Strategy: Depletion of TIA1 (a SG component) can reduce tau-induced granules and associated cellular toxicity in cell lines. We plan to use the same strategy: introduce Tau variants into WT or KO HeLa cells, and then determine if KO cells have reduced number and faster disassembly of tau-positive granules, decreased cell death, and lower cellular sensitivity to secondary stress. Aim 2. Investigate whether and how Top3b mutation can modify the neurodegeneration phenotype of Drosophila tauopathy model. Sub-Aim 2-1. Study whether genetic reduction of Top3b-TDRD3 can modify Tau-induced neurodegeneration Preliminary results: we found that Top3b heterozygous mutation can suppress, whereas TDRD3 mutant can enhance, the neurodegeneration eye phenotype of Tau-V337M mutant. The data imply that an inhibitor of Top3b may alleviate Tau-induced neurodegeneration. We plan to develop such an inhibitor in Aim 4. Sub-Aim 2-2 Studying the mechanism of how Top3b mutation suppresses Tau-induced neurotoxicity Preliminary results: We found that the dissociation of SGs in Top3b KO or TDRD3 KO Drosophila wing disc cells is significantly faster than WT, which is consistent with our findings in Hela cells. Sub-Aim 2-3. Study whether Top3b and Tau act in the same or different pathways in TE silencing. Preliminary Results: We found that Top3b and piRNA machinery interact biochemically and genetically to silence transposons in Drosophila ovary, as Top3b-piRNA double mutant exhibits higher levels of several TEs than each single mutant. Aim 3. Investigate whether Top3b mutations can modify the neurodegeneration phenotype of tauopathy mouse models. We plan to start work on this aim once our Alzheimer's disease Concept is approved by BSC. Aim 4. Investigate whether topoisomerase can be a druggable target for neurodegeneration. We have already obtained some compounds that inhibits topoisomerase activity, but their Kd is still too high (46). We will continue our collaboration to identify compounds with higher potency. Update of Progress: Outline of the original aims of the project We have proposed to use the Drosophila model to examine the roles of Topoisomerase 3beta (Top3b) in Alzheimers disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration (FTLD). The original aims of the project are: 1. Investigate whether and how Top3 mutations can modify the neurodegeneration phenotype of Drosophila Tauopathy model. 2. Study whether and how Top3 mutations can modify the neurodegeneration phenotype of Drosophila TDP-43 model. 3. Study whether FANCM plays a role in heterochromatin formation and TE silencing in Drosophila. 4. Investigate how Top3b/TDRD3 regulates mRNA degradation and translation using Drosophila. Update on current progress Aim1. Progress 1. Top3b and Tdrd3 mutations may modify neurodegeneration induced by Tau-V337M mutant Expression of pathogenic mutant hTau in fly eyes can cause progressive degeneration of optic nerve, resulting in rough eye phenotype (Wittmann et al., Science, 2001). We found that Tdrd3 mutation dominantly enhances Tau-V337M induced rough eye phenotype, whereas addition of Top3b mutant exhibits an inconsistent modification. Our data suggest that TDRD3 can suppress neurodegeneration induced by at least one Tau mutant. Aim1. Progress 2. Top3b-Tdrd3 complex promotes piRNA biogenesis and silencing of transposable elements (TEs). 1. Our unbiased interaction assay (IP-MS) identified that Top3b and Tdrd3 biochemically interact with key piRNA machinery components including: Piwi, Aubergine and Armitage. 2. Based on to reporter assays and RNA-seq, we found that both Top3b and Tdrd3 promote piRNA guided TE silencing. 3. Our genetic interaction screen identified that Top3b specifically coordinates with piRNA biogenesis components in piRNA pathway. Specifically, we generated 15 double mutants of key genes in piRNA pathways in Top3b KO mutant background flies and identified 7 positive hits (Aub, AGO3, vasa, mael, mago, armi, and zuc) (Figure 1A-C). Notably, all positive genetic interactors are involved in piRNA biogenesis. Furthermore, Top3b-/- with piRNA component double mutants show depletion of piRNA levels (Figure 1D,E), suggesting that Top3b promotes TE silencing by enhancing piRNA biogenesis.

我们课题组发现Top3b是真核生物中第一个双活性拓扑异构酶（Xu et al., Nat. Neurosci., 2013）。这一发现极大地拓展了拓扑异构酶的领域，因为它表明不仅DNA，RNA也可能存在需要拓扑异构酶来解决的拓扑问题。此后我们发现 Top3b 是一种 RBP，是哺乳动物细胞中主要的 mRNA 结合拓扑异构酶，它与 TDRD3 形成保守复合物。 Top3b-TDRD3 与 RBP、FMRP（脆性 X 智力迟钝蛋白）相互作用；它们在 SG 中共定位，在多核糖体中共分离，并促进 mRNA 翻译和突触形成。与其他 RBP 和 SG 成分一样，Top3b 突变与神经系统疾病相关。 Top3b-KO 小鼠表现出在精神和认知障碍中观察到的行为和神经缺陷（Joo 等人，Nat. Comm. 2020）。 此外，Top3b-TDRD3 与 RNA 诱导的沉默复合物相互作用，促进果蝇中异染色质的形成和转座子的沉默 (Lee et al., Nat. Comm., 2018)。最近，Top3b-TDRD3已被证明是正链RNA病毒（包括SARS-CoV-2）有效复制所必需的，这表明Top3b可能成为COVID-19和其他由RNA病毒引起的流行病的药物靶点。 三个主要原因促使我们研究 Top3b 在 AD 和 tau 蛋白病中的作用。 首先，Top3b-TDRD3 是 SG 的一个组成部分，它可以充当病理种子，其中错误折叠的蛋白质可以找到低复杂性蛋白质进行聚集，从而导致 AD 和 tau 蛋白病。 Tau、TDP-43和C90orf72诱导的神经元毒性可以通过SG成分的遗传减少来减轻；或通过抑制启动 SG 组装的激酶。因此，SGs 被建议作为神经退行性疾病的药物靶点。重要的是，Top3b-TDRD3 KO 人类细胞和果蝇显示出 SG 的加速解离。此外，Top3b 的基因减少可抑制 Tau 诱导的果蝇神经变性。这些发现支持这样的观点，即 Top3b 可能像其他 SG 成分一样成为 tau 蛋白病的靶标。 其次，我们发现 Top3b-KO 小鼠大脑中表现出转录缺陷的几个基因对于 AD 至关重要，例如 APP 和 Tau (11)。这表明 Top3b 的基因减少可能会降低这些致病蛋白的水平，从而抑制神经退行性变。 第三，我们发现 Top3b 在生化和遗传上与 piRNA 机制相互作用，促进果蝇中转座元件 (TE) 的沉默；这种生化相互作用在小鼠体内是保守的。在 AD 患者和 tau 蛋白病动物模型中观察到 TE 失调。此外，它们被认为是衰老、与年龄相关的炎症和神经退行性疾病的驱动因素。我们计划研究 Top3b 和 Tau 是否可以在 piRNA 和/或其他途径中共同作用以促进 TE 沉默。 具体目标 目标 1. 使用细胞系研究 Top3b-TDRD3 是否调节正常和 tau 诱导的 SG 动力学和细胞毒性 子目标 1-1。研究 Top3b-TDRD3 如何在正常 SG 中发挥作用 我们获得了Top3b和TDRD3-KO HeLa细胞；发现两种KO细胞中SG组装正常，但SG解体加速。我们已经确定了一个新的相互作用伙伴 PRRC2A/C，它是一个已知的 SG 组件；并发现该蛋白在果蝇动物的生存和眼睛发育中与 TDRD3 存在遗传相互作用。 子目标 1-2。研究 Top3b-TDRD3 是否以及如何影响 tau 相关 SG 和毒性 策略：消除 TIA1（SG 成分）可以减少细胞系中 tau 诱导的颗粒和相关的细胞毒性。我们计划使用相同的策略：将 Tau 变体引入 WT 或 KO HeLa 细胞，然后确定 KO 细胞是否减少了 tau 阳性颗粒的数量并加快了分解速度，减少了细胞死亡，并降低了细胞对二次应激的敏感性。 目标 2. 研究 Top3b 突变是否以及如何改变果蝇 tau 蛋白病模型的神经退行性表型。 子目标 2-1。研究 Top3b-TDRD3 的基因减少是否可以改变 Tau 诱导的神经退行性变 初步结果：我们发现Top3b杂合突变可以抑制Tau-V337M突变体的眼部神经退行性表型，而TDRD3突变体可以增强该表型。数据表明 Top3b 抑制剂可以减轻 Tau 诱导的神经变性。我们计划在目标 4 中开发这样的抑制剂。 子目标2-2 研究Top3b突变如何抑制Tau诱导的神经毒性的机制 初步结果：我们发现Top3b KO或TDRD3 KO果蝇翼盘细胞中SG的解离明显快于WT，这与我们在Hela细胞中的发现一致。 子目标 2-3。 研究 Top3b 和 Tau 在 TE 沉默中是否以相同或不同的途径发挥作用。 初步结果：我们发现 Top3b 和 piRNA 机制在生化和遗传上相互作用，从而沉默果蝇卵巢中的转座子，因为 Top3b-piRNA 双突变体比每个单突变体表现出更高水平的多个 TE。 目标 3. 研究 Top3b 突变是否可以改变 tau 蛋白病小鼠模型的神经变性表型。 一旦我们的阿尔茨海默病概念获得 BSC 批准，我们计划开始实现这一目标。 目标 4. 研究拓扑异构酶是否可以成为神经退行性疾病的药物靶点。 我们已经获得了一些抑制拓扑异构酶活性的化合物，但它们的 Kd 仍然太高（46）。我们将继续合作，以确定具有更高效力的化合物。 更新进度： 该项目的最初目标概述 我们建议使用果蝇模型来研究拓扑异构酶 3β (Top3b) 在阿尔茨海默病 (AD)、肌萎缩侧索硬化症 (ALS) 和额颞叶变性 (FTLD) 中的作用。该项目的最初目标是： 1. 研究Top3突变是否以及如何改变果蝇Tau病模型的神经变性表型。 2. 研究Top3突变是否以及如何改变果蝇TDP-43模型的神经退行性表型。 3. 研究FANCM是否在果蝇异染色质形成和TE沉默中发挥作用。 4. 利用果蝇研究 Top3b/TDRD3 如何调节 mRNA 降解和翻译。 更新当前进展 目标1。进展1. Top3b和Tdrd3突变可能改变Tau-V337M突变体诱导的神经退行性变 果蝇眼睛中致病性突变体 hTau 的表达可导致视神经进行性变性，导致眼睛表型粗糙（Wittmann 等人，Science，2001）。 我们发现 Tdrd3 突变显着增强 Tau-V337M 诱导的粗糙眼表型，而添加 Top3b 突变体则表现出不一致的修饰。我们的数据表明 TDRD3 可以抑制至少一种 Tau 突变体引起的神经变性。 目标1。进展 2. Top3b-Tdrd3 复合物促进 piRNA 生物发生和转座元件 (TE) 沉默。 1. 我们的无偏相互作用测定 (IP-MS) 发现 Top3b 和 Tdrd3 与关键 piRNA 机械成分发生生化相互作用，包括：Piwi、Aubergine 和 Armitage。 2.基于报告基因检测和RNA-seq，我们发现Top3b和Tdrd3都促进piRNA引导的TE沉默。 3.我们的遗传相互作用筛选发现Top3b与piRNA途径中的piRNA生物合成成分特异性协调。具体来说，我们在 Top3b KO 突变体背景果蝇中生成了 piRNA 通路中关键基因的 15 个双突变体，并鉴定了 7 个阳性命中（Aub、AGO3、vasa、mael、mago、armi 和 zuc）（图 1A-C）。值得注意的是，所有正向遗传相互作用因子都参与 piRNA 生物发生。此外，具有 piRNA 成分双突变体的 Top3b-/- 显示 piRNA 水平的耗尽（图 1D，E），表明 Top3b 通过增强 piRNA 生物合成来促进 TE 沉默。