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.

我们的小组发现了TOP3B是真核生物中的第一个双活动拓扑酶（Xu等，Nat。Neurosci。，2013）。这一发现大大扩展了拓扑异构酶领域，因为它表明不仅DNA，而且还存在RNA，还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是哺乳动物细胞中的RBP，是哺乳动物细胞中主要的mRNA结合拓扑酶，它与TDRD3形成了保守的复合物。 TOP3B-TDRD3与RBP FMRP（脆弱-X智力低下蛋白）相互作用；它们在SGS中共定位，在多构体中共取分，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有关。和Top3b-ko小鼠在精神病和认知障碍中观察到的行为和神经缺陷（Joo等，Nat。Comm。2020）。 此外，TOP3B-TDRD3与RNA诱导的沉默复合物相互作用，以促进果蝇中转座子的异染色质形成和沉默（Lee等，Nat。Comm。，2018）。最近，已经证明TOP3B-TDRD3是有效复制正链RNA病毒所必需的，包括SARS-COV-2，这表明TOP3B可能是Covid-19的药物靶标，以及由RNA病毒引起的其他大流行病。 有三个主要原因促使我们研究Top3b在AD和Tauopathy中的作用。 首先，TOP3B-TDRD3是SGS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可以找到低复杂性蛋白的聚集，从而导致AD和Tauopathy。 TAU，TDP-43和C90ORF72诱导的神经元毒性可以通过SG成分的遗传降低来缓解。或通过抑制引发SG组装的激酶。因此，已建议SG作为神经退行性的药物靶标。重要的是，TOP3B-TDRD3 KO人类细胞和果蝇表现出SGS的加速解离。此外，TOP3B的遗传降低抑制了果蝇中TAU诱导的神经退行性。这些发现支持这样的观念：Top3b可能像其他SG组件一样成为tauopathy的目标。 其次，我们发现在TOP3B-KO小鼠大脑中显示出有缺陷转录的几个基因对AD和TAU等AD至关重要（11）。这表明TOP3B的遗传还原可能会降低这些致病蛋白的水平，从而导致神经退行性的抑制。 第三，我们发现Top3b在生物化学和遗传上与pirna机械相互作用，以促进蝇中转座元件（TES）的沉默。这种生化相互作用在小鼠中是保守的。在AD患者和陶氏病的动物模型中已经观察到了失调。此外，已建议它们是衰老，与年龄相关的炎症和神经退行性的驱动因素。我们计划研究top3b和tau是否可以在PIRNA和/或其他途径中共同努力以促进沉默。 具体目标 AIM 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 Sub-aim 1-1。研究TOP3B-TDRD3在正常SG中的功能 我们获得了TOP3B和TDRD3-KO HELA细胞；并发现SG组装是正常的，但是在两个KO细胞中，SG拆卸均加速。我们已经确定了一个新的交互合作伙伴PRRC2A/C，这是已知的SG组件。并发现该蛋白质在果蝇动物存活和眼睛发育中与TDRD3相互作用。 Sub-aim 1-2。研究top3b-tdrd3是否以及如何影响tau相关的SG和毒性 策略：TIA1（SG成分）的耗竭可以减少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细胞的数量减少和更快的tau阳性颗粒，降低细胞死亡以及对次应激的细胞敏感性降低。 AIM 2。研究TOP3B突变是否可以修改果蝇Tauopathy模型的神经退行性表型。 Sub-aim 2-1。研究TOP3B-TDRD3的遗传降低是否可以改变Tau诱导的神经变性 初步结果：我们发现TOP3B杂合突变可以抑制，而TDRD3突变体可以增强Tau-V337M突变体的神经变性性眼表型。数据表明，TOP3B的抑制剂可能会减轻TAU诱导的神经变性。我们计划在AIM 4中开发这种抑制剂。 Sub-aim 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果蝇翼圆盘细胞中SG的解离比WT明显快，这与我们在HeLa细胞中的发现一致。 Sub-aim 2-3。 研究Top3b和Tau在沉默中是否在相同或不同的途径中起作用。 初步结果：我们发现Top3b和Pirna机械在生化和遗传上与果蝇卵巢中的沉默转座相互作用，因为Top3b-Pirna双突变体比每个单个突变体都具有更高的几个TES水平。 AIM 3。研究TOP3B突变是否可以修改Tauopathy小鼠模型的神经变性表型。 一旦我们的阿尔茨海默氏病概念得到了BSC批准，我们计划开始以这一目标为目标。 AIM 4。研究拓扑异构酶是否可以成为神经退行性的可药物靶标。 我们已经获得了一些抑制拓扑异构酶活性的化合物，但是它们的KD仍然太高（46）。我们将继续我们的协作，以识别具有更高效力的化合物。 进度更新： 该项目最初目标的概述 我们建议使用果蝇模型检查拓扑异构酶3Beta（TOP3B）在阿尔茨海默氏病（AD），肌萎缩性侧面硬化症（ALS）和额叶叶状洛巴尔变性（FTLD）中的作用。该项目的最初目的是：1。研究TOP3突变是否可以修改果蝇Tauopathy模型的神经变性表型。 2。研究TOP3突变是否可以修改果蝇TDP-43模型的神经退行性表型。 3。研究范例是否在果蝇中的异染色质形成和沉默中起作用。 4。研究TOP3B/TDRD3如何使用果蝇调节mRNA降解和翻译。 更新当前进度 AIM1。进度1。TOP3B和TDRD3突变可能会改变Tau-V337M突变体诱导的神经变性 致病性突变体HTAU在蝇眼中的表达会导致视神经的进行性变性，从而导致眼睛表型粗糙（Wittmann等，Science，Science，2001）。 我们发现TDRD3突变主要增强了Tau-V337M诱导的粗眼表型，而添加TOP3B突变体的修饰不一致。我们的数据表明，TDRD3可以抑制至少一个Tau突变体诱导的神经变性。 AIM1。进展2。TOP3B-TDRD3复合物促进了piRNA生物发生和转座元件的沉默（TES）。 1。我们的无偏相互作用测定（IP-MS）确定TOP3B和TDRD3生化与关键的Pirna机械组件相互作用，包括：Piwi，Aubergine和Armitage。 2。根据记者测定和RNA-Seq，我们发现TOP3B和TDRD3都促进了Pirna引导的TE沉默。 3。我们的遗传相互作用筛选确定TOP3B在PIRNA途径中专门与PIRNA生物发生成分坐标。具体而言，我们在Top3b KO突变体背景苍蝇中生成了PIRNA途径中的关键基因的15个双突变体，并鉴定了7个正命中（AUB，AGO3，VASA，MAEL，MAEL，MAGO，ARMI和ZUC）（图1A-C）。值得注意的是，所有阳性遗传相互作用者均参与PIRNA生物发生。此外，带有piRNA成分双突变体的Top3b - / - 显示出piRNA水平的耗竭（图1D，E），这表明TOP3B通过增强PIRNA生物发生来促进TE沉默。