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

National Institute On Aging

Investigators

Abstract

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.