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Mitochondrial Top3α is required for mitochondrial genome maintenance and energy generation. a M1L flies exhibit lower mtDNA copy numbers than controls at the ages examined. b The decrease of mitochondrial membrane potential is accelerated in M1L flies compared to wildtype and rescued flies. c ATP content in M1L flies declines more rapidly with age when compared to wildtype and rescued counterparts. Two-way ANOVA with Turkey's multiple tests was used for statistical analysis. (NS = not significant, ***P < 0.001, **P < 0.01, *P < 0.05)
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Background
Mitochondria play important roles in providing metabolic energy and key metabolites for synthesis of cellular building blocks. Mitochondria have additional functions in other cellular processes, including programmed cell death and aging. A previous study revealed Drosophila mitochondrial topoisomerase III alpha (Top3α) contributes to the...
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... week-old flies. We found that mitochondrial DNA copy number in the M1L flies was decreased 2.38 and 2-fold at 1 week of age compared with the wildtype and transgene-rescued flies, respectively. By 5 weeks of age, the decrease of mtDNA in M1L flies was enhanced by 4.5 and 3-fold, as compared to the respective controls (wildtype and rescued flies) (Fig. ...
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... also determined the mitochondrial membrane po- tential of the mutant flies by monitoring JC-1 aggrega- tion [17]. As shown in Fig. 2b, the mitochondrial membrane potential of M1L mutant flies was 3.4, 4.6, and 5.3-fold lower than that of wildtype flies at 1-, 3-, and 5-week-old, respectively. As compared with the res- cued flies, the membrane potential in M1L mutants was 2, 2.4, and 5.3-fold lower at 1, 3 and 5 weeks old, re- spectively. Similar to the premature ...
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... is re- quired for ATP production by ATP synthase, we also measured ATP content as an endpoint for ATP gener- ation. Compared with the wildtype and rescued flies, the ATP content of the 1-week-old M1L flies was decreased by 1.05 and 1.07-fold, respectively, while in the 5-week- old M1L flies, the ATP content was decreased by 2.19 and 1.44-fold (Fig. 2c). Consistent with these findings, the age-dependent decrease of ATP content was more apparent in M1L flies (56 %) than that in either wildtype (7 %) or rescued flies (40 %). Our results indicate that mitochondrial Top3α deficiency causes marked declines in mitochondrial DNA copy number, mitochondrial membrane potential, and ATP content ...
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... the E3 ubiquitin ligase Parkin translocates to the mitochondrial outer-membrane [21,22], thereby targeting the dysfunc- tional and damaged mitochondrion for degradation by autophagosomes. Thus, our observation of mitochon- drial membrane debris in M1L flies is consistent with the observed loss of mitochondrial membrane potential during aging (Fig. ...
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... has been shown that mtDNA copy number is nega- tively correlated with age [30]. Decreased mtDNA con- tent is also linked to the decline of mitochondrial function [31]. In the present study, we demonstrated a sharp decrease of mtDNA levels with age in flies lacking mitochondrial Top3α (Fig. 2a). Additionally, mitochon- drial functions in M1L flies are diminished as compared to those of wildtype and rescued flies (Fig. 2b and c). It has been shown that a reduction of mitochondrial activ- ity will not occur until a considerable level (~60 %) of mtDNA deletion has been reached [32]. This so-called "threshold effect" varies ...
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... age [30]. Decreased mtDNA con- tent is also linked to the decline of mitochondrial function [31]. In the present study, we demonstrated a sharp decrease of mtDNA levels with age in flies lacking mitochondrial Top3α (Fig. 2a). Additionally, mitochon- drial functions in M1L flies are diminished as compared to those of wildtype and rescued flies (Fig. 2b and c). It has been shown that a reduction of mitochondrial activ- ity will not occur until a considerable level (~60 %) of mtDNA deletion has been reached [32]. This so-called "threshold effect" varies between different types of cells. Loss of mitochondrial Top3α results in both accumula- tion of mtDNA deletions and a concomitant decrease of ...
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... been shown that a reduction of mitochondrial activ- ity will not occur until a considerable level (~60 %) of mtDNA deletion has been reached [32]. This so-called "threshold effect" varies between different types of cells. Loss of mitochondrial Top3α results in both accumula- tion of mtDNA deletions and a concomitant decrease of mtDNA copy number (Fig. 2a and 5a). Therefore, mul- tiple changes in the mitochondrial genome may lead to the observed mitochondrial dysfunction in M1L ...
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... from NGS analysis demonstrated that the hot- spots for mtDNA deletion in Drosophila are located adjacent to the heavy strand origin, and also to the tRNA clusters, which are possibly the replication ori- gin for the light strand (Fig. 5b and c). The deletion frequency also increases as the mtDNA copy number decreases in the M1L mutant flies (Fig. 5a and 2a). PCR-based assays and Southern blot analysis have demonstrated that mtDNA deletions accumulate with age in Drosophila [34]. However, such analysis did not provide any detailed information on the deletion frequency or the sequence boundaries for deletions. Here, we examined mtDNA deletions with NGS analysis, which not only allowed us to ...
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Citations
... TOP3A can be localized both in the nucleus and mitochondria [131,132]. Mitochondrial TOP3A has been shown to be required for the maintenance of Drosophila mtDNA genome integrity [132,133], decatenation, and segregation of human mtDNA [134]. The type IA human TOP3A in mitochondria contributes to mtDNA replication and mtDNA topology control [135,136] along with the type IB TOP1MT [7]. ...
Topoisomerases regulate the topological state of cellular genomes to prevent impediments to vital cellular processes, including replication and transcription from suboptimal supercoiling of double-stranded DNA, and to untangle topological barriers generated as replication or recombination intermediates. The subfamily of type IA topoisomerases are the only topoisomerases that can alter the interlinking of both DNA and RNA. In this article, we provide a review of the mechanisms by which four highly conserved N-terminal protein domains fold into a toroidal structure, enabling cleavage and religation of a single strand of DNA or RNA. We also explore how these conserved domains can be combined with numerous non-conserved protein sequences located in the C-terminal domains to form a diverse range of type IA topoisomerases in Archaea, Bacteria, and Eukarya. There is at least one type IA topoisomerase present in nearly every free-living organism. The variation in C-terminal domain sequences and interacting partners such as helicases enable type IA topoisomerases to conduct important cellular functions that require the passage of nucleic acids through the break of a single-strand DNA or RNA that is held by the conserved N-terminal toroidal domains. In addition, this review will exam a range of human genetic disorders that have been linked to the malfunction of type IA topoisomerase.
... The mitochondrial isoform of TOP3A has been well studied in Drosophila, where lack of this isoform leads to the loss of mtDNA, neurological diseases, infertility and aging [11,49]. Subsequent studies in human cells showed that TOP3A is required for proper segregation of mtDNA after completion of replication [4] (see Figure 2c). ...
Humans have two Type IA topoisomerases, topoisomerase IIIα (TOP3A) and topoisomerase IIIβ (TOP3B). In this review, we focus on the role of human TOP3A in DNA replication and highlight the recent progress made in understanding TOP3A in the context of replication. Like other topoisomerases, TOP3A acts by a reversible mechanism of cleavage and rejoining of DNA strands allowing changes in DNA topology. By cleaving and resealing single-stranded DNA, it generates TOP3A-linked single-strand breaks as TOP3A cleavage complexes (TOP3Accs) with a TOP3A molecule covalently bound to the 5´-end of the break. TOP3A is critical for both mitochondrial and for nuclear DNA replication. Here, we discuss the formation and repair of irreversible TOP3Accs, as their presence compromises genome integrity as they form TOP3A DNA-protein crosslinks (TOP3A-DPCs) associated with DNA breaks. We discuss the redundant pathways that repair TOP3A-DPCs, and how their defects are a source of DNA damage leading to neurological diseases and mitochondrial disorders.
... All flies used for sleeping and assaying were female virgin flies. The average lifespan of wild-type Drosophila melanogaster is about 2 months in a standard experimental setting [21][22][23]. On the third day after eclosion, Drosophila sleep behavior reaches adult patterns [24]. ...
Aging of the brain usually leads to the decline of neurological processes and is a major risk factor for various neurodegenerative diseases, including sleep disturbances and cognitive decline. Adipose tissue exosomes, as adipocyte-derived vesicles, may mediate the regulatory processes of adipose tissue on other organs, including the brain; however, the regulatory mechanisms remain unclear. We analyzed the sleep-wake behavior of young (10 days) and old (40 days) Drosophila and found that older Drosophila showed increased sleep fragmentation, which is similar to mammalian aging characteristics. To investigate the cross-tissue regulatory mechanisms of adiposity on brain aging, we extracted 10-day and 40-day Drosophila adipose tissue exosomes and identified circRNAs with age-dependent expression differences by RNA-seq and differential analysis. Furthermore, by combining data from 3 datasets of the GEO database (GSE130158, GSE24992, and GSE184559), circ_sxc that was significantly downregulated with age was finally screened out. Moreover, dme-miR-87-3p, a conserved target of circ_sxc, accumulates in the brain with age and exhibits inhibitory effects in predicted binding relationships with neuroreceptor ligand genes. In summary, the current study showed that the Drosophila brain could obtain circ_sxc by uptake of adipose tissue exosomes which crossed the blood-brain barrier. And circ_sxc suppressed brain miR-87-3p expression through sponge adsorption, which in turn regulated the expression of neurological receptor ligand proteins (5-HT1B, GABA-B-R1, Rdl, Rh7, qvr, NaCP60E) and ensured brain neuronal synaptic signaling normal function of synaptic signaling. However, with aging, this regulatory mechanism is dysregulated by the downregulation of the adipose exosome circ_sxc, which contributes to the brain exhibiting sleep disturbances and other “aging” features.
... The TOP3A transcript contains two potential initiation codons, M1 and M26. Translation from the upstream start site generates a longer isoform bearing an N-terminal mitochondrial targeting sequence (MTS) that directs its import into mitochondria, while translation from the downstream initiation codon generates an N-terminally truncated protein that lacks the MTS and is directed for nuclear import (Wang et al, 2002;Wu et al, 2010;Tsai et al, 2016;Nicholls et al, 2018;Menger et al, 2022). Proteolytic cleavage of the MTS during mitochondrial import leads to mature mitochondrial and nuclear isoforms that are almost identical at the N-terminus (Wang et al, 2002). ...
Topoisomerase 3α (TOP3A) is an enzyme that removes torsional strain and interlinks between DNA molecules. TOP3A localises to both the nucleus and mitochondria, with the two isoforms playing specialised roles in DNA recombination and replication respectively. Pathogenic variants in TOP3A can cause a disorder similar to Bloom syndrome, which results from bi-allelic pathogenic variants in BLM, encoding a nuclear-binding partner of TOP3A. In this work, we describe 11 individuals from 9 families with an adult-onset mitochondrial disease resulting from bi-allelic TOP3A gene variants. The majority of patients have a consistent clinical phenotype characterised by bilateral ptosis, ophthalmoplegia, myopathy and axonal sensory-motor neuropathy. We present a comprehensive characterisation of the effect of TOP3A variants, from individuals with mitochondrial disease and Bloom-like syndrome, upon mtDNA maintenance and different aspects of enzyme function. Based on these results, we suggest a model whereby the overall severity of the TOP3A catalytic defect determines the clinical outcome, with milder variants causing adult-onset mitochondrial disease and more severe variants causing a Bloom-like syndrome with mitochondrial dysfunction in childhood.
... In the eukaryotic nucleus, type IIA topoisomerases are the primary enzymes that act in the removal of chromosome interlinks (20,21), while in Escherichia coli both type IIA and type IA topoisomerases can contribute to decatenation (22)(23)(24). The transcript of the human type IA topoisomerase TOP3A contains two translation start sites, generating either a longer isoform bearing a mitochondrial targeting sequence or a shorter isoform that lacks this targeting sequence and is directed to the nucleus (25)(26)(27). The loss of mitochondrial TOP3A leads to the accumulation of catenated mtDNA replication products, indicating that TOP3A is required for decatenation of replicated mtDNA molecules (28,29). ...
Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisa-tion and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the de-catenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.
... the mitochondrial isoform of topoisomerase 3a (tOP3a) 186,368 is required at the end of replication to decatenate the daughter mtDNa molecules, which are intert wined across their OriH regions, a process that is facilitated by mitochondrial tOP1 (tOP1Mt) and is independent of the usual tOP3a-interacting Bloom syndrome protein (BLM)-tOP3a-recQ-mediated genome instability proteins (rMi1/2) (Btr) dissolvasome complex 12 . Mutations in TOP3A have been reported in individuals with combined Bloom and mitochondrial syndromes characterized by dilated cardiomyopathy, mtDNa depletion in muscles and progressive external ophthalmoplegia syndrome 13,187 . in the fruit fly, inactivation of mitochondrial tOP3a results in defective genome integrity and mitochondrial functions with accelerated ageing and infertility 186,193 (TABLe 1). ...
... 188 ), which is consistent with the fact that TOP3A and BLM cooperate in the dissolvasome complex 6,189 to suppress sister chromatid exchanges 161,190 and HDR 191 and contribute to the resolution of stalled replication forks 192 . The mitochondrial isoform of TOP3A is crucial for mtDNA replication 12 , fertility 186,193 and, owing to its mitochondrial functions, the viability of postmitotic cells 185 (BOx 1; TABLe 1). TOP3B is not essential, but mice lacking TOP3B have a shortened lifespan, a higher incidence of aneuploidy in germ cells, increased autoimmunity [194][195][196] , abnormal synapse formation 197 and behavioural impairments 198 (TABLe 1). ...
Human topoisomerases comprise a family of six enzymes: two type IB (TOP1 and mitochondrial TOP1 (TOP1MT), two type IIA (TOP2A and TOP2B) and two type IA (TOP3A and TOP3B) topoisomerases. In this Review, we discuss their biochemistry and their roles in transcription, DNA replication and chromatin remodelling, and highlight the recent progress made in understanding TOP3A and TOP3B. Because of recent advances in elucidating the high-order organization of the genome through chromatin loops and topologically associating domains (TADs), we integrate the functions of topoisomerases with genome organization. We also discuss the physiological and pathological formation of irreversible topoisomerase cleavage complexes (TOPccs) as they generate topoisomerase DNA–protein crosslinks (TOP-DPCs) coupled with DNA breaks. We discuss the expanding number of redundant pathways that repair TOP-DPCs, and the defects in those pathways, which are increasingly recognized as source of genomic damage leading to neurological diseases and cancer. Topoisomerases have essential roles in transcription, DNA replication, chromatin remodelling and, as recently revealed, 3D genome organization. However, topoisomerases also generate DNA–protein crosslinks coupled with DNA breaks, which are increasingly recognized as a source of disease-causing genomic damage.
... Mitochondrial Top3α works independently without its nuclear cofactors such as BLM-Top3α-RMI1-RMI2 (BTR) complex to maintain the distribution of single mtDNA minicircle throughout the mitochondrial network (Nicholls et al., 2018a). The mitochondrial Top3α knockout in Drosophila renders decreased mtDNA copy number along with the accumulation of mtDNA deletions which progressively declines mitochondrial function with age, decrease of lifespan (Tsai et al., 2016), while female germline flies lacking mitochondrial Top3α lay sterile eggs (Wu et al., 2010). Patients with biallelic mutation of Top3α suffering from a Bloom's syndrome-like disorder show clinical features of mtDNA depletion in skeletal muscle consistent with its role in decatenation of mtDNA hemicatenane (Martin et al., 2018). ...
Topoisomerases regulate DNA topology, organization of the intracellular DNA, the transmission of genetic materials, and gene expressions. Other than the nuclear genome, mitochondria also harbor the small, circular DNA (mtDNA) that encodes a critical subset of proteins for the production of cellular ATP; however, mitochondria are solely dependent on the nucleus for all the mitochondrial proteins necessary for mtDNA replication, repair, and maintenance. Mitochondrial genome compiles topological stress from bidirectional transcription and replication, therefore imports four nuclear encoded topoisomerases (Top1mt, Top2α, Top2β, and Top3α) in the mitochondria to relax mtDNA supercoiling generated during these processes. Trapping of topoisomerase on DNA results in the formation of protein-linked DNA adducts (PDAs), which are widely exploited by topoisomerase-targeting anticancer drugs. Intriguingly mtDNA is potentially exposed to DNA damage that has been attributed to a variety of human diseases, including neurodegeneration, cancer, and premature aging. In this review, we focus on the role of different topoisomerases in the mitochondria and our current understanding of the mitochondrial DNA damage through trapped protein-DNA complexes, and the progress in the molecular mechanisms of the repair for trapped topoisomerase covalent complexes (Topcc). Finally, we have discussed how the pathological DNA lesions that cause mtDNA damage, trigger mitochondrial fission and mitophagy, which serve as quality control events for clearing damaged mtDNA.
... [45] Topo IIIα is also localised to the mitochondria, and mutation of the mitochondrial import sequence in Drosophila melanogaster caused premature aging, mobility defects, and impaired fertility, caused by mitochondrial degeneration due to degradation of mitochondrial DNA (mtDNA). [46,47] A Met100Val mutation in human topo IIIα was identified in a patient with a mitochondrial disorder and was demonstrated to prevent resolution of mtDNA replication-specific hemicatenanes ( Figure 1A). [48] ...
DNA topoisomerases, capable of manipulating DNA topology, are ubiquitous and indispensable for cellular survival due to the numerous roles they play during DNA metabolism. As we review here, current structural approaches have revealed unprecedented insights into the complex DNA‐topoisomerase interaction and strand passage mechanism, helping to advance our understanding of their activities in vivo. This has been complemented by single‐molecule techniques, which have facilitated the detailed dissection of the various topoisomerase reactions. Recent work has also revealed the importance of topoisomerase interactions with accessory proteins and other DNA‐associated proteins, supporting the idea that they often function as part of multi‐enzyme assemblies in vivo. In addition, novel topoisomerases have been identified and explored, such as topo VIII and Mini‐A. These new findings are advancing our understanding of DNA‐related processes and the vital functions topos fulfil, demonstrating their indispensability in virtually every aspect of DNA metabolism.
... Here, flies are gently knocked to the bottom of a vial and are recorded by video as they instinctively climb upward (Ganetzky and Flanagan, 1978;Gargano et al., 2005). Climbing performance is often reported as some measure of the flies' position versus time, such as mean position at a time cut-off (Gargano et al., 2005;Lavoy et al., 2018) or time until a percentage of flies reach a set height (Ma et al., 2014;Podratz et al., 2013;Tsai et al., 2016;Xu et al., 2008). ...
Negative geotaxis (climbing) performance is a useful metric for quantifying Drosophila health. Manual methods to quantify climbing performance are tedious and often biased, while many available computational methods have challenging hardware or software requirements. We present an alternative: FreeClimber. This open source, Python-based platform subtracts a video's static background to improve detection for flies moving across heterogeneous backgrounds. FreeClimber calculates a cohort's velocity as the slope of the most linear portion of a mean-vertical position vs. time curve. It can run from a graphical user interface for optimization or a command line interface for high-throughput and automated batch processing, improving accessibility for users with different expertise. FreeClimber outputs calculated slopes, spot locations for follow up analyses (e.g. tracking), and several visualizations and plots. We demonstrate FreeClimber's utility in a longitudinal study for endurance exercise performance in Drosophila mitonuclear genotypes using six distinct mitochondrial haplotypes paired with a common w 1118 nuclear background.
... The observed increased of ATP intracellular levels in keratinocytes clearly demonstrates that, at least in vitro, PMCE enhances cell viability, cell proliferation and energy metabolism of keratinocytes. According to previous reports, this increase is associated with higher levels of mitochondrial activity, energy metabolism and cell proliferation 14,15 and is indicative of a lack of cytotoxicity 14 In line with this, PMCE confers mitochondrial functionality under oxidative stress which is associated with the attenuated aging process as well as with the maintenance of significant cellular functions 16,17 . ...
Abstract Natural ingredients have been used to improve the state of health in humans. The genus Paeonia has been studied only limited yet it’s reported to have many activities such as antioxidant and anti-inflammatory. To this context, here we focused on an endemic Paeonia species in Attica. This study aims to present the development of the Paeonia mascula subsp. hellenica callus extract and its pleiotropic bioactivity on human primary keratinocytes exploring its potential application as an active agent in skin-related products. This extract showed a high scavenging activity with high phenolic content and an interesting metabolic profile. At a molecular level, the study on the transcript accumulation of genes revealed that this extract exhibits in vitro skin-related protection properties by mediating mitochondrial energy, cell proliferation, immune and inflammatory response and positively regulates genes involved in epidermal and in stratum corneum function. Besides, the extract is proven not skin irritant on reconstructed human skin model. These findings indicate that the specific P. mascula subsp. hellenica extract possesses significant in vitro protection activity on human epidermis and provides new insights into its beneficial role in skin confirming that the advent of biotechnology contribution the past few decades.
