Publications by authors named "Elsa Logarinho"

31 Publications

Modulates Cell Non-Autonomous Response in Zebrafish Skeletal Muscle Homeostasis.

Cells 2021 05 18;10(5). Epub 2021 May 18.

i3S-Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135 Porto, Portugal.

is a master regulator of the cell cycle, contributing to cell proliferation. Recent data have shown that this transcription factor also modulates gene networks associated with other cellular mechanisms, suggesting non-proliferative functions that remain largely unexplored. In this study, we used CRISPR/Cas9 to disrupt in the zebrafish terminally differentiated fast-twitching muscle cells. genomic disruption increased myofiber death and clearance. Interestingly, this contributed to non-autonomous satellite cell activation and proliferation. Moreover, we observed that Cas9 expression alone was strongly deleterious to muscle cells. Our report shows that modulates a muscle non-autonomous response to myofiber death and highlights underreported toxicity to high expression of Cas9 in vivo.
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http://dx.doi.org/10.3390/cells10051241DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158134PMC
May 2021

FOXM1 repression increases mitotic death upon antimitotic chemotherapy through BMF upregulation.

Cell Death Dis 2021 05 25;12(6):542. Epub 2021 May 25.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135, Porto, Portugal.

Inhibition of spindle microtubule (MT) dynamics has been effectively used in cancer treatment. Although the mechanisms by which MT poisons elicit mitotic arrest are fairly understood, efforts are still needed towards elucidating how cancer cells respond to antimitotic drugs owing to cytotoxicity and resistance side effects. Here, we identified the critical G2/M transcription factor Forkhead box M1 (FOXM1) as a molecular determinant of cell response to antimitotics. We found FOXM1 repression to increase death in mitosis (DiM) due to upregulation of the BCL-2 modifying factor (BMF) gene involved in anoikis, an apoptotic process induced upon cell detachment from the extracellular matrix. FOXM1 binds to a BMF intronic cis-regulatory element that interacts with both the BMF and the neighbor gene BUB1B promoter regions, to oppositely regulate their expression. This mechanism ensures that cells treated with antimitotics repress BMF and avoid DiM when FOXM1 levels are high. In addition, we show that this mechanism is partly disrupted in anoikis/antimitotics-resistant tumor cells, with resistance correlating with lower BMF expression but in a FOXM1-independent manner. These findings provide a stratification biomarker for antimitotic chemotherapy response.
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http://dx.doi.org/10.1038/s41419-021-03822-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8149823PMC
May 2021

Candidate Alzheimer's Disease Biomarker miR-483-5p Lowers TAU Phosphorylation by Direct ERK1/2 Repression.

Int J Mol Sci 2021 Apr 1;22(7). Epub 2021 Apr 1.

Laboratory of Preclinical Testing of Higher Standard, Nencki Institute of Experimental Biology of Polish Academy of Sciences, Pasteur 3, 02-093 Warsaw, Poland.

MicroRNAs have been demonstrated as key regulators of gene expression in the etiology of a range of diseases including Alzheimer's disease (AD). Recently, we identified miR-483-5p as the most upregulated miRNA amongst a panel of miRNAs in blood plasma specific to prodromal, early-stage Alzheimer's disease patients. Here, we investigated the functional role of miR-483-5p in AD pathology. Using TargetScan and miRTarBase, we identified the microtubule-associated protein MAPT, often referred to as TAU, and the extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), known to phosphorylate TAU, as predicted direct targets of miR-483-5p. Employing several functional assays, we found that miR-483-5p regulates ERK1 and ERK2 at both mRNA and protein levels, resulting in lower levels of phosphorylated forms of both kinases. Moreover, miR-483-5p-mediated repression of ERK1/2 resulted in reduced phosphorylation of TAU protein at epitopes associated with TAU neurofibrillary pathology in AD. These results indicate that upregulation of miR-483-5p can decrease phosphorylation of TAU via ERK pathway, representing a compensatory neuroprotective mechanism in AD pathology. This miR-483-5p/ERK1/TAU axis thus represents a novel target for intervention in AD.
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http://dx.doi.org/10.3390/ijms22073653DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8037306PMC
April 2021

SOGA1 and SOGA2/MTCL1 are CLASP-interacting proteins required for faithful chromosome segregation in human cells.

Chromosome Res 2021 06 15;29(2):159-173. Epub 2021 Feb 15.

Chromosome Instability & Dynamics Group, i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Rua Alfredo Allen 208, 4200-135, Porto, Portugal.

CLASPs are key modulators of microtubule dynamics throughout the cell cycle. During mitosis, CLASPs independently associate with growing microtubule plus-ends and kinetochores and play essential roles in chromosome segregation. In a proteomic survey for human CLASP1-interacting proteins during mitosis, we have previously identified SOGA1 and SOGA2/MTCL1, whose mitotic roles remained uncharacterized. Here we performed an initial functional characterization of human SOGA1 and SOGA2/MTCL1 during mitosis. Using specific polyclonal antibodies raised against SOGA proteins, we confirmed their expression and reciprocal interaction with CLASP1 and CLASP2 during mitosis. In addition, we found that both SOGA1 and SOGA2/MTCL1 are phospho-regulated during mitosis by CDK1. Immunofluorescence analysis revealed that SOGA2/MTCL1 co-localizes with mitotic spindle microtubules and spindle poles throughout mitosis and both SOGA proteins are enriched at the midbody during mitotic exit/cytokinesis. GFP-tagging of SOGA2/MTCL1 further revealed a microtubule-independent localization at kinetochores. Live-cell imaging after siRNA-mediated knockdown of SOGA1 and SOGA2/MTCL1 showed that they are independently required for distinct aspects of chromosome segregation. Thus, SOGA1 and SOGA2/MTCL1 are bona fide CLASP-interacting proteins during mitosis required for faithful chromosome segregation in human cells.
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http://dx.doi.org/10.1007/s10577-021-09651-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7610860PMC
June 2021

Small-molecule inhibition of aging-associated chromosomal instability delays cellular senescence.

EMBO Rep 2020 05 5;21(5):e49248. Epub 2020 Mar 5.

i3S - Instituto de Investigação e Inovação em Saúde, Universidade do Porto, Porto, Portugal.

Chromosomal instability (CIN) refers to the rate at which cells are unable to properly segregate whole chromosomes, leading to aneuploidy. Besides its prevalence in cancer cells and postulated implications in promoting tumorigenesis, studies in aneuploidy-prone mouse models uncovered an unanticipated link between CIN and aging. Using young to old-aged human dermal fibroblasts, we observed a dysfunction of the mitotic machinery arising with age that mildly perturbs chromosome segregation fidelity and contributes to the generation of fully senescent cells. Here, we investigated mitotic mechanisms that contribute to age-associated CIN. We found that elderly cells have an increased number of stable kinetochore-microtubule (k-MT) attachments and decreased efficiency in the correction of improper k-MT interactions. Chromosome mis-segregation rates in old-aged cells decreased upon both genetic and small-molecule enhancement of MT-depolymerizing kinesin-13 activity. Notably, restored chromosome segregation accuracy inhibited the phenotypes of cellular senescence. Therefore, we provide mechanistic insight into age-associated CIN and disclose a strategy for the use of a small-molecule to inhibit age-associated CIN and to delay the cellular hallmarks of aging.
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http://dx.doi.org/10.15252/embr.201949248DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7202060PMC
May 2020

Tissue engineering strategies for human hair follicle regeneration: How far from a hairy goal?

Stem Cells Transl Med 2020 03 26;9(3):342-350. Epub 2019 Dec 26.

Aging and Aneuploidy Group, IBMC, Instituto de Biologia Molecular e Celular, Porto, Portugal.

The demand for an efficient therapy for alopecia disease has fueled the hair research field in recent decades. However, despite significant improvements in the knowledge of key processes of hair follicle biology such as genesis and cycling, translation into hair follicle replacement therapies has not occurred. Great expectation has been recently put on hair follicle bioengineering, which is based on the development of fully functional hair follicles with cycling activity from an expanded population of hair-inductive (trichogenic) cells. Most bioengineering approaches focus on in vitro reconstruction of folliculogenesis by manipulating key regulatory molecular/physical features of hair follicle growth/cycling in vivo. Despite their great potential, no cell-based product is clinically available for hair regeneration therapy to date. This is mainly due to demanding issues that still hinder the functionality of cultured human hair cells. The present review comprehensively compares emergent strategies using different cell sources and tissue engineering approaches, aiming to successfully achieve a clinical cure for hair loss. The hurdles of these strategies are discussed, as well as the future directions to overcome the obstacles and fulfill the promise of a "hairy" feat.
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http://dx.doi.org/10.1002/sctm.19-0301DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7031632PMC
March 2020

α-Fodrin is required for the organization of functional microtubules during mitosis.

Cell Cycle 2019 Oct 27;18(20):2713-2726. Epub 2019 Aug 27.

Cancer Research Program-III, Rajiv Gandhi Centre for Biotechnology, University of Kerala , Thiruvananthapuram , India.

The cytoskeleton protein α-fodrin plays a major role in maintaining structural stability of membranes. It was also identified as part of the brain γ-tubulin ring complex, the major microtubule nucleator. Here, we investigated the requirement of α-fodrin for microtubule spindle assembly during mitotic progression. We found that α-fodrin depletion results in abnormal mitosis with uncongressed chromosomes, leading to prolonged activation of the spindle assembly checkpoint and a severe mitotic delay. Further, α-fodrin repression led to the formation of shortened spindles with unstable kinetochore-microtubule attachments. We also found that the mitotic kinesin CENP-E had reduced levels at kinetochores to likely account for the chromosome misalignment defects in α-fodrin-depleted cells. Importantly, we showed these cells to exhibit reduced levels of detyrosinated α-tubulin, which primarily drives CENP-E localization. Since proper microtubule dynamics and chromosome alignment are required for completion of normal mitosis, this study reveals an unforeseen role of α-fodrin in regulating mitotic progression. Future studies on these lines of observations should reveal important mechanistic insight for fodrin's involvement in cancer.
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http://dx.doi.org/10.1080/15384101.2019.1656476DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6773225PMC
October 2019

Chromosomal instability and pro-inflammatory response in aging.

Mech Ageing Dev 2019 09 15;182:111118. Epub 2019 May 15.

Ageing and Aneuploidy Laboratory, IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135, Porto, Portugal; i3S, Instituto de Investigação e Inovação em Saúde, Universidade do Porto, 4200-135, Porto, Portugal. Electronic address:

Aging refers to the progressive deterioration of tissue and organ function over time. Increasing evidence points to the accumulation of highly damaged cell cycle-arrested cells with age (cellular senescence) as major reason for the development of certain aging-associated diseases. Recent studies have independently shown that aneuploidy, an abnormal chromosome set, occurs in senescent cells, and that the accumulation of cytoplasmic DNA driven by faulty chromosome segregation during mitosis aids in the establishment of senescence and its associated secretory phenotype known as SASP. Here we review the emerging link between chromosomal instability (CIN) and senescence in the context of aging, with emphasis on the cGAS-STING pathway activation and its role in the development of the SASP. Based on current evidence, we propose that age-associated CIN in mitotically active cells contributes to aging and its associated diseases, and we discuss the inhibition of CIN as a potential strategy to prevent the generation of aneuploid senescent cells and thereby to delay aging.
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http://dx.doi.org/10.1016/j.mad.2019.111118DOI Listing
September 2019

The FOXM1 Inhibitor RCM-1 Decreases Carcinogenesis and Nuclear β-Catenin.

Mol Cancer Ther 2019 07 30;18(7):1217-1229. Epub 2019 Apr 30.

Perinatal Institute, Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

The oncogenic transcription factor FOXM1 has been previously shown to play a critical role in carcinogenesis by inducing cellular proliferation in multiple cancer types. A small-molecule compound, Robert Costa Memorial drug-1 (RCM-1), has been recently identified from high-throughput screen as an inhibitor of FOXM1 and in mouse model of allergen-mediated lung inflammation. In the present study, we examined antitumor activities of RCM-1 using tumor models. Treatment with RCM-1 inhibited tumor cell proliferation as evidenced by increased cell-cycle duration. Confocal imaging of RCM-1-treated tumor cells indicated that delay in cellular proliferation was concordant with inhibition of FOXM1 nuclear localization in these cells. RCM-1 reduced the formation and growth of tumor cell colonies in the colony formation assay. In animal models, RCM-1 treatment inhibited growth of mouse rhabdomyosarcoma Rd76-9, melanoma B16-F10, and human H2122 lung adenocarcinoma. RCM-1 decreased FOXM1 protein in the tumors, reduced tumor cell proliferation, and increased tumor cell apoptosis. RCM-1 decreased protein levels and nuclear localization of β-catenin, and inhibited protein-protein interaction between β-catenin and FOXM1 in cultured tumor cells and Altogether, our study provides important evidence of antitumor potential of the small-molecule compound RCM-1, suggesting that RCM-1 can be a promising candidate for anticancer therapy.
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http://dx.doi.org/10.1158/1535-7163.MCT-18-0709DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7341442PMC
July 2019

NOL12 Repression Induces Nucleolar Stress-Driven Cellular Senescence and Is Associated with Normative Aging.

Mol Cell Biol 2019 06 28;39(12). Epub 2019 May 28.

Instituto de Investigação e Inovação em Saúde (i3S), Universidade do Porto, Porto, Portugal

The nucleolus is a subnuclear compartment with key roles in rRNA synthesis and ribosome biogenesis, complex processes that require hundreds of proteins and factors. Alterations in nucleolar morphology and protein content have been linked to the control of cell proliferation and stress responses and, recently, further implicated in cell senescence and ageing. In this study, we report the functional role of NOL12 in the nucleolar homeostasis of human primary fibroblasts. NOL12 repression induces specific changes in nucleolar morphology, with increased nucleolar area but reduced nucleolar number, along with nucleolar accumulation and increased levels of fibrillarin and nucleolin. Moreover, NOL12 repression leads to stabilization and activation of p53 in an RPL11-dependent manner, which arrests cells at G phase and ultimately leads to senescence. Importantly, we found NOL12 repression in association with nucleolar stress-like responses in human fibroblasts from elderly donors, disclosing it as a biomarker in human chronological aging.
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http://dx.doi.org/10.1128/MCB.00099-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6549468PMC
June 2019

Impact of Preservation Solutions on the Trichogenicity of Hair Micrografts Ascertained by Dermal Papilla Gene Expression.

Dermatol Surg 2019 12;45(12):1649-1659

Aging and Aneuploidy Group, IBMC, Instituto de Biologia Molecular e Celular, Porto, Portugal.

Background: Appropriate storage of human hair follicle (HF) grafts during follicular unit excision (FUE) is crucial toward successful hair shaft implantation. Several commercial storage solutions are currently used to ensure ex vivo maintenance of follicular grafts viability and trichogenicity. However, quantitative experimental evidence demonstrating molecular changes in HF cells associated with the usage of different storage solutions is largely missing.

Objective: To identify gene expression changes in HF cells caused by ex vivo storage of hair grafts in different preservation conditions.

Methods: The authors performed gene expression analysis in dermal papilla (DP) isolated from HF stored under different temperatures and solutions. The expression signature of key genes controlling hair growth and cycling, apoptosis, inflammation, and senescence was assessed for (1) chilled versus room temperature (RT) and (2) DP cell medium, saline, Hypothermosol, platelet-rich plasma, and ATPv-supplemented saline.

Results: The authors found chilled versus RT to prevent inflammatory cytokine signaling. Under chilled conditions, ATPv-supplemented saline was the best condition to preserve the expression of the trichogenic genes HEY1 and LEF1.

Conclusion: Data disclose DP gene expression analysis as a useful methodology to ascertain the efficacy of preserving solutions and elucidate about the best currently available option for FUE clinical practice.
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http://dx.doi.org/10.1097/DSS.0000000000001895DOI Listing
December 2019

Approaches towards Longevity: Reprogramming, Senolysis, and Improved Mitotic Competence as Anti-Aging Therapies.

Int J Mol Sci 2019 Feb 21;20(4). Epub 2019 Feb 21.

Ageing and Aneuploidy Laboratory, IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135 Porto, Portugal.

Mainstream approaches that are currently used as anti-aging therapies primarily explore the senescence and epigenetic drift aging hallmarks and they are at two ends of the spectrum. While senolytic therapies include either the selective elimination of senescent cells or the disruption of their secretome with the use of drugs or natural compounds, cellular reprogramming uses genetic manipulation to revert cells all the way back to pluripotency. Here, we describe the progress that has been made on these therapies, while highlighting the major challenges involved. Moreover, based on recent findings elucidating the impact of mitotic shutdown and aneuploidy in cellular senescence, we discuss the modulation of mitotic competence as an alternative strategy to delay the hallmarks of aging. We propose that a regulated rise in mitotic competence of cells could circumvent certain limitations that are present in the senolytic and reprogramming approaches, by acting to decelerate senescence and possibly restore the epigenetic landscape.
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http://dx.doi.org/10.3390/ijms20040938DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6413205PMC
February 2019

The antitumor efficacy of monomeric disintegrin obtustatin in S-180 sarcoma mouse model.

Invest New Drugs 2019 10 25;37(5):1044-1051. Epub 2019 Jan 25.

Aging and Aneuploidy Laboratory, Instituto de Biologia Molecular e Celular, Instituto de Investigação e Inovação em Saúde - i3S, Universidade do Porto, Rua Alfredo Allen 208, 4200-135, Porto, Portugal.

Obtustatin, isolated from the Levantine Viper snake venom (Macrovipera lebetina obtusa -MLO), is the shortest known monomeric disintegrin shown to specifically inhibit the binding of the α1β1 integrin to collagen IV. Its oncostatic effect is due to the inhibition of angiogenesis, likely through α1β1 integrin inhibition in endothelial cells. To explore the therapeutic potential of obtustatin, we studied its effect in S-180 sarcoma-bearing mice model in vivo as well as in human dermal microvascular endothelial cells (HMVEC-D) in vitro, and tested anti-angiogenic activity in vivo using the chick embryo chorioallantoic membrane assay (CAM assay). Our in vivo results show that obtustatin inhibits tumour growth by 33%. The expression of vascular endothelial growth factor (VEGF) increased after treatment with obtustatin, but the level of expression of caspase 8 did not change. In addition, our results demonstrate that obtustatin inhibits FGF2-induced angiogenesis in the CAM assay. Our in vitro results show that obtustatin does not exhibit cytotoxic activity in HMVEC-D cells in comparison to in vivo results. Thus, our findings disclose that obtustatin might be a potential candidate for the treatment of sarcoma in vivo with low toxicity.
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http://dx.doi.org/10.1007/s10637-019-00734-2DOI Listing
October 2019

FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescence.

Nat Commun 2018 07 19;9(1):2834. Epub 2018 Jul 19.

Aging and Aneuploidy Laboratory, IBMC, Instituto de Biologia Molecular e Celular, Universidade do Porto, 4200-135, Porto, Portugal.

Aneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison-Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors' cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes.
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http://dx.doi.org/10.1038/s41467-018-05258-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053425PMC
July 2018

Mitotic Dysfunction Associated with Aging Hallmarks.

Adv Exp Med Biol 2017 ;1002:153-188

Aging and Aneuploidy Laboratory, Instituto de Biologia Molecular e Celular, Instituto de Investigação e Inovação em Saúde - i3S, Universidade do Porto, Rua Alfredo Allen 208, 4200-135, Porto, Portugal.

Aging is a biological process characterized by the progressive deterioration of physiological functions known to be the main risk factor for chronic diseases and declining health. There has been an emerging connection between aging and aneuploidy, an aberrant number of chromosomes, even though the molecular mechanisms behind age-associated aneuploidy remain largely unknown. In recent years, several genetic pathways and biochemical processes controlling the rate of aging have been identified and proposed as aging hallmarks. Primary hallmarks that cause the accumulation of cellular damage include genomic instability, telomere attrition, epigenetic alterations and loss of proteostasis (López-Otín et al., Cell 153:1194-1217, 2013). Here we review the provocative link between these aging hallmarks and the loss of chromosome segregation fidelity during cell division, which could support the correlation between aging and aneuploidy seen over the past decades. Secondly, we review the systemic impacts of aneuploidy in cell physiology and emphasize how these include some of the primary hallmarks of aging. Based on the evidence, we propose a mutual causality between aging and aneuploidy, and suggest modulation of mitotic fidelity as a potential means to ameliorate healthy lifespan.
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http://dx.doi.org/10.1007/978-3-319-57127-0_7DOI Listing
September 2017

Selective advantage of trisomic human cells cultured in non-standard conditions.

Sci Rep 2016 Mar 9;6:22828. Epub 2016 Mar 9.

Department of Biological Sciences, Blacksburg, VA 24061 - USA.

An abnormal chromosome number, a condition known as aneuploidy, is a ubiquitous feature of cancer cells. A number of studies have shown that aneuploidy impairs cellular fitness. However, there is also evidence that aneuploidy can arise in response to specific challenges and can confer a selective advantage under certain environmental stresses. Cancer cells are likely exposed to a number of challenging conditions arising within the tumor microenvironment. To investigate whether aneuploidy may confer a selective advantage to cancer cells, we employed a controlled experimental system. We used the diploid, colorectal cancer cell line DLD1 and two DLD1-derived cell lines carrying single-chromosome aneuploidies to assess a number of cancer cell properties. Such properties, which included rates of proliferation and apoptosis, anchorage-independent growth, and invasiveness, were assessed both under standard culture conditions and under conditions of stress (i.e., serum starvation, drug treatment, hypoxia). Similar experiments were performed in diploid vs. aneuploid non-transformed human primary cells. Overall, our data show that aneuploidy can confer selective advantage to human cells cultured under non-standard conditions. These findings indicate that aneuploidy can increase the adaptability of cells, even those, such as cancer cells, that are already characterized by increased proliferative capacity and aggressive tumorigenic phenotypes.
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http://dx.doi.org/10.1038/srep22828DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4783771PMC
March 2016

Chromosome mis-segregation and cytokinesis failure in trisomic human cells.

Elife 2015 May 5;4. Epub 2015 May 5.

Department of Biological Sciences, Virginia Tech, Blacksburg, United States.

Cancer cells display aneuploid karyotypes and typically mis-segregate chromosomes at high rates, a phenotype referred to as chromosomal instability (CIN). To test the effects of aneuploidy on chromosome segregation and other mitotic phenotypes we used the colorectal cancer cell line DLD1 (2n = 46) and two variants with trisomy 7 or 13 (DLD1+7 and DLD1+13), as well as euploid and trisomy 13 amniocytes (AF and AF+13). We found that trisomic cells displayed higher rates of chromosome mis-segregation compared to their euploid counterparts. Furthermore, cells with trisomy 13 displayed a distinctive cytokinesis failure phenotype. We showed that up-regulation of SPG20 expression, brought about by trisomy 13 in DLD1+13 and AF+13 cells, is sufficient for the cytokinesis failure phenotype. Overall, our study shows that aneuploidy can induce chromosome mis-segregation. Moreover, we identified a trisomy 13-specific mitotic phenotype that is driven by up-regulation of a gene encoded on the aneuploid chromosome.
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http://dx.doi.org/10.7554/eLife.05068DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4443816PMC
May 2015

Dominant negative effect of polyglutamine expansion perturbs normal function of ataxin-3 in neuronal cells.

Hum Mol Genet 2015 Jan 20;24(1):100-17. Epub 2014 Aug 20.

Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710-057 Braga ICVS/3B's-PT Government Associate Laboratory, Braga/Guimarães and

The physiological function of Ataxin-3 (ATXN3), a deubiquitylase (DUB) involved in Machado-Joseph Disease (MJD), remains elusive. In this study, we demonstrate that ATXN3 is required for neuronal differentiation and for normal cell morphology, cytoskeletal organization, proliferation and survival of SH-SY5Y and PC12 cells. This cellular phenotype is associated with increased proteasomal degradation of α5 integrin subunit (ITGA5) and reduced activation of integrin signalling and is rescued by ITGA5 overexpression. Interestingly, silencing of ATXN3, overexpression of mutant versions of ATXN3 lacking catalytic activity or bearing an expanded polyglutamine (polyQ) tract led to partially overlapping phenotypes. In vivo analysis showed that both Atxn3 knockout and MJD transgenic mice had decreased levels of ITGA5 in the brain. Furthermore, abnormal morphology and reduced branching were observed both in cultured neurons expressing shRNA for ATXN3 and in those obtained from MJD mice. Our results show that ATXN3 rescues ITGA5 from proteasomal degradation in neurons and that polyQ expansion causes a partial loss of this cellular function, resulting in reduced integrin signalling and neuronal cytoskeleton modifications, which may be contributing to neurodegeneration.
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http://dx.doi.org/10.1093/hmg/ddu422DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262494PMC
January 2015

Mitotic spindle multipolarity without centrosome amplification.

Nat Cell Biol 2014 May;16(5):386-94

Mitotic spindle bipolarity is essential for faithful segregation of chromosomes during cell division. Multipolar spindles are often seen in human cancers and are usually associated with supernumerary centrosomes that result from centrosome overduplication or cytokinesis failure. A less-understood path to multipolar spindle formation may arise due to loss of spindle pole integrity in response to spindle and/or chromosomal forces. Here we discuss the different routes leading to multipolar spindle formation, focusing on spindle multipolarity without centrosome amplification. We also present the distinct and common features between these pathways and discuss their therapeutic implications.
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http://dx.doi.org/10.1038/ncb2958DOI Listing
May 2014

Spindle assembly checkpoint robustness requires Tpr-mediated regulation of Mad1/Mad2 proteostasis.

J Cell Biol 2013 Dec;203(6):883-93

Tpr is a conserved nuclear pore complex (NPC) protein implicated in the spindle assembly checkpoint (SAC) by an unknown mechanism. Here, we show that Tpr is required for normal SAC response by stabilizing Mad1 and Mad2 before mitosis. Tpr coimmunoprecipitated with Mad1 and Mad2 (hereafter designated as Tpr/Mad1/Mad2 or TM2 complex) during interphase and mitosis, and is required for Mad1–c-Mad2 recruitment to NPCs. Interestingly, Tpr was normally undetectable at kinetochores and dispensable for Mad1, but not for Mad2, kinetochore localization, which suggests that SAC robustness depends on Mad2 levels at kinetochores. Protein half-life measurements demonstrate that Tpr stabilizes Mad1 and Mad2, ensuring normal Mad1–c-Mad2 production in an mRNA- and kinetochore-independent manner. Overexpression of GFP-Mad2 restored normal SAC response and Mad2 kinetochore levels in Tpr-depleted cells. Mechanistically, we provide evidence that Tpr might spatially regulate SAC proteostasis through the SUMO-isopeptidases SENP1 and SENP2 at NPCs. Thus, Tpr is a kinetochore-independent, rate-limiting factor required to mount and sustain a robust SAC response.
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http://dx.doi.org/10.1083/jcb.201309076DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3871433PMC
December 2013

CLASPs prevent irreversible multipolarity by ensuring spindle-pole resistance to traction forces during chromosome alignment.

Nat Cell Biol 2012 Feb 5;14(3):295-303. Epub 2012 Feb 5.

Instituto de Biologia Molecular e Celular, Universidade do Porto, Rua do Campo Alegre 823, 4150-180 Porto, Portugal.

Loss of spindle-pole integrity during mitosis leads to multipolarity independent of centrosome amplification. Multipolar-spindle conformation favours incorrect kinetochore-microtubule attachments, compromising faithful chromosome segregation and daughter-cell viability. Spindle-pole organization influences and is influenced by kinetochore activity, but the molecular nature behind this critical force balance is unknown. CLASPs are microtubule-, kinetochore- and centrosome-associated proteins whose functional perturbation leads to three main spindle abnormalities: monopolarity, short spindles and multipolarity. The first two reflect a role at the kinetochore-microtubule interface through interaction with specific kinetochore partners, but how CLASPs prevent spindle multipolarity remains unclear. Here we found that human CLASPs ensure spindle-pole integrity after bipolarization in response to CENP-E- and Kid-mediated forces from misaligned chromosomes. This function is independent of end-on kinetochore-microtubule attachments and involves the recruitment of ninein to residual pericentriolar satellites. Distinctively, multipolarity arising through this mechanism often persists through anaphase. We propose that CLASPs and ninein confer spindle-pole resistance to traction forces exerted during chromosome congression, thereby preventing irreversible spindle multipolarity and aneuploidy.
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http://dx.doi.org/10.1038/ncb2423DOI Listing
February 2012

Absence of ataxin-3 leads to enhanced stress response in C. elegans.

PLoS One 2011 Apr 19;6(4):e18512. Epub 2011 Apr 19.

Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

Ataxin-3, the protein involved in Machado-Joseph disease, is able to bind ubiquitylated substrates and act as a deubiquitylating enzyme in vitro, and it has been involved in the modulation of protein degradation by the ubiquitin-proteasome pathway. C. elegans and mouse ataxin-3 knockout models are viable and without any obvious phenotype in a basal condition however their phenotype in stress situations has never been described.Considering the role of ataxin-3 in the protein degradation pathway, we analyzed the effects of heat shock, a known protein homeostasis stressor, in C. elegans ataxin-3 (ATX-3) knockout animals. We found that ATX-3 mutants have an exacerbated stress response and survive significantly better than wild type animals when subjected to a noxious heat shock stimulus. This increased thermotolerance of mutants was further enhanced by pre-exposure to a mild heat shock. At a molecular level, ATX-3 mutants have a distinct transcriptomic and proteomic profile with several molecular chaperones abnormally up-regulated during heat shock and recovery, consistent with the observed resistance phenotype.The improved thermotolerance in ATX-3 mutants is independent of heat shock factor 1, the maestro of the heat shock response, but fully dependent on DAF-16, a critical stress responsive transcription factor involved in longevity and stress resistance. We also show that the increased thermotolerance of ATX-3 mutants is mainly due to HSP-16.2, C12C8.1 and F44E5.5 given that the knockdown of these heat shock proteins using RNA interference causes the phenotype to revert. This report suggests that the absence of ATX-3 activates the DAF-16 pathway leading to an overexpression of molecular chaperones, which yields knockout animals with an improved capacity for dealing with deleterious stimuli.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0018512PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3079722PMC
April 2011

Ataxin-3 plays a role in mouse myogenic differentiation through regulation of integrin subunit levels.

PLoS One 2010 Jul 23;5(7):e11728. Epub 2010 Jul 23.

Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

Background: During myogenesis several transcription factors and regulators of protein synthesis and assembly are rapidly degraded by the ubiquitin-proteasome system (UPS). Given the potential role of the deubiquitinating enzyme (DUB) ataxin-3 in the UPS, and the high expression of the murine ataxin-3 homolog in muscle during embryogenesis, we sought to define its role in muscle differentiation.

Methodology/principal Findings: Using immunofluorescence analysis, we found murine ataxin-3 (mATX3) to be highly expressed in the differentiated myotome of E9.5 mouse embryos. C2C12 myoblasts depleted of mATX3 by RNA interference exhibited a round morphology, cell misalignment, and a delay in differentiation following myogenesis induction. Interestingly, these cells showed a down-regulation of alpha5 and alpha7 integrin subunit levels both by immunoblotting and immunofluorescence. Mouse ATX3 was found to interact with alpha5 integrin subunit and to stabilize this protein by repressing its degradation through the UPS. Proteomic analysis of mATX3-depleted C2C12 cells revealed alteration of the levels of several proteins related to integrin signaling.

Conclusions: Ataxin-3 is important for myogenesis through regulation of integrin subunit levels.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0011728PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2909204PMC
July 2010

Absence of ataxin-3 leads to cytoskeletal disorganization and increased cell death.

Biochim Biophys Acta 2010 Oct 15;1803(10):1154-63. Epub 2010 Jul 15.

Life and Health Sciences Research Institute, University of Minho, Braga, Portugal.

Ataxin-3 (ATXN3) is a widely expressed protein that binds to ubiquitylated proteins, has deubiquitylating activity in vitro and is thought to modulate substrate degradation through the ubiquitin-proteasome pathway. Expansion of a polyglutamine tract in ATXN3 causes Machado-Joseph disease, a late-onset neurodegenerative disorder characterized by ubiquitin-positive aggregate formation and specific neuronal death. Although ATXN3 has been involved in transcriptional repression and in the ubiquitin-proteasome pathway, its biological function is still unknown. In this work, we show that depletion of ATXN3 using small-interference RNA (siRNA) causes a prominent phenotype in both human and mouse cell lines. A mild increase in ubiquitylation occurs and cells exhibit ubiquitin-positive foci, which is consistent with ATXN3 putative function as a deubiquitylating enzyme. In addition, siATXN3-silenced cells exhibit marked morphological changes such as rounder shape and loss of adhesion protrusions. At a structural level, the microtubule, microfilament and intermediate filament networks are severely compromised and disorganized. This cytoskeletal phenotype is reversible and dependent on ATXN3 levels. Cell-extracellular matrix connection is also affected in ATXN3-depleted cells as talin expression is reduced in the focal adhesions and lower levels of alpha-1 integrin subunit are expressed at their surface. Although the cytoskeletal and adhesion problems do not originate any major change in the cell cycle of siATXN3-depleted cells, cell death is increased in siATXN3 cultures compared to controls. In summary, in this work we show that the absence of ATXN3 leads to an overt cytoskeletal/adhesion defect raising the possibility that this protein may play a role in the cytoskeleton.
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http://dx.doi.org/10.1016/j.bbamcr.2010.07.004DOI Listing
October 2010

IFN-gamma-dependent activation of macrophages during experimental infections by Mycobacterium ulcerans is impaired by the toxin mycolactone.

J Immunol 2010 Jan 11;184(2):947-55. Epub 2009 Dec 11.

Life and Health Sciences Research Institute, School of Health Sciences, University of Minho, Braga, Portugal.

Buruli ulcer, caused by Mycobacterium ulcerans infections, is a necrotizing skin disease whose pathogenesis is associated with the exotoxin mycolactone. Despite the relevance of this emergent disease, little is known on the immune response against the pathogen. Following the recent demonstration of an intramacrophage growth phase for M. ulcerans, we investigated the biological relevance of IFN-gamma and the antimycobacterial mechanisms activated by this cytokine in M. ulcerans-infected macrophages. Three M. ulcerans strains were tested: 5114 (mutant mycolactone-negative, avirulent strain); 94-1327 (intermediate virulence); and 98-912 (high virulence). We show in this study that IFN-gamma is expressed in mouse-infected tissues and that IFN-gamma-deficient mice display increased susceptibility to infection with strains 5114 and, to a lesser extent, 94-1327, but not with the highly virulent strain. Accordingly, IFN-gamma-activated cultured macrophages controlled the proliferation of the avirulent and the intermediate virulent strains. Addition of mycolactone purified from strain 98-912 to cultures of IFN-gamma-activated macrophages infected with the mycolactone-negative strain led to a dose-dependent inhibition of the IFN-gamma-induced protective mechanisms, involving phagosome maturation/acidification and increased NO production, therefore resulting in increased bacterial burdens. Our findings suggest that the protection mediated by IFN-gamma in M. ulcerans-infected macrophages is impaired by the local buildup of mycolactone.
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http://dx.doi.org/10.4049/jimmunol.0902717DOI Listing
January 2010

ATX-3, CDC-48 and UBXN-5: a new trimolecular complex in Caenorhabditis elegans.

Biochem Biophys Res Commun 2009 Sep 21;386(4):575-81. Epub 2009 Jun 21.

Life and Health Sciences Research Institute (ICVS), School of Health Sciences, Campus de Gualtar, University of Minho, Braga, Portugal.

Ataxin-3 is the protein involved in Machado-Joseph disease, a neurodegenerative disorder caused by a polyglutamine expansion. Ataxin-3 binds ubiquitylated proteins and acts as a deubiquitylating enzyme in vitro. It was previously proposed that ataxin-3, along with the VCP/p97 protein, escorts ubiquitylated substrates for proteasomal degradation, although other players of this escort complex were not identified yet. In this work, we show that the Caenorhabditis elegans ataxin-3 protein (ATX-3) interacts with both VCP/p97 worm homologs, CDC-48.1 and CDC-48.2 and we map the interaction domains. We describe a motility defect in both ATX-3 and CDC-48.1 mutants and, in addition, we identify a new protein interactor, UBXN-5, potentially an adaptor of the CDC-48-ATX-3 escort complex. CDC-48 binds to both ATX-3 and UBXN-5 in a non-competitive manner, suggesting the formation of a trimolecular complex. Both CDC-48 and ATX-3, but not UBXN-5, were able to bind K-48 polyubiquitin chains, the standard signal for proteasomal degradation. Additionally, we describe several common interactors of ATX-3 and UBXN-5, some of which can be in vivo targets of this complex.
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http://dx.doi.org/10.1016/j.bbrc.2009.06.092DOI Listing
September 2009

Kinetochore-microtubule interactions "in check" by Bub1, Bub3 and BubR1: The dual task of attaching and signalling.

Cell Cycle 2008 Jun 20;7(12):1763-8. Epub 2008 Jun 20.

Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Braga, Portugal.

The spindle assembly checkpoint (SAC) prevents anaphase onset until all chromosomes accomplish proper bipolar attachments to the mitotic spindle and come under tension, thereby ensuring the fidelity of chromosome segregation. Despite significant advances in our understanding of SAC signalling, a clear link between checkpoint signalling and the molecular mechanisms underlying chromosome attachment to microtubules has not been established so far. However, independent studies from many groups have interestingly found that the bone-a-fide Bub1, BubR1 and Bub3 SAC proteins are themselves required for proper kinetochoremicrotubule (K-MT) interactions. Here, we review these findings and discuss the specific contribution of each of these proteins in the regulation of K-MT attachment, taking into consideration their interdependencies for kinetochore localization as well as their relationship with other proteins with a known role in chromosome attachment and congression.
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http://dx.doi.org/10.4161/cc.7.12.6180DOI Listing
June 2008

The human spindle assembly checkpoint protein Bub3 is required for the establishment of efficient kinetochore-microtubule attachments.

Mol Biol Cell 2008 Apr 16;19(4):1798-813. Epub 2008 Jan 16.

Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal.

The spindle assembly checkpoint monitors the status of kinetochore-microtubule (K-MT) attachments and delays anaphase onset until full metaphase alignment is achieved. Recently, the role of spindle assembly checkpoint proteins was expanded with the discovery that BubR1 and Bub1 are implicated in the regulation of K-MT attachments. One unsolved question is whether Bub3, known to form cell cycle constitutive complexes with both BubR1 and Bub1, is also required for proper chromosome-to-spindle attachments. Using RNA interference and high-resolution microscopy, we analyzed K-MT interactions in Bub3-depleted cells and compared them to those in Bub1- or BubR1-depleted cells. We found that Bub3 is essential for the establishment of correct K-MT attachments. In contrast to BubR1 depletion, which severely compromises chromosome attachment and alignment, we found Bub3 and Bub1 depletions to produce defective K-MT attachments that, however, still account for significant chromosome congression. After Aurora B inhibition, alignment defects become severer in Bub3- and Bub1-depleted cells, while partially rescued in BubR1-depleted cells, suggesting that Bub3 and Bub1 depletions perturb K-MT attachments distinctly from BubR1. Interestingly, misaligned chromosomes in Bub3- and Bub1-depleted cells were found to be predominantly bound in a side-on configuration. We propose that Bub3 promotes the formation of stable end-on bipolar attachments.
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http://dx.doi.org/10.1091/mbc.e07-07-0633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2291436PMC
April 2008

NEDD8: a new ataxin-3 interactor.

Biochim Biophys Acta 2007 Nov 24;1773(11):1619-27. Epub 2007 Aug 24.

UnIGENe-IBMC, Instituto de Biologia Molecular e Celular, University of Porto, 4150-180 Porto, Portugal.

Machado-Joseph disease (MJD/SCA3) is an autosomal dominant neurodegenerative disease caused by the expansion of a CAG tract in the coding portion of the ATXN3 gene. The presence of ubiquitin-positive aggregates of the defective protein in affected neurons is characteristic of this and most of the polyglutamine disorders. Recently, the accumulation of the neural precursor cell expressed developmentally downregulated 8 (NEDD8), a ubiquitin-like protein, in the inclusions of MJD brains was reported. Here, we report a new molecular interaction between wild-type ataxin-3 and NEDD8, using in vitro and in situ approaches. Furthermore, we show that this interaction is not dependent on the ubiquitin-interacting motifs in ataxin-3, since the presence of the Josephin domain is sufficient for the interaction to occur. The conservation of the interaction between the Caenorhabditis elegans ataxin-3 homologue (atx-3) and NEDD8 suggests its biological and functional relevance. Molecular docking studies of the NEDD8 molecule to the Josephin domain of ataxin-3 suggest that NEDD8 interacts with ataxin-3 in a substrate-like mode. In agreement, ataxin-3 displays deneddylase activity against a fluorogenic NEDD8 substrate.
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http://dx.doi.org/10.1016/j.bbamcr.2007.07.012DOI Listing
November 2007
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