Quando ganhar é perder : estudo da função da ataxina-3 e da sua perturbação no contexto da doença de Machado-Joseph

Tese de doutoramento em Ciências da Saúde.Ataxin-3 (ATXN3) is the protein involved in Machado-Joseph Disease (MJD), one of the nine neurodegenerative disorders known to be caused by a polyglutamine (polyQ) expansion. This polyQ tract causes the appearance of misfolded protein species, protein aggregates, neuronal dysfunction and cell death. ATXN3 is known to interact with polyubiquitin chains and to have deubiquitylating (DUB) activity in vitro, but its substrates and its cellular and physiological role(s) remain unknown, specially in neurons. Since the leading hypothesis concerning the pathogenesis of MJD is that the expanded polyQ tract confers a toxic gain of function of ATXN3, not much attention has been dedicated to its normal function. However, it is believed that a partial loss of the normal function of ATXN3 may also contribute to and modulate disease progression. This study was based on the idea that understanding the normal physiological role of ATXN3 will be of relevance for our understanding of the pathogenesis of MJD. In this work, we explored the function of ATXN3 in neuronal cells and its perturbation in the context of MJD. We found that ATXN3 is required for neuronal differentiation and for normal cellular morphology, cytoskeleton organization, proliferation and survival. This phenotype is associated with increased proteasomal degradation of alpha5-integrin subunit (ITGA5) and reduced activation of integrin signaling. Interestingly, we show that silencing of ATXN3, overexpression of a catalytically inert version of the protein or a mutant protein bearing an expanded polyQ tract led to partially overlapping phenotypes, suggesting that a loss of the neuronal function of ATXN3 may be contributing to neurodegeneration. Consistent with a wider role of ATXN3 in the regulation of the cytoskeleton network, we found that loss of function of ATXN3 also leads to a deregulation of tau expression, namely a deregulation of tau exon 10 splicing. This event has a negative impact in neuronal morphology and differentiation. Additionally, we found that ATXN3 interacts with SFRS7, a regulator of tau splicing, and regulates its ubiquitylation levels. As similar alterations were found in the brain of a mouse model of MJD, it is likely that this mechanism is contributing to pathogenesis of this disorder. Hence, this work establishes for the first time a functional link between two key proteins involved in different neurodegenerative diseases. Lastly, we characterized the ubiquitome of neuronal cells lacking ATXN3 in an attempt to identify potential substrates of its DUB activity. We found that a large proportion of these proteins were involved in RNA posttranscriptional modification. Considering this, we analyzed by transcriptomic analysis and using reporter minigenes the global splicing pattern in neuronal cells upon silencing of ATXN3 and found that splicing was globally altered in these cells. These findings lead us to propose for the first time that ATXN3 plays a role in splicing regulation in neurons, a novel function for this protein. In summary, this work adds new knowledge about the relevance for neurons of one specific DUB, ataxin- 3, and provides new clues about its biological functions and the pathways in which it is involved. It reinforces ATXN3’s involvement with the UPP and it also raises new hypotheses for its role in cytoskeleton organization and in splicing regulation. Additionally, it provides evidence for perturbation of the normal function of ATXN3 in the context of disease, through a dominant negative effect, which may have relevance for the development of future therapeutical strategies.A Ataxina-3 (ATXN3) é a proteína envolvida na Doença de Machado-Joseph (DMJ), uma das nove doenças neurodegenerativas que se sabe serem causadas por uma expansão de poliglutaminas (poliQ). Este trato de poliQ causa o aparecimento de espécies proteicas com uma conformação anormal, agregados proteicos, disfunção neuronal e morte celular. A ATXN3 interage com cadeias de poliubiquitina e tem atividade de ubiquitina hidrolase (DUB) in vitro, mas os seus substratos e a(s) sua(s) função(ões) fisiológica(s) permanecem desconhecidos, especialmente em neurónios. Dado que a hipótese actualmente mais aceite relativa ao mecanismo patogénico da DMJ considera que o trato de poliQ expandido confere um ganho tóxico de função à ATXN3, não tem sido dedicada muita atenção à sua função normal. Contudo, acredita-se que a perda parcial da função normal da ATXN3 também pode contribuir e modular a progressão da doença. Este estudo baseou-se na ideia de que conhecer a função fisiológica normal da ATXN3 será relevante para conseguirmos compreender a patogénese da doença. Neste estudo, explorámos a função da ATXN3 em neurónios e a sua perturbação pela expansão poliQ em DMJ. Descobrimos que a ATXN3 é necessária para a diferenciação neuronal e para a normal morfologia celular, organização do citosqueleto, proliferação e sobrevivência. Este fenótipo está associado a um aumento da degradação da subunidade 5-alpha da integrina (ITGA5) pelo proteossoma e uma ativação diminuída da sinalização pela via das integrinas. Curiosamente, demonstrámos que o silenciamento da ATXN3, a sobreexpressão de uma versão cataliticamente inerte da proteína ou de uma proteína mutante contendo um trato de poliQ expandido conduzem a fenótipos parcialmente sobreponíveis, sugerindo que a perda da função neuronal da ATXN3 pode contribuir para a neurodegeneração. De forma consistente com um papel mais abrangente da ATXN3 na regulação da organização do citosqueleto, descobrimos que a perda de função da ATXN3 também origina uma desregulação da expressão da tau, nomeadamente uma desregulação do splicing do exão 10 da tau em células neuronais. Este evento tem um impacto negativo na sua morfologia e diferenciação. Além disso, descobrimos que a ATXN3 interage com o SFRS7, um factor regulador do splicing da tau, e regula os seus níveis de ubiquitilação. Considerando que alterações semelhantes foram encontradas no cérebro do modelo de DMJ em ratinho, este mecanismo parece contribuir para a patogénese. Este trabalho estabelece assim, pela primeira vez, uma ligação funcional entre duas proteínas chave envolvidas em diferentes doenças neurodegenerativas. Por fim, caracterizámos o ubiquitoma de células neuronais silenciadas para a ATXN3, com o objectivo de identificar potenciais candidatos para a sua atividade DUB. Concluímos que uma grande proporção destas proteínas estão envolvidas na modificação pós-transcripcional do RNA. Tendo este dado em consideração, analisámos o padrão global do splicing por análises de transcriptómica e usando minigenes repórteres, e descobrimos que o splicing estava globalmente afetado nestas células. Estes achados levaram-nos a propor pela primeira vez que a ATXN3 desempenha um papel na regulação do splicing em neurónios, uma nova função para esta proteína. Em sumário, este trabalho amplia o conhecimento acerca da relevância para os neurónios de uma DUB específica, a ataxina-3, e fornece indicativos sobre as suas funções biológicas e as vias celulares onde está envolvida. Além disso, reforça o envolvimento da ATXN3 com a UPP, levantando também novas hipóteses para o seu papel na regulação do citosqueleto e na regulação do splicing. Adicionalmente, são apresentadas evidências para a perturbação da função normal da ATXN3 no contexto da doença através de um efeito dominante negativo, facto que poderá ser relevante para o desenvolvimento de futuras estratégias terapêuticas.This work was supported by Fundação para a Ciência e Tecnologia (FCT) and COMPETE through a Bolsa de Doutoramento (SFRH/BD/51059/2010) and the project (PTDC/SAUGMG/101572/2008) and through National Institutes of Health (NHI) [R01NS038712]

Selective impact of Tau loss on nociceptive primary afferents and pain sensation

Tau protein hyperphosphorylation and consequent malfunction are hallmarks of Alzheimer's disease pathology; importantly, pain perception is diminished in these patients. In physiological conditions, Tau contributes to cytoskeletal dynamics and in this way, influences a number of cellular mechanisms including axonal trafficking, myelination and synaptic plasticity, processes that are also implicated in pain perception. However, there is no in vivo evidence clarifying the role of Tau in nociception. Thus, we tested Tau-null (Tau-/-) and Tau+/+ mice for acute thermal pain (Hargreaves' test), acute and tonic inflammatory pain (formalin test) and mechanical allodynia (Von Frey test). We report that Tau-/- animals presented a decreased response to acute noxious stimuli when compared to Tau+/+ while their pain-related behavior is augmented under tonic painful stimuli. This increased reactivity to tonic pain was accompanied by enhanced formalin-evoked c-fos staining of second order nociceptive neurons at Tau-null dorsal horn. In addition, we analyzed the primary afferents conveying nociceptive signals, estimating sciatic nerve fiber density, myelination and nerve conduction. Ultrastructural analysis revealed a decreased C-fiber density in the sciatic nerve of Tau-null mice and a hypomyelination of myelinated fibers (Ad-fibers) - also confirmed by western blot analysis - followed by altered conduction properties of Tau-null sciatic nerves. To our knowledge, this is the first in vivo study that demonstrates that Tau depletion negatively affects the main systems conveying nociceptive information to the CNS, adding to our knowledge about Tau function(s) that might also be relevant for understanding peripheral neurological deficits in different Tauopathies.We would like to thank Drs Joao Relvas, Joana Paes de Faria Monteiro and Nuno Dias for their comments in this work. Many thanks to Dr Joao Relvas for the MBP antibody. The work was supported by grants "SFRH/BPD/80118/2011", "PTDC/SAU-NMC/113934/2009" funded by FCT - Portuguese Foundation for Science and Technology and project DoIT - Desenvolvimento e Operacionalizacao da Investigacao de Translacao (No. do projeto 13853), funded by Fund Europeu de Desenvolvimento Regional (FEDER) through the Programa Operacional Fatores de Competitividade (POFC). Author's contributions: experimental design - IS, HA, VP, AA, and NS; performed research - IS, HA, VP, AL, SL, SS, SP, AC, FPR, and RF; data analyses - IS, HA, AL, VP, SC, and FPR; and manuscript preparation - IS, HA, VP, and NS

Increased thermotolerance in the absence of ataxin-3 in C. elegans

Dissertação de mestrado em Genética MolecularAtaxin-3 is the protein involved in Machado-Joseph disease, one of the nine neurodegenerative disorders caused by a polyglutamine (polyQ) expansion. This polyQ expansion causes the appearance of misfolded protein species, aggregates, neuronal dysfunction and cell death. Ataxin-3 is a deubiquitylating (DUB) enzyme in vitro, is able to bind ubiquitin and ubiquitylated substrates and has been involved in the protein degradation by the ubiquitin-proteasome pathway (UPP). Accumulation of damaged or unneeded proteins due to the absence of a processing enzyme such as ATX-3 can disrupt cellular homeostasis. However, besides its putative role in the UPP, little is known about ataxin-3 cellular function. The possibility to carry out functional studies in a multicellular yet simple organism, and the availability of a C. elegans atx-3 knockout strain in our lab, led us to proceed our studies in this model. C. elegans lacking ATX-3 are viable and with no overt phenotype in basal conditions. However, considering the role of ataxin-3 in protein quality control we decided to analyze the effects of ataxin-3 absence in protein homeostasis stress conditions. In this work, we studied an ATX-3 knockout mutant after a heat stress insult that compromises the protein homeostasis of the whole organism. We showed that the mutant worms have an exacerbated stress response and survive significantly better than wild type animals upon a heat shock stimulus. The increased stress resistance is further enhanced by a previous mild heat shock. At a molecular level, ATX-3 mutants have a distinct profile with several molecular chaperones up-regulated at 25ºC (stress-threshold temperature), such as HSP-4 and HSP-16 family members – HSP-16.1 and HSP-16.49 – as analyzed by western blot and mRNA expression studies. These results suggested to us that the absence of ataxin-3 throughout C. elegans development might lead to a mild cellular stress, probably due to an imbalance of ATX-3 substrate degradation, having global consequences on protein homeostasis. To test this hypothesis, we used temperature-sensitive (ts) strains that constitute highly sensitive indicators of disruption in protein homeostasis, because ts mutant proteins are very dependent on the cellular folding environment. However, we have found no differences between wild type and mutant animals, in muscle and neuronal cells. Finally, we showed that the stress-resistance phenotype is dependent on the DAF-16 pathway, known to modulate aging. In summary, this work gathered significant insight into ataxin-3 biological function and pathways where it is involved.A ataxina-3 é a proteína envolvida na doença de Machado-Joseph, uma das nove doenças neurodegenerativas causadas por uma expansão de poliglutaminas (poliQ). Esta expansão de poliQ está associada a uma alteração de conformação das proteínas, com consequente formação de agregados, disfunção neuronal e morte celular. A ataxina-3 humana é uma ubiquitina hidrolase in vitro e liga-se a ubiquitina e a substratos ubiquitilados, sugerindo um papel na degradação proteica através da via ubiquitina-proteassoma (UPP). A ausência de uma enzima processadora como a ataxina-3, pode perturbar a homeostasia celular. Contudo, além do seu potencial papel na via UPP, pouco se sabe acerca da função celular da ataxina-3. A possibilidade de desenvolver estudos funcionais num animal multicelular simples, e o facto de existir uma estirpe knockout para a ataxina-3 (ATX-3) no nosso laboratório, permitiu-nos prosseguir os estudos neste modelo. Estes animais são viáveis e não apresentam um fenótipo visível em condições fisiológicas normais. Contudo, considerando o papel da ataxina-3 no controlo de qualidade das proteínas, decidimos analisar os efeitos da ausência da ATX-3 numa situação de stress. Neste trabalho, estudámos um mutante ATX-3 após um choque térmico, que compromete a homeostasia celular proteica de todo o organismo. Mostrámos que os nemátodes mutantes têm uma resposta exacerbada ao stress e sobrevivem significantemente melhor do que os animais selvagens. Este aumento na termoresistência é ainda maior após um prévio choque térmico não letal. A nível molecular, os mutantes ATX-3 têm um perfil proteómico distinto, com algumas chaperonas moleculares sobre-expressas a 25ºC (a temperatura limite para induzir stress), tal como a HSP-4 e membros da família HSP-16 – HSP-16.1 e HSP-16.49 – como analisado por western-blot e estudos de expressão de mRNA. Estes resultados sugeriram-nos que a ausência da ATX-3, ao longo do desenvolvimento do C. elegans, pode levar a um stress celular, que resulta provavelmente de uma alteração na degradação de substratos da ATX-3, com consequências na homeostasia proteica celular global. Para testar esta hipótese, usámos estirpes termo-sensíveis (ts), que constituem indicadores muito sensíveis da perturbação da homeostasia proteica, uma vez que as proteínas ts mutantes dependem da homeostasia celular proteica. Contudo, não encontrámos diferenças entre a estirpe selvagem e os animais mutantes, em células musculares e neuronais. Por fim, mostrámos que o fenótipo termo-resistente é dependente da via do DAF-16, associada à modulação da longevidade. Em sumário, este trabalho originou pistas acerca da função biológica da ataxina-3 e das vias onde esta proteína está envolvida

Neotropical xenarthrans: a dataset of occurrence of xenarthran species in the Neotropics.

International audienceXenarthrans—anteaters, sloths, and armadillos—have essential functions forecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosys-tem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts withdomestic dogs, these species have been threatened locally, regionally, or even across their fulldistribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths.Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae(3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data onDasypus pilo-sus(Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized,but new genetic studies have revealed that the group is represented by seven species. In thisdata paper, we compiled a total of 42,528 records of 31 species, represented by occurrence andquantitative data, totaling 24,847 unique georeferenced records. The geographic range is fromthe southern United States, Mexico, and Caribbean countries at the northern portion of theNeotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regardinganteaters,Myrmecophaga tridactylahas the most records (n=5,941), andCyclopessp. havethe fewest (n=240). The armadillo species with the most data isDasypus novemcinctus(n=11,588), and the fewest data are recorded forCalyptophractus retusus(n=33). Withregard to sloth species,Bradypus variegatushas the most records (n=962), andBradypus pyg-maeushas the fewest (n=12). Our main objective with Neotropical Xenarthrans is to makeoccurrence and quantitative data available to facilitate more ecological research, particularly ifwe integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, andNeotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure,habitat loss, fragmentation effects, species invasion, and climate change effects will be possiblewith the Neotropical Xenarthrans data set. Please cite this data paper when using its data inpublications. We also request that researchers and teachers inform us of how they are usingthese data

NEOTROPICAL XENARTHRANS: a data set of occurrence of xenarthran species in the Neotropics

Xenarthrans—anteaters, sloths, and armadillos—have essential functions for ecosystem maintenance, such as insect control and nutrient cycling, playing key roles as ecosystem engineers. Because of habitat loss and fragmentation, hunting pressure, and conflicts with domestic dogs, these species have been threatened locally, regionally, or even across their full distribution ranges. The Neotropics harbor 21 species of armadillos, 10 anteaters, and 6 sloths. Our data set includes the families Chlamyphoridae (13), Dasypodidae (7), Myrmecophagidae (3), Bradypodidae (4), and Megalonychidae (2). We have no occurrence data on Dasypus pilosus (Dasypodidae). Regarding Cyclopedidae, until recently, only one species was recognized, but new genetic studies have revealed that the group is represented by seven species. In this data paper, we compiled a total of 42,528 records of 31 species, represented by occurrence and quantitative data, totaling 24,847 unique georeferenced records. The geographic range is from the southern United States, Mexico, and Caribbean countries at the northern portion of the Neotropics, to the austral distribution in Argentina, Paraguay, Chile, and Uruguay. Regarding anteaters, Myrmecophaga tridactyla has the most records (n = 5,941), and Cyclopes sp. have the fewest (n = 240). The armadillo species with the most data is Dasypus novemcinctus (n = 11,588), and the fewest data are recorded for Calyptophractus retusus (n = 33). With regard to sloth species, Bradypus variegatus has the most records (n = 962), and Bradypus pygmaeus has the fewest (n = 12). Our main objective with Neotropical Xenarthrans is to make occurrence and quantitative data available to facilitate more ecological research, particularly if we integrate the xenarthran data with other data sets of Neotropical Series that will become available very soon (i.e., Neotropical Carnivores, Neotropical Invasive Mammals, and Neotropical Hunters and Dogs). Therefore, studies on trophic cascades, hunting pressure, habitat loss, fragmentation effects, species invasion, and climate change effects will be possible with the Neotropical Xenarthrans data set. Please cite this data paper when using its data in publications. We also request that researchers and teachers inform us of how they are using these data

Characterisation of microbial attack on archaeological bone

As part of an EU funded project to investigate the factors influencing bone preservation in the archaeological record, more than 250 bones from 41 archaeological sites in five countries spanning four climatic regions were studied for diagenetic alteration. Sites were selected to cover a range of environmental conditions and archaeological contexts. Microscopic and physical (mercury intrusion porosimetry) analyses of these bones revealed that the majority (68%) had suffered microbial attack. Furthermore, significant differences were found between animal and human bone in both the state of preservation and the type of microbial attack present. These differences in preservation might result from differences in early taphonomy of the bones. © 2003 Elsevier Science Ltd. All rights reserved

Evaluation of a quality improvement intervention to reduce anastomotic leak following right colectomy (EAGLE): pragmatic, batched stepped-wedge, cluster-randomized trial in 64 countries

Background Anastomotic leak affects 8 per cent of patients after right colectomy with a 10-fold increased risk of postoperative death. The EAGLE study aimed to develop and test whether an international, standardized quality improvement intervention could reduce anastomotic leaks. Methods The internationally intended protocol, iteratively co-developed by a multistage Delphi process, comprised an online educational module introducing risk stratification, an intraoperative checklist, and harmonized surgical techniques. Clusters (hospital teams) were randomized to one of three arms with varied sequences of intervention/data collection by a derived stepped-wedge batch design (at least 18 hospital teams per batch). Patients were blinded to the study allocation. Low- and middle-income country enrolment was encouraged. The primary outcome (assessed by intention to treat) was anastomotic leak rate, and subgroup analyses by module completion (at least 80 per cent of surgeons, high engagement; less than 50 per cent, low engagement) were preplanned. Results A total 355 hospital teams registered, with 332 from 64 countries (39.2 per cent low and middle income) included in the final analysis. The online modules were completed by half of the surgeons (2143 of 4411). The primary analysis included 3039 of the 3268 patients recruited (206 patients had no anastomosis and 23 were lost to follow-up), with anastomotic leaks arising before and after the intervention in 10.1 and 9.6 per cent respectively (adjusted OR 0.87, 95 per cent c.i. 0.59 to 1.30; P = 0.498). The proportion of surgeons completing the educational modules was an influence: the leak rate decreased from 12.2 per cent (61 of 500) before intervention to 5.1 per cent (24 of 473) after intervention in high-engagement centres (adjusted OR 0.36, 0.20 to 0.64; P < 0.001), but this was not observed in low-engagement hospitals (8.3 per cent (59 of 714) and 13.8 per cent (61 of 443) respectively; adjusted OR 2.09, 1.31 to 3.31). Conclusion Completion of globally available digital training by engaged teams can alter anastomotic leak rates. Registration number: NCT04270721 (http://www.clinicaltrials.gov)

ISARIC-COVID-19 dataset: A Prospective, Standardized, Global Dataset of Patients Hospitalized with COVID-19

The International Severe Acute Respiratory and Emerging Infection Consortium (ISARIC) COVID-19 dataset is one of the largest international databases of prospectively collected clinical data on people hospitalized with COVID-19. This dataset was compiled during the COVID-19 pandemic by a network of hospitals that collect data using the ISARIC-World Health Organization Clinical Characterization Protocol and data tools. The database includes data from more than 705,000 patients, collected in more than 60 countries and 1,500 centres worldwide. Patient data are available from acute hospital admissions with COVID-19 and outpatient follow-ups. The data include signs and symptoms, pre-existing comorbidities, vital signs, chronic and acute treatments, complications, dates of hospitalization and discharge, mortality, viral strains, vaccination status, and other data. Here, we present the dataset characteristics, explain its architecture and how to gain access, and provide tools to facilitate its use

Guidelines for the use and interpretation of assays for monitoring autophagy (4th edition)

In 2008, we published the first set of guidelines for standardizing research in autophagy. Since then, this topic has received increasing attention, and many scientists have entered the field. Our knowledge base and relevant new technologies have also been expanding. Thus, it is important to formulate on a regular basis updated guidelines for monitoring autophagy in different organisms. Despite numerous reviews, there continues to be confusion regarding acceptable methods to evaluate autophagy, especially in multicellular eukaryotes. Here, we present a set of guidelines for investigators to select and interpret methods to examine autophagy and related processes, and for reviewers to provide realistic and reasonable critiques of reports that are focused on these processes. These guidelines are not meant to be a dogmatic set of rules, because the appropriateness of any assay largely depends on the question being asked and the system being used. Moreover, no individual assay is perfect for every situation, calling for the use of multiple techniques to properly monitor autophagy in each experimental setting. Finally, several core components of the autophagy machinery have been implicated in distinct autophagic processes (canonical and noncanonical autophagy), implying that genetic approaches to block autophagy should rely on targeting two or more autophagy-related genes that ideally participate in distinct steps of the pathway. Along similar lines, because multiple proteins involved in autophagy also regulate other cellular pathways including apoptosis, not all of them can be used as a specific marker for bona fide autophagic responses. Here, we critically discuss current methods of assessing autophagy and the information they can, or cannot, provide. Our ultimate goal is to encourage intellectual and technical innovation in the field