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]

Combined therapy with m-TOR-dependent and -independent autophagy inducers causes neurotoxicity in a mouse model of Machado-Joseph disease

A major pathological hallmark in several neurodegenerative disorders, like polyglutamine disorders (polyQ), including Machado-Joseph disease (MJD), is the formation of protein aggregates. MJD is caused by a CAG repeat expansion in the ATXN3 gene, resulting in an abnormal protein, which is prone to misfolding and forms cytoplasmic and nuclear aggregates within neurons, ultimately inducing neurodegeneration. Treatment of proteinopathies with drugs that up-regulate autophagy has shown promising results in models of polyQ diseases. Temsirolimus (CCI-779) inhibits the mammalian target of rapamycin (m-TOR), while lithium chloride (LiCl) acts by inhibiting inositol monophosphatase, both being able to induce autophagy. We have previously shown that chronic treatment with LiCl (10.4 mg/kg) had limited effects in a transgenic MJD mouse model. Also, others have shown that CCI-779 had mild positive effects in a different mouse model of the disease. It has been suggested that the combination of mTOR-dependent and -independent autophagy inducers could be a more effective therapeutic approach. To further explore this avenue toward therapy, we treated CMVMJD135 transgenic mice with a conjugation of CCI-779 and LiCl, both at concentrations known to induce autophagy and not to be toxic. Surprisingly, this combined treatment proved to be deleterious to both wild-type (wt) and transgenic animals, failing to rescue their neurological symptoms and actually exerting neurotoxic effects. These results highlight the possible dangers of manipulating autophagy in the nervous system and suggest that a better understanding of the potential disruption in the autophagy pathway in MJD is required before successful long-term autophagy modulating therapies can be developed.Fundação para a Ciência e Tecnologia through the projects [FEDER/FCT, POCI/SAU-MMO/60412/2004], [PTDC/SAU-GMG/64076/2006]. This work was supported by Fundação para a Ciência e Tecnologi

The secretome of bone marrow and wharton jelly derived mesenchymal stem cells induces differentiation and neurite outgrowth in Sh-SY5Y cells

The goal of this study was to determine and compare the effects of the secretome of mesenchymal stem cells (MSCs) isolated from human bone-marrow (BMSCs) and the Wharton jelly surrounding the vein and arteries of the umbilical cord (human umbilical cord perivascular cells (HUCPVCs)) on the survival and differentiation of a human neuroblastoma cell line (SH-SY5Y). For this purpose, SH-SY5Y cells were differentiated with conditioned media (CM) from the MSCs populations referred above. Retinoic acid cultured cells were used as control for neuronal differentiated SH-SY5Y cells. SH-SY5Y cells viability assessment revealed that the secretome of BMSCs and HUCPVCs, in the form of CM, was able to induce their survival. Moreover, immunocytochemical experiments showed that CM from both MSCs was capable of inducing neuronal differentiation of SH-SY5Y cells. Finally, neurite lengths assessment and quantitative real-time reverse-transcription polymerase chain reaction (RT-PCR) analysis demonstrated that CM from BMSCs and HUCPVCs differently induced neurite outgrowth and mRNA levels of neuronal markers exhibited by SH-SY5Y cells. Overall, our results show that the secretome of both BMSCs and HUCPVCs was capable of supporting SH-SY5Y cells survival and promoting their differentiation towards a neuronal phenotype.The authors would like to thank Foundation Calouste de Gulbenkian for the funds attributed to Antonio J. Salgado, Portuguese Science Foundation of Science and Technology (FCT) for the Ph.D. fellowship attributed to Ana O. Pires (reference: SFRH/BD/33900/2009), and IF Development Grant to Antonio J. Salgado. They also would like to acknowledge Professor J. E. Davies (University of Toronto, Canada) and Professor Patricia Maciel (University of Minho, Portugal) for kindly providing HUCPVCs and SH-SY5Y cells, respectively. Finally, the authors would like to acknowledge the contribution of Fabio Rodrigues, Nuno Silva, Silvina Samy, and Vera Cardoso to this work

Secretome of Mesenchymal Progenitors from the Umbilical Cord Acts as Modulator of Neural/Glial Proliferation and Differentiation

It was recently shown that the conditioned media (CM) of Human Umbilical Cord Perivascular Cells (HUCPVCs), a mesenchymal progenitor population residing within the Wharton Jelly of the umbilical cord, was able to modulate in vitro the survival and viability of different neuronal and glial cells populations. In the present work, we aimed to assess if the secretome of HUCPVCs is able to 1) induce the differentiation of human telencephalon neural precursor cells (htNPCs) in vitro, and 2) modulate neural/glial proliferation, differentiation and survival in the dentate gyrus (DG) of adult rat hippocampus. For this purpose, two separate experimental setups were performed: 1) htNPCs were incubated with HUCPVCs-CM for 5 days after which neuronal differentiation was assessed and, 2) HUCPVCs, or their respective CM, were injected into the DG of young adult rats and their effects assessed 7 days later. Results revealed that the secretome of HUCPVCs was able to increase neuronal cell differentiation in vitro; indeed, higher densities of immature (DCX+ cells) and mature neurons (MAP-2(+) cells) were observed when htNPCs were incubated with the HUCPVCs-CM. Additionally, when HUCPVCs and their CM were injected in the DG, results revealed that both cells or CM were able to increase the endogenous proliferation (BrdU(+) cells) 7 days after injection. It was also possible to observe an increased number of newborn neurons (DCX+ cells), upon injection of HUCPVCs or their respective CM. Finally western blot analysis revealed that after CM or HUCPVCs transplantation, there was an increase of fibroblast growth factor-2 (FGF-2) and, to a lesser extent, of nerve growth factor (NGF) in the DG tissue. Concluding, our results have shown that the transplantation of HUCPVCs or the administration of their secretome were able to potentiate neuronal survival and differentiation in vitro and in vivo.Foundation Calouste Gulbenkian for funds under the scope of the Gulbenkian Programme to Support Cutting Edge Research in Life Sciences; Portuguese Foundation for Science and Technology (FCT) for Ciência 2007 program and IF Development Grant (A.J. Salgado), and pre-doctoral fellowship to F.G. Teixeira (SFRH / BD / 69637 / 2010); John E. Davies for kindly providing the HUCPVCs used in this workinfo:eu-repo/semantics/publishedVersio

The Notch Ligand Delta-Like 4 Regulates Multiple Stages of Early Hemato-Vascular Development

Background: In mouse embryos, homozygous or heterozygous deletions of the gene encoding the Notch ligand Dll4 result in early embryonic death due to major defects in endothelial remodeling in the yolk sac and embryo. Considering the close developmental relationship between endothelial and hematopoietic cell lineages, which share a common mesodermderived precursor, the hemangioblast, and many key regulatory molecules, we investigated whether Dll4 is also involved in the regulation of early embryonic hematopoiesis. Methodology/Principal Findings: Using Embryoid Bodies (EBs) derived from embryonic stem cells harboring hetero- or homozygous Dll4 deletions, we observed that EBs from both genotypes exhibit an abnormal endothelial remodeling in the vascular sprouts that arise late during EB differentiation, indicating that this in vitro system recapitulates the angiogenic phenotype of Dll4 mutant embryos. However, analysis of EB development at early time points revealed that the absence of Dll4 delays the emergence of mesoderm and severely reduces the number of blast-colony forming cells (BL-CFCs), the in vitro counterpart of the hemangioblast, and of endothelial cells. Analysis of colony forming units (CFU) in EBs and yolk sacs from Dll4 +/2 and Dll4 2/2 embryos, showed that primitive erythropoiesis is specifically affected by Dll4 insufficiency. In Dll4 mutant EBs, smooth muscle cells (SMCs) were seemingly unaffected and cardiomyocyte differentiation was increased, indicating that SMC specification is Dll4-independent while a normal dose of this Notch ligand is essential for th

Profiling microglia in a mouse model of Machado-Joseph disease

Microglia have been increasingly implicated in neurodegenerative diseases (NDs), and specific disease associated microglia (DAM) profiles have been defined for several of these NDs. Yet, the microglial profile in Machado–Joseph disease (MJD) remains unexplored. Here, we characterized the profile of microglia in the CMVMJD135 mouse model of MJD. This characterization was performed using primary microglial cultures and microglial cells obtained from disease-relevant brain regions of neonatal and adult CMVMJD135 mice, respectively. Machine learning models were implemented to identify potential clusters of microglia based on their morphological features, and an RNA-sequencing analysis was performed to identify molecular perturbations and potential therapeutic targets. Our findings reveal morphological alterations that point to an increased activation state of microglia in CMVMJD135 mice and a disease-specific transcriptional profile of MJD microglia, encompassing a total of 101 differentially expressed genes, with enrichment in molecular pathways related to oxidative stress, immune response, cell proliferation, cell death, and lipid metabolism. Overall, these results allowed us to define the cellular and molecular profile of MJD-associated microglia and to identify genes and pathways that might represent potential therapeutic targets for this disorder.This work was supported by Fundação para a Ciência e a Tecnologia (FCT) (PTDC/NEUNMC/3648/2014) and COMPETE-FEDER (POCI-01-0145-FEDER-016818). It was also supported by Portuguese funds through FCT in the framework of the Project POCI-01-0145-FEDER-031987 (PTDC/MED-OUT/31987/2017). A.B.C. was supported by a doctoral fellowship from FCT (PD/BD/ 127828/2016). S.P.N. was also supported by FCT (PD/BD/114120/2015). Work in the JBR laboratory was financed by FEDER—Fundo Europeu de Desenvolvimento Regional funds through the COMPETE 2020—Operational Programme for Competitiveness and Internationalization (POCI), Portugal 2020, and by Portuguese funds through FCT in the framework of the Project POCI-01-0145- FEDER030647 (PTDC/MED-NEU/31318/2017). This work was funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI (Portuguese Platform of Bioimaging) (PPBIPOCI-01-0145-FEDER-022122), and by National funds, through FCT—project UIDB/50026/2020 and UIDP/50026/2020

Genetic ablation of inositol 1,4,5-Trisphosphate receptor type 2 (IP3R2) fails to modify disease progression in a mouse model of Spinocerebellar Ataxia type 3

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP3R2) is the predominant IP3R in mediating astrocyte somatic calcium signals, and genetically ablation of IP3R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP3R2 in the onset and progression of SCA3. For this, we tested whether IP3R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP3R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP3R2 deletion in astrocytes does not modify SCA3 progression.This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020, PTDC/NEUNMC/3648/2014 and COMPETE-FEDER (POCI-01-0145-FEDER-016818); fellowships to DCG (2021.08121.BD), DMF (SFRH/BD/147947/2019), JSC (SFRH/BD/140624/2018), ANC (SFRH/BPD/118779/2016), AVF (UMINHO/BIL-CNCG/2022/11), SGG (SFRH/BD/101298/2014), and JFV (2020.05109.BD); FCT Scientific Employment Stimulus (CEEC)—Individual Call position to SDS (CEECIND/00685/2020); grants from the Bial Foundation (037/18) and “the la Caixa” Foundation (LCF/PR/HR21/52410024) to JFO; and by the projects NORTE-01-0145-FEDER-000013 and NORTE-01-0145-FEDER-000023, supported by the Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). It was also supported by grants from the ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122 and national funds through the Foundation for Science and Technology (FCT)

Students’ interests in biodiversity: links with health and sustainability

The COVID-19 pandemic has shown that sustainable actions to preserve biodiversity are critical to preventing new microorganisms from harming human health. In this sense, education and encouraging young people’s interest in local biodiversity are crucial to promoting its preservation and sustainability. This research studied the biodiversity interests of 14–15-year-old students in São Paulo State, focusing on the links between biodiversity and human health. The criterion of maximum variation was used to constitute a heterogeneous sample of students. Students answered a four-point Likert questionnaire. The items in this questionnaire were divided into categories related to the interest of young people in biodiversity, and these were analyzed using descriptive and inferential statistics (Wilcoxon test). Categories of biodiversity linked to “health or human utility” were of higher interest to young people than those with no links to human benefits, such as “diversity of organisms”. These findings, along with the literature, showed that young people are interested in biodiversity issues associated with human health. Therefore, teaching biodiversity should reflect on new possibilities for making a more sustainable environment and promoting social and environmental justice, fundamental aspects of promoting and guaranteeing human health.This research was funded by the São Paulo Research Foundation (FAPESP)(grant #2018/21756-0, São Paulo Research Foundation (FAPESP), grant#2019/08689-4, São Paulo Research Foundation (FAPESP), grant# 2016/05843-4, São Paulo Research Foundation (FAPESP)), the FOUNDATION FOR SCIENCE AND TECHNOLOGY (FCT), grant number SFRH/BD/79512/2011 and RESEARCH CENTRE ON CHILD STUDIES (R&D Unit 317 of FCT; projects UIDB/00317/2020 and UIDP/00317/2020) and the CAPES (Coordination for the Improvement of Higher Education—Personnel)

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