Identificação de modificadores da proteotoxicidade da ataxina-3 em modelos animais para a doença de Machado-Joseph

Tese doutoramento em Ciências da Saúde (especialidade de Ciências da Saúde)At least nine human neurodegenerative diseases are caused by the expansion of CAG repeats within otherwise unrelated genes. In these diseases, including Machado-Joseph disease (MJD), polyglutamine (polyQ) expansions cause the appearance of misfolded protein species, that ultimately lead to the formation of aggregates and neuronal loss. Along with the pathogenic motif, all these diseases have in common the fact that the associated gene products are widely expressed but affect only specific subsets of neurons. This specificity suggests that protein misfolding and its toxic outcomes may be determined by the polyQ-flanking sequences of the specific disease-associated proteins. Ataxin- 3 (ATXN3) is a polyQ protein and expansion of its repetitive glutamine tract causes MJD. MJD is characterized by the formation of ubiquitylated intra-neuronal inclusions but the mechanism underlying mutant ATXN3-mediated neuronal dysfunction still remains unsolved. Caenorhabditis elegans offers unique advantages for examining the aggregation dynamics of aggregation-prone proteins and its toxic effects on individual neurons, since the transparency of all 959 cells allows easy detection of fluorescent proteins in live animals. Despite having relatively few neurons, C. elegans display a wide array of complex behaviors and a clear link exists between the behavior and the function of neuronal subsets. In this study, we established a novel pan-neuronal C. elegans model for the study of ATXN3 pathogenesis. Pan-neuronal expression of mutant ATXN3 leads to a polyQ-length dependent, neuron subtype-specific aggregation and neuronal dysfunction. Analysis of different neurons revealed a pattern of dorsal nerve cord and sensory neuron processes susceptibility to mutant ATXN3 that was distinct from the aggregation and toxicity profiles of polyQ-alone proteins. This suggests that the sequences flanking the polyQ-stretch in ATXN3 have a dominant influence on cell-intrinsic neuronal factors that modulate polyQ-mediated pathogenesis. We investigated the role of the wild-type (WT) ATXN3 in polyQ-related pathogenesis and found that WT ATXN3 is irreversibly recruited into polyQ-containing cellular aggregates, aggravating the animals’ motor dysfunction. In contrast, genetic ablation of endogenous C. elegans ATX-3 did not modulate MJD pathogenesis. Our findings support the idea that, unlike what happens in other polyQ disorders, WT ATXN3 does not seem to display a neuroprotective role in MJD. We have also found that in C. elegans mutations reducing insulin/insulin growth factor (IGF)-1- like signaling (IIS) pathway partially rescues mutant ATXN3-mediated aggregation and toxicity. Strikingly, other longevity-related pathways showed different effects on ATXN3 proteotoxicity: dietary restricted animals succumbed to neuronal ATXN3 pathogenesis at similar rates to those of regularly fed animals. In turn, mutations leading to altered mitochondrial function and known to lead to increased longevity showed heterogeneous effects: clk-1 mutation severely aggravated mutant ATXN3 pathogenesis, whereas isp-1 mutation caused a significant delay in the appearance of aggregates. These results suggest that, in spite of improving global organism survival, aging-related pathways may not always show a positive effect on conformational disorders. Heat shock factor 1 (HSF-1) plays a neuroprotective role in ATXN3-mediated pathology in C. elegans. However, in mice, genetic reduction of Hsf-1 resulted in comparable motor uncoordination and pathology, when compared with MJD transgenic mice with two copies of Hsf-1 gene, suggesting that one copy of Hsf-1 is sufficient to cope with ATXN3(Q94) proteotoxicity in mice. Lastly, we validated our novel C. elegans model as a tool for identification of potential therapeutic compounds for MJD and established five compounds, potentially involved in heat shock response, autophagy, transcription regulation and longevity, as good candidates to test in higher model organisms for MJD. In summary, this work provided new clues for the study of ATXN3 pathogenesis and the role of the WT protein in disease. It raised new hypotheses regarding the mechanistic link(s) between aging determinants and proteotoxicity. It also made available a valuable C. elegans model/tool for drug discovery and target identification that can be very useful in future therapy development in MJD.Diferentes doenças neurodegenerativas humanas são causadas por uma expansão de uma repeticão CAG em genes que, de outra forma, não estão relacionados. Neste tipo de doenças, nomeadamente na doença de Machado-Joseph (DMJ), a expansão de poliglutaminas (poliQ) está associada a uma alteração da conformação das proteínas, com consequente formação de agregados e perda de células neuronais. Além do domínio patogénico, todas estas doenças têm em comum o facto de as suas proteínas causadoras terem uma expressão ubíqua, mas somente afectarem populações específicas de neurónios características de cada uma das doenças. Esta especificidade sugere que a agregação proteica e os seus efeitos tóxicos podem ser determinados pela sequência aminoacídica de cada proteína. A ataxina-3 (ATXN3) contém um segmento de poliQ cuja expansão está na origem da DMJ. A DMJ, assim como outras doenças de poliQ, é caracterizada pela formação de inclusões intraneuronais ubiquitiladas, mas o mecanismo associado à disfunção neuronal causada pela expressão da ATXN3 mutante não é totalmente compreendido. O nemátode Caenorhabditis elegans proporciona grandes vantagens no estudo dos efeitos tóxicos de proteínas poliQ em neurónios, uma vez que a transparência das suas 959 células facilita a detecção de proteínas fluorescentes in vivo. Apesar de apresentarem um número reduzido de neurónios, os C. elegans apresentam inúmeros comportamentos complexos, existindo uma relação clara entre a função de determinados subtipos neuronais e o comportamentos regulados por esses grupos de neurónios. Neste estudo, estabelecemos um novo modelo animal com expressão da ATXN3 humana em todas as células do sistema nervoso dos C. elegans, para o estudo da patogénese da DMJ. A expressão da ATXN3 mutada resulta no aparecimento de agregados e em disfunção neurológica. Ambos os fenótipos dependem do tamanho da sequência de poliQ da ATXN3. A análise de neurónios específicos revelou que os processos de neurónios sensoriais e do cordão nervoso dorsal são especificamente afectados em animais que expressam a ATXN3 mutante e não em animais que expressam proteínas poliQ. Estes resultados sugerem que o efeito das sequências flanqueantes da ATXN3 se sobrepõe a factores intrínsecos ao ambiente neuronal, modulando a patogénese mediada pelo tracto de poliQ. O papel da ATXN3 normal na patogénese da DMJ foi também investigado. Verificámos que a proteína normal é irreversivelmente recrutada para agregados de poliQ, provocando um agravamento do fenótipo motor destes animais. Em contraste, a deleção da ataxina-3 endógena (ATX-3) não modifica o fenótipo do modelo da DMJ em C. elegans. Estes resultados sugerem que, ao contrário do que acontece noutras doenças de poliQ, a ATXN3 WT não parece ter um papel neuroprotector na DMJ. Em C. elegans, mutações que reduzem a sinalização da via da insulina/factor de crescimento da insulina 1 (IIS) revertem parcialmente a agregação e toxicidade causada pela ATXN3 mutante. Surpreendentemente, outras vias que afectam a longevidade de organismos mostraram efeitos diversos na proteotoxicidade da ATXN3: animais em restrição calórica não apresentaram diferenças na patogénese da mediada pela ATXN3 mutada relativamente a animais com uma dieta ad libitum. Mutações que alteram a função mitocondrial e causam aumento da longevidade em C. elegans apresentaram efeitos heterogéneos: a mutação do gene clk-1 agravou o fenótipo de agregação, enquanto que a mutação do gene isp-1 atrasou o aparecimento dos agregados. Estes resultados sugerem que as vias da longevidade, embora aumentem a sobrevivência global dos organismos, nem sempre têm um impacto positivo no tratamento de doenças associadas a conformações proteicas. O factor de choque térmico 1 (HSF-1) desempenha um papel neuro-protector relativamente à proteotoxicidade da ATXN3 em C. elegans. Contudo, em ratinhos esse efeito não é claro. A redução dos níveis do gene Hsf-1 em ratinho resultou num fenótipo de descoordenação motora semelhante ao de ratinhos contendo duas cópias do gene. Este resultado sugere que uma cópia do Hsf-1 é suficiente para combater a proteotoxicidade causada pela ATXN3 quando mutada. Finalmente, validámos o nosso modelo em C. elegans como uma ferramenta para a identificação de potenciais compostos terapêuticos para a DMJ e identificámos cinco compostos, potencialmente envolvidos na regulação da resposta ao choque térmico nas células, na autofagia, na regulação da transcrição e na longevidade, como bons candidatos para testar em modelos da DMJ desenvolvidos em organismos evolutivamente mais próximos do humano, como é o caso do ratinho. Em resumo, este trabalho trouxe novas pistas para o estudo da patogénese da DMJ e para o papel da ATXN3 normal na doença. Também levantou novas possíveis hipóteses no que diz respeito a ligações mecanísticas entre factores que determinam a longevidade dos organismos e a proteotoxicidade. Este novo modelo passou a estar disponível para a comunidade como uma ferramenta para a potencial descoberta de novas drogas e identificação de alvos que podem ser úteis para o desenvolvimento de terapias para a DMJ

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

Burnout among Portuguese healthcare workers during the COVID-19 pandemic

During COVID-19 pandemic, healthcare workers (HCWs) have had high workload and have been exposed to multiple psychosocial stressors. The aim of this study was to evaluate HCWs in terms of the relative contributions of socio-demographic and mental health variables on three burnout dimensions: personal, workrelated, and client-related burnout. Methods: A cross-sectional study was performed using an online questionnaire spread via social networks. A snowball technique supported by health care institutions and professional organizations was applied. Results: A total of 2008 subjects completed the survey. Gender, parental status, marriage status, and salary reduction were found to be significant factors for personal burnout. Health problems and direct contact with infected people were significantly associated with more susceptibility to high personal and work-related burnout. Frontline working positions were associated with all three dimensions. Higher levels of stress and depression in HCWs were significantly associated with increased levels of all burnout dimensions. Higher levels of satisfaction with life and resilience were significantly associated with lower levels of all burnout dimensions. Conclusions: All three burnout dimensions were associated with a specific set of covariates. Consideration of these three dimensions is important when designing future burnout prevention programs for HCWs.info:eu-repo/semantics/publishedVersio

Learning the biochemical basis of axonal guidance: using Caenorhabditis elegans as a model

Aim: Experimental models are a powerful aid in visualizing molecular phenomena. This work reports how the worm Caenorhabditis elegans (C. elegans) can be effectively explored for students to learn how molecular cues dramatically condition axonal guidance and define nervous system structure and behavior at the organism level. Summary of work: A loosely oriented observational activity preceded detailed discussions on molecules implied in axonal migration. C. elegans mutants were used to introduce second-year medical students to the deleterious effects of gene malfunctioning in neuron response to extracellular biochemical cues and to establish links between molecular function, nervous system structure, and animal behavior. Students observed C. elegans cultures and associated animal behavior alterations with the lack of function of specific axon guidance molecules (the soluble cue netrin/UNC-6 or two receptors, DCC/UNC-40 and UNC-5H). Microscopical observations of these strains, in combination with pan-neuronal GFP expression, allowed optimal visualization of severely affected neurons. Once the list of mutated genes in each strain was displayed, students could also relate abnormal patterns in axon migration/ventral and dorsal nerve cord neuron formation in C. elegans with mutated molecular components homologous to those in humans. Summary of results: Students rated the importance and effectiveness of the activity very highly. Ninety-three percent found it helpful to grasp human axonal migration, and all students were surprised with the power of the model in helping to visualize the phenomenon.This work has been funded by National funds, through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020 and by the projects, NORTE01-0145-FEDER-000039 and NORTE-01-0145-FEDER-085468, supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF). This work has been also funded by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). Additionally, C.V. and D.V.C. were supported by the FCT individual fellowships 2022.11176.BD and SFRH/BD/147826/2019, respectively

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)

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

Preclinical assessment of mesenchymal-stem-cell-based therapies in spinocerebellar ataxia type 3

The low regeneration potential of the central nervous system (CNS) represents a challenge for the development of new therapeutic strategies for neurodegenerative diseases, including spinocerebellar ataxias. Spinocerebellar ataxia type 3 (SCA3)—or Machado–Joseph disease (MJD)—is the most common dominant ataxia, being mainly characterized by motor deficits; however, SCA3/MJD has a complex and heterogeneous pathophysiology, involving many CNS brain regions, contributing to the lack of effective therapies. Mesenchymal stem cells (MSCs) have been proposed as a potential therapeutic tool for CNS disorders. Beyond their differentiation potential, MSCs secrete a broad range of neuroregulatory factors that can promote relevant neuroprotective and immunomodulatory actions in different pathophysiological contexts. The objective of this work was to study the effects of (1) human MSC transplantation and (2) human MSC secretome (CM) administration on disease progression in vivo, using the CMVMJD135 mouse model of SCA3/MJD. Our results showed that a single CM administration was more beneficial than MSC transplantation—particularly in the cerebellum and basal ganglia—while no motor improvement was observed when these cell-based therapeutic approaches were applied in the spinal cord. However, the effects observed were mild and transient, suggesting that continuous or repeated administration would be needed, which should be further tested.This research was funded by the National Ataxia Foundation (NAF) and by Portuguese national funds, through the Foundation for Science and Technology (FCT)—projects UIDB/50026/2020, UIDP/50026/2020, POCI-01-0145-FEDER-029206, and through the Santa Casa Neuroscience Awards (Santa Casa da Misericórdia Lisboa)—project MC-04/17. Additionally, this project was funded by the ICVS Scientific Microscopy Platform, a member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122). S.C.S. received an individual fellowship within the project TUBITAK/0007/2014. The FCT funded individual fellowships to J.S C., A.N.-C., B.M.- P., F.G.T., R.L., S.M., N.A.S., C.S.-C., and S.D.-S. (SFRH/BD/140624/2018, SFRH/BPD/118779/2016, SFRH/BD/120124/2016, SFRH/BPD/118408/2016, PD/BDE/127836/2016, CEECIND/01902/2017, CEECIND/04794/2017, CEECIND/03887/2017, and CEECIND/00685/2020)

Aripiprazole offsets mutant ATXN3-induced motor dysfunction by targeting dopamine D2 and serotonin 1A and 2A receptors in C. elegans

The atypical antipsychotic aripiprazole is a Food and Drug Administration-approved drug for the treatment of psychotic, mood, and other psychiatric disorders. Previous drug discovery efforts pinpointed aripiprazole as an effective suppressor of Machado–Joseph disease (MJD) pathogenesis, as its administration resulted in a reduced abundance and aggregation of mutant Ataxin-3 (ATXN3) proteins. Dopamine partial agonism and functional selectivity have been proposed as the main pharmacological mechanism of action of aripiprazole in the treatment of psychosis; however, this mechanism remains to be determined in the context of MJD. Here, we focus on confirming the efficacy of aripiprazole to reduce motor dysfunction in vivo, using a Caenorhabditis elegans (C. elegans) model of MJD, and on unveiling the drug targets required for its positive action against mutant ATXN3 pathogenesis. We employed pharmacogenetics and pharmacological approaches to identify which dopamine and serotonin receptors are critical for aripiprazole-mediated improvements in motor function. We demonstrated that dopamine D2-like and serotonin 5-HT1A and 5-HT2A receptors play important roles in this process. Our findings strengthen the relevance of dopaminergic and serotoninergic signaling modulation against mutant ATXN3-mediated pathogenesis. The identification of aripiprazole’s cellular targets, relevant for MJD and perhaps other neurodegenerative diseases, may pave the way for prospective drug discovery and development campaigns aiming to improve the features of this prototypical compound and reduce side effects not negligible in the case of aripiprazole.This work was funded by FEDER through the Competitiveness Internationalization Operational Program (POCI) and by National funds through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-0 31987, NORTE-01-0145-FEDER-000013, and NORTE-01-0145-FEDER-000023, supported by the Northern Portugal Regional Operational Program (NORTE 2020), under the Portugal 2020 Partnership Agreement through the European Regional Development Fund (ERDF) and by ICVS Scientific Microscopy Platform, member of the national infrastructure PPBI—Portuguese Platform of Bioimaging (PPBI-POCI-01-0145-FEDER-022122; by National funds through the Foundation for Science and Technology (FCT)—project UIDB/50026/2020 and UIDP/50026/2020). Additionally, this project was supported by the National Ataxia Foundation (NAF). A.J., J.P.-S., D.V.-C., and J.D.S. were supported by the FCT individual fellowships SFRH/BD/76613/2011, PD/BDE/127834/2016, SFRH/BD/147826/2019, and PD/BD/128074/2016, respectively

From pathogenesis to novel therapeutics for spinocerebellar ataxia Type 3: Evading potholes on the way to translation

Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is a neurodegenerative disorder caused by a polyglutamine expansion in the ATXN3 gene. In spite of the identification of a clear monogenic cause 25 years ago, the pathological process still puzzles researchers, impairing prospects for an effective therapy. Here, we propose the disruption of protein homeostasis as the hub of SCA3 pathogenesis, being the molecular mechanisms and cellular pathways that are deregulated in SCA3 downstream consequences of the misfolding and aggregation of ATXN3. Moreover, we attempt to provide a realistic perspective on how the translational/clinical research in SCA3 should evolve. This was based on molecular findings, clinical and epidemiological characteristics, studies of proposed treatments in other conditions, and how that information is essential for their (re-)application in SCA3. This review thus aims i) to critically evaluate the current state of research on SCA3, from fundamental to translational and clinical perspectives; ii) to bring up the current key questions that remain unanswered in this disorder; and iii) to provide a frame on how those answers should be pursued.The authors thank all the members of the Maciel lab for their helpful tips and discussion. This work was funded by the European Regional Development Fund (FEDER), through the Competitiveness Internationalization Operational Programme (POCI), and by national funds, through the Foundation for Science and Technology (FCT), under the scope of the project POCI-01-0145-FEDER-0 31987. Moreover, the work was supported by the Northern Portugal Regional Operational Programme (NORTE 2020), under the Portugal 2020 Partnership Agreement, through the FEDER (project NORTE-01-0145-FEDER-000013). A fellowship supporting the development of this work was attributed by FCT to J. D. Da S. (PD/BD/128074/2016)

An image processing application for quantification of protein ag-gregates in Caenorhabditis elegans

Protein aggregation became a widely accepted marker of many polyQ disorders, including Machado-Joseph disease (MJD), and is often used as readout for disease progression and development of therapeutic strategies. The lack of good platforms to rapidly quantify protein aggregates in a wide range of disease animal models prompted us to generate a novel image processing application that automatically identifies and quantifies the aggregates in a standardized and operator-independent manner. We propose here a novel image processing tool to quantify the protein aggregates in a Caenorhabditis elegans (C. elegans) model of MJD. Confocal microscopy images were obtained from animals of different genetic conditions. The image processing application was developed using MeVisLab as a platform to process, analyse and visualize the images obtained from those animals. All segmentation algorithms were based on intensity pixel levels.The quantification of area or numbers of aggregates per total body area, as well as the number of aggregates per animal were shown to be reliable and reproducible measures of protein aggregation in C. elegans. The results obtained were consistent with the levels of aggregation observed in the images. In conclusion, this novel imaging processing application allows the non-biased, reliable and high throughput quantification of protein aggregates in a C. elegans model of MJD, which may contribute to a significant improvement on the prognosis of treatment effectiveness for this group of disorders.The authors acknowledge to Foundation for Science and Technology (FCT) - Portugal for the fellowships with the references: SFRH/BD/27258/2006; SFRH/ BPD/51058/2010; SFRH/BPD/66151/2009; and, SFRH/BPD/46851/2008. This work was also supported by FCT R&D project PTDC/SAU-BEB/103368/2008 and through a grant from National Ataxia Foundation (2010)