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  • Unraveling the role of ataxin-2 in metabolism
    Publication . Carmo-Silva, Sara; Nóbrega, Clévio; de Almeida, Luís Pereira; Cavadas, Cláudia
    Ataxin-2 is a polyglutamine protein implicated in several biological processes such as RNA metabolism and cytoskeleton reorganization. Ataxin-2 is highly expressed in various tissues including the hypothalamus, a brain region that controls food intake and energy balance. Ataxin-2 expression is influenced by nutritional status. Emerging studies discussed here now show that ataxin-2 deficiency correlates with insulin resistance and dyslipidemia, an action mediated via the mTOR pathway, suggesting that ataxin-2 might play key roles in metabolic homeostasis including body weight regulation, insulin sensitivity, and cellular stress responses. In this review we also discuss the relevance of ataxin-2 in the hypothalamic regulation of energy balance, and its potential as a therapeutic target in metabolic disorders such as obesity.
  • Restoring brain cholesterol turnover improves autophagy and has therapeutic potential in mouse models of spinocerebellar ataxia
    Publication . Nóbrega, Clévio; Mendonca, Liliana; Marcelo, Adriana; Lamaziere, Antonin; Tome, Sandra; Despres, Gaetan; Matos, Carlos A; Mechmet, Fatich; Langui, Dominique; den Dunnen, Wilfred; de Almeida, Luis Pereira; Cartier, Nathalie; Alves, Sandro
    Spinocerebellar ataxias (SCAs) are devastating neurodegenerative disorders for which no curative or preventive therapies are available. Deregulation of brain cholesterol metabolism and impaired brain cholesterol turnover have been associated with several neurodegenerative diseases. SCA3 or Machado-Joseph disease (MJD) is the most prevalent ataxia worldwide. We show that cholesterol 24-hydroxylase (CYP46A1), the key enzyme allowing efflux of brain cholesterol and activating brain cholesterol turnover, is decreased in cerebellar extracts from SCA3 patients and SCA3 mice. We investigated whether reinstating CYP46A1 expression would improve the disease phenotype of SCA3 mouse models. We show that administration of adeno-associated viral vectors encoding CYP46A1 to a lentiviral-based SCA3 mouse model reduces mutant ataxin-3 accumulation, which is a hallmark of SCA3, and preserves neuronal markers. In a transgenic SCA3 model with a severe motor phenotype we confirm that cerebellar delivery of AAVrh10-CYP46A1 is strongly neuroprotective in adult mice with established pathology. CYP46A1 significantly decreases ataxin-3 protein aggregation, alleviates motor impairments and improves SCA3-associated neuropathology. In particular, improvement in Purkinje cell number and reduction of cerebellar atrophy are observed in AAVrh10-CYP46A1-treated mice. Conversely, we show that knocking-down CYP46A1 in normal mouse brain impairs cholesterol metabolism, induces motor deficits and produces strong neurodegeneration with impairment of the endosomal-lysosomal pathway, a phenotype closely resembling that of SCA3. Remarkably, we demonstrate for the first time both in vitro, in a SCA3 cellular model, and in vivo, in mouse brain, that CYP46A1 activates autophagy, which is impaired in SCA3, leading to decreased mutant ataxin-3 deposition. More broadly, we show that the beneficial effect of CYP46A1 is also observed with mutant ataxin-2 aggregates. Altogether, our results confirm a pivotal role for CYP46A1 and brain cholesterol metabolism in neuronal function, pointing to a key contribution of the neuronal cholesterol pathway in mechanisms mediating clearance of aggregate-prone proteins. This study identifies CYP46A1 as a relevant therapeutic target not only for SCA3 but also for other SCAs.
  • The contribution of genetics and environment to obesity
    Publication . Albuquerque, David; Nóbrega, Clévio; Manco, Licínio; Padez, Cristina
    Background: Obesity is a global health problem mainly attributed to lifestyle changes such as diet, low physical activity or socioeconomics factors. However, several evidences consistently showed that genetics contributes significantly to the weight-gain susceptibility. Sources of data: A systematic literature search of most relevant original, review and meta-analysis, restricted to English was conducted in PubMed, Web of Science and Google scholar up to May 2017 concerning the contribution of genetics and environmental factors to obesity. Areas of agreement: Several evidences suggest that obesogenic environments contribute to the development of an obese phenotype. However, not every individual from the same population, despite sharing the same obesogenic environment, develop obesity. Areas of controversy: After more than 10 years of investigation on the genetics of obesity, the variants found associated with obesity represent only 3% of the estimated BMI-heritability, which is around 47-80%. Moreover, genetic factors per se were unable to explain the rapid spread of obesity prevalence. Growing points: The integration of multi-omics data enables scientists having a better picture and to elucidate unknown pathways contributing to obesity. Areas timely for developing research: New studies based on case-control or gene candidate approach will be important to identify new variants associated with obesity susceptibility and consequently unveiling its genetic architecture. This will lead to an improvement of our understanding about underlying mechanisms involved in development and origin of the actual obesity epidemic. The integration of several omics will also provide insights about the interplay between genes and environments contributing to the obese phenotype.
  • Machado-Joseph disease: from pathogenic mechanisms to therapeutic strategies
    Publication . Nóbrega, Clévio; Matos, C. A.; Carmo-Silva, S.; Tome, S.; Mendoca, L.; Almeida, Luis Pereira de; Marcelo, A.
  • Machado-Joseph disease/spinocerebellar ataxia type 3: lessons from disease pathogenesis and clues into therapy
    Publication . Matos, Carlos A.; Pereira De Almeida, Luis; Nóbrega, Clévio
    Machado-Joseph disease (MJD), also known as spinocerebellar ataxia type 3 (SCA3), is an incurable disorder, widely regarded as the most common form of spinocerebellar ataxia in the world. MJD/SCA3 arises from mutation of the ATXN3 gene, but this simple monogenic cause contrasts with the complexity of the pathogenic mechanisms that are currently admitted to underlie neuronal dysfunction and death. The aberrantly expanded protein product - ataxin-3 - is known to aggregate and generate toxic species that disrupt several cell systems, including autophagy, proteostasis, transcription, mitochondrial function and signalling. Over the years, research into putative therapeutic approaches has often been devoted to the development of strategies that counteract disease at different stages of cellular pathogenesis. Silencing the pathogenic protein, blocking aggregation, inhibiting toxic proteolytic processing and counteracting dysfunctions of the cellular systems affected have yielded promising ameliorating results in studies with cellular and animal models. The current review analyses the available studies dedicated to the investigation of MJD/SCA3 pathogenesis and the exploration of possible therapeutic strategies, focusing primarily on gene therapy and pharmacological approaches rooted on the molecular and cellular mechanisms of disease.
  • Polyglutamine disorders preface
    Publication . Nóbrega, Clévio; Almeida, Luís Pereira de; Nóbrega, Clévio; Almeida, Luís Pereira de
    Polyglutamine (polyQ) diseases are a group of rare neurodegenerative disorders that share a common genetic cause: they arise as a result of abnormal expansions of CAG trinucleotide sequences occurring at particular genome loci. In contrast with other repeat-related disorders, the repeat-bearing tracts associated with polyQ diseases are present at the codifying region of genes, being translated as expanded polyQ tracts in their respective protein products. Although these genes and proteins are otherwise unrelated and share no significant homology outside the CAG/polyQ tract, proteins carrying an abnormally expanded polyQ tract tend to aggregate, forming insoluble protein aggregates that constitute a key neuropathological feature of polyQ disorders. The group currently includes nine disorders: Huntington’s disease (HD), dentatorubral-pallidoluysian atrophy (DRPLA), spinal and bulbar muscular atrophy (SBMA), and six different types of spinocerebellar ataxia: SCA 1, 2, 3, 6, 7, and 17. PolyQ diseases are highly incapacitating and, to this date, no therapy able to modify disease progression is available for any of them.
  • Cordycepin activates autophagy through AMPK phosphorylation to reduce abnormalities in Machado-Joseph disease models
    Publication . Marcelo, Adriana; Brito, Filipa; Carmo-Silva, Sara; Matos, Carlos A.; Alves-Cruzeiro, Joao; Vasconcelos-Ferreira, Ana; Koppenol, Rebekah; Mendonca, Liliana; de Almeida, Luis Pereira; Nóbrega, Clévio
    Machado-Joseph disease (MJD) is a neurodegenerative disorder caused by an abnormal expansion of citosine-adenine-guanine trinucleotide repeats in the disease-causing gene. This mutation leads to an abnormal polyglutamine tract in the protein ataxin-3 (Atx3), resulting in formation of mutant Atx3 aggregates. Despite several attempts to develop a therapeutic option for MJD, currently there are no available therapies capable of delaying or stopping disease progression. Recently, our group reported that reducing the expression levels of mutant Atx3 lead to a mitigation of several MJD-related behavior and neuropathological abnormalities. Aiming a more rapid translation to the human clinics, in this study we investigate a pharmacological inhibitor of translation-cordycepin-in several preclinical models. We found that cordycepin treatment significantly reduced (i) the levels of mutant Atx3, (ii) the neuropathological abnormalities in a lentiviral mouse model, (iii) the motor and neuropathological deficits in a transgenic mouse model and (iv) the number of ubiquitin aggregates in a human neural model. We hypothesize that the effect of cordycepin is mediated by the increase of phosphorylated adenosine monophosphate-activated protein kinase (AMPK) levels, which is accompanied by a reduction in the global translation levels and by a significant activation of the autophagy pathway. Overall, this study suggests that cordycepin might constitute an effective and safe therapeutic approach for MJD, and probably for the other polyglutamine diseases.
  • Motor dysfunctions and neuropathology in mouse models of spinocerebellar ataxia type 2: A comprehensive review
    Publication . Alves-Cruzeiro, João M. da Conceição; Mendonça, Liliana; Almeida, Luís Pereira de; Nóbrega, Clévio
    Spinocerebellar ataxia type 2 (SCA2) is an autosomal dominant ataxia caused by an expansion of CAG repeats in the exon 1 of the gene ATXN2, conferring a gain of toxic function that triggers the appearance of the disease phenotype. SCA2 is characterized by several symptoms including progressive gait ataxia and dysarthria, slow saccadic eye movements, sleep disturbances, cognitive impairments, and psychological dysfunctions such as insomnia and depression, among others. The available treatments rely on palliative care, which mitigate some of the major symptoms but ultimately fail to block the disease progression. This persistent lack of effective therapies led to the development of several models in yeast, C. elegans, D. melanogaster, and mice to serve as platforms for testing new therapeutic strategies and to accelerate the research on the complex disease mechanisms. In this work, we review 4 transgenic and 1 knock-in mouse that exhibit a SCA2-related phenotype and discuss their usefulness in addressing different scientific problems. The knock-in mice are extremely faithful to the human disease, with late onset of symptoms and physiological levels of mutant ataxin-2, while the other transgenic possess robust and well-characterized motor impairments and neuropathological features. Furthermore, a new BAC model of SCA2 shows promise to study the recently explored role of non-coding RNAs as a major pathogenic mechanism in this devastating disorder. Focusing on specific aspects of the behavior and neuropathology, as well as technical aspects, we provide a highly practical description and comparison of all the models with the purpose of creating a useful resource for SCA2 researchers worldwide.
  • MSGP: the first database of the protein components of the mammalian stress granules
    Publication . Nunes, Catarina; Mestre, Isa; Marcelo, Adriana; Koppenol, Rebekah; Matos, Carlos A.; Nóbrega, Clévio
    In response to different stress stimuli, cells transiently form stress granules (SGs) in order to protect themselves and re-establish homeostasis. Besides these important cellular functions, SGs are now being implicated in different human diseases, such as neurodegenerative disorders and cancer. SGs are ribonucleoprotein granules, constituted by a variety of different types of proteins, RNAs, factors involved in translation and signaling molecules, being capable of regulating mRNA translation to facilitate stress response. However, until now a complete list of the SG components has not been available. Therefore, we aimer at identifying and linting in an open access database all the proteins described so far as components of SGs. The identification was made through an exhaustive search of studies listed in PubMed and double checked. Moreover, for each identified protein several details were also gathered from public databases, such as the molecular function, the cell types in which they were detected, the type of stress stimuli used to induce SG formation and the reference of the study describing the recruitment of the component to SGs. Expression levels in the context of different neurodegenerative diseases were also obtained and are also described in the database. The Mammalian Stress Granules Proteome is available at https://msgp.pt/, being a new and unique open access online database, the first to list all the protein components of the SGs identified so far. The database constitutes an important and valuable tool for researchers in this research area of growing interest.
  • RNA interference therapy for Machado-Joseph disease: Long-term safety profile of lentiviral vectors encoding short hairpin RNAs targeting mutant ataxin-3
    Publication . Nóbrega, Clévio; Codesso, Jose Miguel; Mendonca, Liliana; de Almeida, Luis Pereira
    Machado-Joseph disease (MJD) or spinocerebellar ataxia type 3 is a neurodegenerative disorder caused by an abnormal repetition of a CAG codon in the MJD1 gene. This expansion translates into a long polyglutamine tract, leading to the misfolding of the mutant protein ataxin-3, which abnormally accumulates in the nucleus, thus leading to neurodegeneration in specific brain regions. No treatment able to modify the progression of the disease is available. However, it has previously been shown that specific silencing of mutant ataxin-3 by RNA interference with viral vectors is a promising therapeutic strategy for MJD. Nevertheless, reports of cytotoxic effects of this technology led to the safety profile of the previously tested lentiviral vectors encoding short hairpin (sh)RNAs (LV-shmutatx3) targeting mutant ataxin-3 upon brain injection being investigated. For this purpose, the vectors were injected in the mouse striata, and neuronal dysfunction, degeneration, gliosis, off-target effects, and saturation of the RNA interference machinery were evaluated. It was found that: (1) LV-shmutatx3 mediated stable and long-term expression of the shRNA in neurons of the mouse striatum; (2) neuronal dysfunction evaluated by darpp-32, NeuN, and cresyl violet staining, initially more pronounced, became indistinguishable from the phosphate-buffered saline group at 8 weeks and resolved within 20 weeks; (3) astrocytic activation was present, which resolved within 8 weeks; (4) microglial activity and proinflammatory cytokines release were present, which resolved and normalized within 20 weeks; and (5) there were no off-target effects or saturation of the endogenous RNA interference processing machinery in the mouse striatum. The data show that injection of lentiviral vectors encoding a shRNA targeting mutant ataxin-3 in the mouse brain induce transient dysfunctions, which resolve within 20 weeks. Importantly, long-term expression (up to 20 weeks post injection) of this shRNA (driven by H1 promoter) led to no toxic effect in vivo. This study thus constitutes an additional step in a future translation of gene silencing as a therapy for MJD.