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Machado-Joseph disease (MJD)/spinocerebellar ataxia type 3 (SCA3) is the most common autosomal dominantly inherited ataxia worldwide. It is caused by an over-repetition of the trinucleotide CAG within the ATXN3 gene, which confers toxic properties to ataxin-3 (ATXN3) species. RNA interference technology has shown promising therapeutic outcomes but still lacks a non-invasive delivery method to the brain. Extracellular vesicles (EVs) emerged as promising delivery vehicles due to their capacity to deliver small nucleic acids, such as microRNAs (miRNAs). miRNAs were found to be enriched into EVs due to specific signal motifs designated as ExoMotifs. In this study, we aimed at investigating whether ExoMotifs would promote the packaging of artificial miRNAs into EVs to be used as non-invasive therapeutic delivery vehicles to treat MJD/SCA3. We found that miRNA-based silencing sequences, associated with ExoMotif GGAG and ribonucleoprotein A2B1 (hnRNPA2B1), retained the capacity to silence mutant ATXN3 (mutATXN3) and were 3-fold enriched into EVs. Bioengineered EVs containing the neuronal targeting peptide RVG on the surface significantly decreased mutATXN3 mRNA in primary cerebellar neurons from MJD YAC 84.2 and in a novel dual-luciferase MJD mouse model upon daily intranasal administration. Altogether, these findings indicate that bioengineered EVs carrying miRNA-based silencing sequences are a promising delivery vehicle for brain therapy.
Assuntos
Doença de Machado-Joseph , MicroRNAs , Camundongos , Animais , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/terapia , MicroRNAs/genética , Ataxina-3/genética , Interferência de RNA , Peptídeos/genéticaRESUMO
AIMS: Machado-Joseph disease (MJD) is the most frequent dominantly inherited cerebellar ataxia worldwide. Expansion of a CAG trinucleotide in the MJD1 gene translates into a polyglutamine tract within ataxin-3, which upon proteolysis may lead to MJD. The aim of this work was to understand the in vivo contribution of calpain proteases to the pathogenesis of MJD. Therefore, we investigated (a) the calpain cleavage sites in ataxin-3 protein, (b) the most toxic ataxin-3 fragment generated by calpain cleavage and (c) whether targeting calpain cleavage sites of mutant ataxin-3 could be a therapeutic strategy for MJD. METHODS: We generated truncated and calpain-resistant constructs at the predicted cleavage sites of ataxin-3 using inverse PCR mutagenesis. Lentiviral vectors encoding these constructs were transduced in the adult mouse brain prior to western blot and immunohistochemical analysis 5 and 8 weeks later. RESULTS: We identified the putative calpain cleavage sites for both wild-type and mutant ataxin-3 proteins. The mutation of these sites eliminated the formation of the toxic fragments, namely, the 26-kDa fragment, the major contributor for striatal degeneration. Nonetheless, reducing the formation of both the 26- and 34-kDa fragments was required to preclude the intranuclear localisation of ataxin-3. A neuroprotective effect was observed upon mutagenesis of calpain cleavage sites within mutant ataxin-3 protein. CONCLUSIONS: These findings suggest that the calpain system should be considered a target for MJD therapy. The identified calpain cleavage sites will contribute to the design of targeted drugs and genome editing systems for those specific locations.
Assuntos
Doença de Machado-Joseph , Animais , Ataxina-3/genética , Ataxina-3/metabolismo , Calpaína/genética , Calpaína/metabolismo , Corpo Estriado/metabolismo , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/terapia , Camundongos , MutaçãoRESUMO
Nonalcoholic fatty liver disease (NAFLD), a condition strongly associated with obesity and insulin resistance, is characterized by hepatic lipid accumulation and activation of the endoplasmic reticulum (ER) stress response. The sirtuin 2 (SIRT2) protein deacetylase is emerging as a new player in metabolic homeostasis, but its role in the development of hepatic steatosis and its link with ER stress activation remains unknown. SIRT2-knockout (SIRT2-KO) and wild-type mice were fed either a control or a high-fat diet (HFD) for 4 weeks. Genetic manipulation of SIRT2 levels was performed in human hepatic cells. Although apparently normal under a control diet, SIRT2-KO mice showed accelerated body weight gain and adiposity on a HFD, accompanied by severe insulin resistance. Importantly, SIRT2-KO mice exhibited worsened hepatic steatosis independently from diet, consistent with upregulated gene expression of lipogenic enzymes and increased expression of ER stress markers. Exposure of hepatic cells to palmitate induced lipid accumulation, increased ER stress, and decreased SIRT2 expression. Moreover, SIRT2-silenced cells showed enhanced lipid accumulation and ER stress activation under basal conditions, whereas SIRT2 overexpression abrogated palmitate-induced lipid deposition and ER stress activation. Our findings reveal a role for SIRT2 in the regulation of hepatic lipid homeostasis, potentially through the ER stress response, suggesting that SIRT2 activation might constitute a therapeutic strategy against obesity and its metabolic complications.
Assuntos
Resistência à Insulina , Hepatopatia Gordurosa não Alcoólica , Sirtuína 2/metabolismo , Animais , Dieta Hiperlipídica , Estresse do Retículo Endoplasmático/fisiologia , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Hepatopatia Gordurosa não Alcoólica/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Obesidade/complicações , Obesidade/genética , Obesidade/metabolismo , Palmitatos/metabolismo , Sirtuína 2/genéticaRESUMO
Machado-Joseph disease (MJD) is a genetic neurodegenerative disease caused by an expanded polyglutamine tract within the protein ataxin-3 (ATXN3). Despite current efforts, MJD's mechanism of pathogenesis remains unclear and no disease-modifying treatment is available. Therefore, in this study, we investigated (1) the role of the 3' UTR of ATXN3, a putative microRNA (miRNA) target, (2) whether miRNA biogenesis and machinery are dysfunctional in MJD, and (3) which specific miRNAs target ATXN3-3' UTR and whether they can alleviate MJD neuropathology in vivo. Our results demonstrate that endogenous miRNAs, by targeting sequences in the 3' UTR, robustly reduce ATXN3 expression and aggregation in vitro and neurodegeneration and neuroinflammation in vivo. Importantly, we found an abnormal MJD-associated downregulation of genes involved in miRNA biogenesis and silencing activity. Finally, we identified three miRNAs-mir-9, mir-181a, and mir-494-that interact with the ATXN3-3' UTR and whose expression is dysregulated in human MJD neurons and in other MJD cell and animal models. Furthermore, overexpression of these miRNAs in mice resulted in reduction of mutATXN3 levels, aggregate counts, and neuronal dysfunction. Altogether, these findings indicate that endogenous miRNAs and the 3' UTR of ATXN3 play a crucial role in MJD pathogenesis and provide a promising opportunity for MJD treatment.
Assuntos
Regulação da Expressão Gênica , Doença de Machado-Joseph/genética , MicroRNAs/genética , Regiões 3' não Traduzidas , Animais , Ataxina-3/genética , Linhagem Celular , Modelos Animais de Doenças , Expressão Gênica , Ordem dos Genes , Genes Reporter , Vetores Genéticos/genética , Humanos , Lentivirus/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Camundongos , Camundongos Transgênicos , Mutação , Neurônios/metabolismo , Agregação Patológica de Proteínas , Interferência de RNA , Estabilidade de RNARESUMO
Polyglutamine diseases are hereditary degenerative disorders of the nervous system that have remained, to this date, untreatable. Promisingly, investigation into their molecular etiology and the development of increasingly perfected tools have contributed to the design of novel strategies with therapeutic potential. Encouraging studies have explored gene therapy as a means to counteract cell demise and loss in this context. The current chapter addresses the two main focuses of research in the area: the characteristics of the systems used to deliver nucleic acids to cells and the molecular and cellular actions of the therapeutic agents. Vectors used in gene therapy have to satisfyingly reach the tissues and cell types of interest, while eliciting the lowest toxicity possible. Both viral and non-viral systems have been developed for the delivery of nucleic acids to the central nervous system, each with its respective advantages and shortcomings. Since each polyglutamine disease is caused by mutation of a single gene, many gene therapy strategies have tried to halt degeneration by silencing the corresponding protein products, usually recurring to RNA interference. The potential of small interfering RNAs, short hairpin RNAs and microRNAs has been investigated. Overexpression of protective genes has also been evaluated as a means of decreasing mutant protein toxicity and operate beneficial alterations. Recent gene editing tools promise yet other ways of interfering with the disease-causing genes, at the most upstream points possible. Results obtained in both cell and animal models encourage further delving into this type of therapeutic strategies and support the future use of gene therapy in the treatment of polyglutamine diseases.
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Edição de Genes/métodos , Terapia Genética/métodos , Transtornos Heredodegenerativos do Sistema Nervoso/genética , Transtornos Heredodegenerativos do Sistema Nervoso/terapia , Animais , Transtornos Heredodegenerativos do Sistema Nervoso/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Mutação , Peptídeos/genética , Peptídeos/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismoRESUMO
Alzheimer's disease (AD) is the most common form of dementia worldwide, characterized by progressive memory impairment, behavioral changes, and, ultimately, loss of consciousness and death. Recently, microRNA (miRNA) dysfunction has been associated with increased production and impaired clearance of amyloid-ß (Aß) peptides, whose accumulation is one of the most well-known pathophysiological markers of this disease. In this study, we identified several miRNAs capable of targeting key proteins of the amyloidogenic pathway. The expression of one of these miRNAs, miR-31, previously found to be decreased in AD patients, was able to simultaneously reduce the levels of APP and Bace1 mRNA in the hippocampus of 17-month-old AD triple-transgenic (3xTg-AD) female mice, leading to a significant improvement of memory deficits and a reduction in anxiety and cognitive inflexibility. In addition, lentiviral-mediated miR-31 expression significantly ameliorated AD neuropathology in this model, drastically reducing Aß deposition in both the hippocampus and subiculum. Furthermore, the increase of miR-31 levels was enough to reduce the accumulation of glutamate vesicles in the hippocampus to levels found in non-transgenic age-matched animals. Overall, our results suggest that miR-31-mediated modulation of APP and BACE1 can become a therapeutic option in the treatment of AD.
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Cellulose nanocrystals (CNC) were obtained from macauba and gravata fibers. Macauba (or Bocaiuva) is a palm tree found throughout most of Brazil and Gravata is an abundant kind of bromelia with 1-2m long leaves, found in Brazilian Pantanal and Cerrado. The raw fibers of both fibers were mercerized with NaOH solutions and bleached; they were then submitted to acid hydrolysis using H2SO4 at 45 °C, varying the hydrolysis time from 15 up to 75 min. The fibers were analyzed by X-ray diffraction (XRD), FTIR Spectroscopy, scanning electron microscopy (SEM) and thermal stability by thermogravimetric analysis (TG). XRD patterns did not present changes in the crystal structure of cellulose after mercerization, but it was observed a decrease of hemicellulose and lignin contents, and consequently an increase of cellulose content with the increase of NaOH solution concentration in the mercerization. After acid hydrolysis, the cellulose nanocrystals (CNC) were also analyzed by transmission electron microscopy (TEM) which showed an acicular or rod-like aspect and nanometric dimensions of CNC from both fibers, but the higher values of aspect ratio (L/D) were found on CNC obtained from gravata after 45 min of acid hydrolysis. The mercerization and subsequent bleaching of fibers influenced the crystallinity index and thermal stability of the resulting CNC, but their properties are mainly influenced by the hydrolysis time, i. e., there is an increase in crystallinity and thermal stability up to 45 min of hydrolysis, after this time, both properties decrease, probably due to the cellulose degradation by the sulfuric acid.
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Extracellular vesicles (EVs) are cell-derived membrane vesicles virtually secreted by all cells, including brain cells. EVs are a major term that includes apoptotic bodies, microvesicles and exosomes. The release of EVs has been recognized as an important modulator in cross-talking between neurons, astrocytes, microglia and oligodendrocytes, not only in central nervous system (CNS) physiology but also in neurodegenerative and neuroinflammatory disease states as well as in brain tumors, such as glioma. EVs are able to cross the blood brain barrier (BBB), spread to body fluids and reach distant tissues. This prominent spreading ability has suggested that EVs can be exploited into several different clinical applications ranging from biomarkers to therapeutic carriers. Exosomes, the well-studied group of EVs, have been emerging as a promising tool for therapeutic delivery strategies due to their intrinsic features, such as the stability, biocompatibility and stealth capacity when circulating in bloodstream, the ability to overcome natural barriers and inherent targeting properties. Over the last years, it became apparent that EVs can be loaded with specific cargoes directly in isolated EVs or by modulation of producer cells. In addition, the engineering of its membrane for targeting purposes is expected to allow generating carriers with unprecedented abilities for delivery in specific organs or tissues. Nevertheless, some challenges remain regarding the loading and targeting of EVs for which more research is necessary, and will be discussed in this review. Recently-emerged promising derivations are also discussed, such as exosome associated with adeno-associated virus (AAV) vectors (vexosomes), enveloped protein nanocages (EPNs) and exosome-mimetic nanovesicles. This article provides an updated review of this fast-progressing field of EVs and their role in brain diseases, particularly focusing in their therapeutic applications.
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Encefalopatias/tratamento farmacológico , Sistemas de Liberação de Medicamentos , Vesículas Extracelulares , Animais , Encéfalo/metabolismo , Humanos , Nanopartículas/administração & dosagem , Nanopartículas/uso terapêuticoRESUMO
Biodegradable blends of urea plasticized thermoplastic starch (UTPS) and poly(ε-caprolactone) (PCL) were prepared in a co-rotating twin screw extruder. The UTPS and PCL content varied in a range of 25wt%. The materials were characterized by capillary rheometry, scanning electron microscopy (SEM), termogravimetry (TGA), differential scanning calorimetry (DSC) and tensile tests. Capillary rheometry showed better interaction between UTPS and PCL at 110°C than at 130°C. SEM showed immiscibility of all blends and good dispersion of UTPS in PCL matrix up to 50wt%. However, a co-continuous morphology was found for UTPS/PCL 75/25. Thermal analysis showed that introducing PCL in UTPS, increased Tonset due to higher thermal stability of PCL, and blends presented an intermediate behavior of neat polymers. The presence of PCL in blends improved significantly the mechanical properties of neat UTPS. Because they are totally biodegradable, these blends can be vehicles for controlled or slow release of nutrients to the soil while degraded.
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Alteration of corticostriatal glutamatergic function is an early pathophysiological change associated with Huntington's disease (HD). The factors that regulate the maintenance of corticostriatal glutamatergic synapses post-developmentally are not well understood. Recently, the striatum-enriched transcription factor Foxp2 was implicated in the development of these synapses. Here, we show that, in mice, overexpression of Foxp2 in the adult striatum of two models of HD leads to rescue of HD-associated behaviors, while knockdown of Foxp2 in wild-type mice leads to development of HD-associated behaviors. We note that Foxp2 encodes the longest polyglutamine repeat protein in the human reference genome, and we show that it can be sequestered into aggregates with polyglutamine-expanded mutant Huntingtin protein (mHTT). Foxp2 overexpression in HD model mice leads to altered expression of several genes associated with synaptic function, genes that present additional targets for normalization of corticostriatal dysfunction in HD.
Assuntos
Corpo Estriado/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Doença de Huntington/metabolismo , Proteínas Repressoras/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Técnica Indireta de Fluorescência para Anticorpo , Fatores de Transcrição Forkhead/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Humanos , Doença de Huntington/genética , Masculino , Camundongos , Fenótipo , Proteínas Repressoras/genéticaRESUMO
Machado-Joseph disease (MJD) is a neurodegenerative disorder characterized by an abnormal expansion of the CAG triplet in the ATXN3 gene, translating into a polyglutamine tract within the ataxin-3 protein. The available treatments only ameliorate symptomatology and do not block disease progression. In this study we find that caloric restriction dramatically rescues the motor incoordination, imbalance and the associated neuropathology in transgenic MJD mice. We further show that caloric restriction rescues SIRT1 levels in transgenic MJD mice, whereas silencing SIRT1 is sufficient to prevent the beneficial effects on MJD pathology. In addition, the re-establishment of SIRT1 levels in MJD mouse model, through the gene delivery approach, significantly ameliorates neuropathology, reducing neuroinflammation and activating autophagy. Furthermore, the pharmacological activation of SIRT1 with resveratrol significantly reduces motor incoordination of MJD mice. The pharmacological SIRT1 activation could provide important benefits to treat MJD patients.