RESUMO
AIMS: Tay-Sachs and Sandhoff diseases (GM2 gangliosidosis) are autosomal recessive disorders of lysosomal function that cause progressive neurodegeneration in infants and young children. Impaired hydrolysis catalysed by ß-hexosaminidase A (HexA) leads to the accumulation of GM2 ganglioside in neuronal lysosomes. Despite the storage phenotype, the role of autophagy and its regulation by mTOR has yet to be explored in the neuropathogenesis. Accordingly, we investigated the effects on autophagy and lysosomal integrity using skin fibroblasts obtained from patients with Tay-Sachs and Sandhoff diseases. RESULTS: Pathological autophagosomes with impaired autophagic flux, an abnormality confirmed by electron microscopy and biochemical studies revealing the accelerated release of mature cathepsins and HexA into the cytosol, indicating increased lysosomal permeability. GM2 fibroblasts showed diminished mTOR signalling with reduced basal mTOR activity. Accordingly, provision of a positive nutrient signal by L-arginine supplementation partially restored mTOR activity and ameliorated the cytopathological abnormalities. INNOVATION: Our data provide a novel molecular mechanism underlying GM2 gangliosidosis. Impaired autophagy caused by insufficient lysosomal function might represent a new therapeutic target for these diseases. CONCLUSIONS: We contend that the expression of autophagy/lysosome/mTOR-associated molecules may prove useful peripheral biomarkers for facile monitoring of treatment of GM2 gangliosidosis and neurodegenerative disorders that affect the lysosomal function and disrupt autophagy.
Assuntos
Arginina/farmacologia , Autofagia , Gangliosidoses GM2/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Catepsinas/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Hexosaminidase A/química , Hexosaminidase A/metabolismo , Hexosaminidase B/química , Hexosaminidase B/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Mutação/genética , Permeabilidade , Proteínas Proto-Oncogênicas c-akt/metabolismo , Doença de Sandhoff/patologia , Transdução de Sinais/efeitos dos fármacos , Doença de Tay-Sachs/patologia , Transcriptoma/genéticaRESUMO
Inflammation is a hallmark of aging and accelerated aging syndromes such as Hutchinson-Gilford progeria syndrome (HGPS). In this study, we present evidence of increased expression of the components of the NLRP3 inflammasome pathway in HGPS skin fibroblasts, an outcome that was associated with morphological changes of the nuclei of the cells. Lymphoblasts from HGPS patients also showed increased basal levels of NLRP3 and caspase 1. Consistent with these results, the expression of caspase 1 and Nlrp3, but not of the other inflammasome receptors was higher in the heart and liver of Zmpste24-/- mice, which phenocopy the human disease. These data were further corroborated in LmnaG609G/G609G mice, another HGPS animal model. We also showed that pharmacological inhibition of the NLRP3 inflammasome by its selective inhibitor, MCC950, improved cellular phenotype, significantly extended the lifespan of progeroid animals, and reduced inflammasome-dependent inflammation. These findings suggest that inhibition of the NLRP3 inflammasome is a potential therapeutic approach for the treatment of HGPS.
Assuntos
Progéria , Animais , Modelos Animais de Doenças , Humanos , Inflamassomos , Lamina Tipo A/genética , Longevidade , Camundongos , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Progéria/genéticaRESUMO
Inflammation is a hallmark of aging and is negatively affecting female fertility. In this study, we evaluate the role of the NLRP3 inflammasome in ovarian aging and female fertility. Age-dependent increased expression of NLRP3 in the ovary was observed in WT mice during reproductive aging. High expression of NLRP3, caspase-1, and IL-1ß was also observed in granulosa cells from patients with ovarian insufficiency. Ablation of NLRP3 improved the survival and pregnancy rates and increased anti-Müllerian hormone levels and autophagy rates in ovaries. Deficiency of NLRP3 also reduced serum FSH and estradiol levels. Consistent with these results, pharmacological inhibition of NLRP3 using a direct NLRP3 inhibitor, MCC950, improved fertility in female mice to levels comparable to those of Nlrp3-/- mice. These results suggest that the NLRP3 inflammasome is implicated in the age-dependent loss of female fertility and position this inflammasome as a potential new therapeutic target for the treatment of infertility.
RESUMO
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.
Assuntos
Autofagia , Animais , Autofagossomos , Autofagia/fisiologia , Proteínas Relacionadas à Autofagia/metabolismo , Bioensaio/normas , Biomarcadores , Humanos , LisossomosRESUMO
Primary ovarian insufficiency (POI) is a critical fertility defect characterized by anticipated impairment of the follicular reserve, which pathophysiological mechanisms have not yet been identified. We have investigated the possible relationship between AMP-activated protein kinase (AMPK) and the pathophysiology of POI. We studied 35 POI patients with altered levels of follicle-stimulating hormone (FSH) and estradiol and increased percentage of overweight compared with 20 healthy women. Blood mononuclear cells from POI patients showed reduced levels of phosphorylated AMPK, adenosine triphosphate, and high serum levels of leptin and tumor necrosis factor-alpha. All these alterations were also observed in granulosa cells, a more closer environment of the ovary. Significant negative correlations between AMPK and body mass index, FSH and leptin levels, and a positive correlation between AMPK and estradiol levels were observed. Oral metformin supplementation restored biochemical parameters. Interestingly, AMPK levels from patients were negatively correlated with age, suggesting an accelerated aging in POI mediated by the impairment of AMPK. These results lead to the hypothesis that an impairment of AMPK could be implicated in the metabolic pathophysiology of POI indicating at AMPK induction as a possible new therapeutic target.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Suscetibilidade a Doenças , Metabolismo Energético , Insuficiência Ovariana Primária/etiologia , Insuficiência Ovariana Primária/metabolismo , Biomarcadores , Estudos de Casos e Controles , Gerenciamento Clínico , Metabolismo Energético/efeitos dos fármacos , Feminino , Hormônio Foliculoestimulante/sangue , Hormônio Foliculoestimulante/metabolismo , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Metformina/farmacologia , Metformina/uso terapêutico , Folículo Ovariano/metabolismo , Insuficiência Ovariana Primária/diagnóstico , Insuficiência Ovariana Primária/tratamento farmacológicoRESUMO
Aging is the major risk factor for many metabolic chronic diseases. Several metabolic pathways suffer a progressive impairment during aging including body composition and insulin resistance which are associated to autophagy dysfunction and increased inflammation. Many of these alterations are aggravated by non-healthy lifestyle such as obesity and hypercaloric diet which have been shown to accelerate aging. Here, we show that the deleterious effect of hypercaloric diets is reverted by the NLRP3 inflammasome inhibition. NLRP3 deficiency extends mean lifespan of adult mice fed a high-fat diet. This lifespan extension is accompanied by metabolic health benefits including reduced liver steatosis and cardiac damage, improved glucose and lipid metabolism, and improved protein expression profiles of SIRT-1, mTOR, autophagic flux, and apoptosis. These findings suggest that the suppression of NLRP3 prevented many age-associated changes in metabolism impaired by the effect of hypercaloric diets.
Assuntos
Inflamassomos , Longevidade , Proteína 3 que Contém Domínio de Pirina da Família NLR , Animais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos ObesosRESUMO
While NLRP3-inflammasome has been implicated in cardiovascular diseases, its role in physiological cardiac aging is largely unknown. During aging, many alterations occur in the organism, which are associated with progressive impairment of metabolic pathways related to insulin resistance, autophagy dysfunction, and inflammation. Here, we investigated the molecular mechanisms through which NLRP3 inhibition may attenuate cardiac aging. Ablation of NLRP3-inflammasome protected mice from age-related increased insulin sensitivity, reduced IGF-1 and leptin/adiponectin ratio levels, and reduced cardiac damage with protection of the prolongation of the age-dependent PR interval, which is associated with atrial fibrillation by cardiovascular aging and reduced telomere shortening. Furthermore, old NLRP3 KO mice showed an inhibition of the PI3K/AKT/mTOR pathway and autophagy improvement, compared with old wild mice and preserved Nampt-mediated NAD+ levels with increased SIRT1 protein expression. These findings suggest that suppression of NLRP3 prevented many age-associated changes in the heart, preserved cardiac function of aged mice and increased lifespan.
Assuntos
Sistema Cardiovascular/fisiopatologia , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Animais , Longevidade , Masculino , Camundongos , Transdução de SinaisRESUMO
The NLRP3 inflammasome has emerged as an important regulator of metabolic disorders and age-related diseases in NLRP3-deficient mice. In this article, we determine whether, in old mice C57BL6J, the NLRP3 inflammasome inhibitor MCC950 is able to attenuate age-related metabolic syndrome to providing health benefits. We report that MCC950 attenuates metabolic and hepatic dysfunction in aged mice. In addition, MCC950 inhibited the Pi3K/AKT/mTOR pathway, enhanced autophagy, and activated peroxisome proliferator-activated receptor-α in vivo and in vitro. The data suggest that MCC950 mediates the protective effects by the mammalian target of rapamycin inhibition, thus activating autophagy and peroxisome proliferator-activated receptor-α. In conclusion, pharmacological inhibition of NLRP3 in aged mice has a significant impact on health. Thus, NLRP3 may be a therapeutic target of human age-related metabolic syndrome.
Assuntos
Autofagia/efeitos dos fármacos , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , PPAR alfa/efeitos dos fármacos , Sulfonas/farmacologia , Envelhecimento , Animais , Fígado Gorduroso/prevenção & controle , Furanos , Expressão Gênica , Indenos , Lipídeos/sangue , Fígado/metabolismo , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-akt/efeitos dos fármacos , Sulfonamidas , Serina-Treonina Quinases TOR/efeitos dos fármacosRESUMO
Inflammasomes are multiprotein complexes formed and activated after exposure to pathogenic microbes and host danger signals that control the maturation and production of IL-1ß and IL-18. Their implication in different diseases such as cardiovascular, neurodegenerative, psychiatric, and metabolic diseases opens a door to developing new therapeutic perspectives. However, the rapid increase in the knowledge about inflammasomes is associated with their involvement in clinical practice. Two topics open the way to future lines of research: a clinical trial with the new specific inhibitors and the development of diagnostic tools.
Assuntos
Inflamassomos , Humanos , Interleucina-18/imunologia , Interleucina-1beta/imunologiaRESUMO
Activation of the NLRP3 inflammasome, a multiprotein complex, leading to caspase activation with production of proinflammatory IL-1ß represents a major pathway of inflammation. Recent, studies in mice and human patients uncovered several gain-of- function (GOF) mutations in inflammasome sensor proteins that allow inflammasome assembly in the absence of cognate ligands to trigger autoinflammatory syndromes. Cryopyrin-associated periodic syndromes (CAPS) are rare autoinflammatory diseases, comprising a broad disease spectrum with varying severity. CAPS are associated with GOF mutations in the NLRP3 inflammasome and activation of IL-1ß leading to episodes of fever, cutaneous, musculoskeletal, articular, ocular, and neurological symptoms. Here, we review current knowledge on different mutations leading to CAPS and related clinical syndromes. Homologous gene mutations in mice provide insights into the regulation and consequences of the activation of different inflammasomes in several autoinflammatory syndrome. In view of the critical role of IL-1ß in the pathogenesis of autoinflammatory GOF mutations such as CAPS, blockade of the action of IL-1ß is critical. Therapeutic administration of recombinant IL-1 receptor antagonists or monoclonal anti-IL-1ß antibody had a beneficial effect. Furthermore, novel inhibitors of inflammasome complex formation such as MCC950 and related compounds attenuate experimental and clinical disease. The discovery of new GOF mutants of inflammasomes leading to further insights in pathomechanisms and the development of novel inhibitors represent a great challenge.
Assuntos
Síndromes Periódicas Associadas à Criopirina/genética , Mutação com Ganho de Função/genética , Inflamassomos/genética , Multimerização Proteica/genética , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/uso terapêutico , Autoimunidade , Síndromes Periódicas Associadas à Criopirina/imunologia , Síndromes Periódicas Associadas à Criopirina/terapia , Modelos Animais de Doenças , Humanos , Inflamassomos/metabolismo , Inflamação , Interleucina-1beta/metabolismo , Camundongos , Receptores de Interleucina-1/antagonistas & inibidores , Receptores de Interleucina-1/imunologiaRESUMO
The NLRP3-inflammasome complex has emerged as an important component of inflammatory processes in metabolic dysfunction induced by high-caloric diets. In this study, we investigate the molecular mechanisms by which NLRP3 inhibition may attenuate diet-induced cardiac injury. Here we show the cardiac damage induced by high sugar diet (HSD), high fat diet (HFD) or high sugar/fat diet (HSFD) over 15 weeks. Genetic ablation of NLRP3 protected against this damage by autophagy induction and apoptotic control. Furthermore, NLRP3 inhibition by the selective small molecule MCC950 resulted in similar autophagy induction and apoptotic control in hearts after diets. These data were reproduced in THP-1 cells treated with MCC950 and cultured in media supplemented with serum from mice dosed with MCC950 and fed with diets. NLRP3 inhibition exerted beneficial metabolic, and autophagic adaptations in hearts from obesogenic diets. The inhibition of NLRP3 activation may hold promise in the treatment of metabolic and cardiovascular diseases.
Assuntos
Transtorno Depressivo Maior/metabolismo , Transtorno Depressivo Maior/psicologia , Proteína 12 Relacionada à Autofagia/metabolismo , Transtorno Depressivo Maior/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Proteínas Associadas aos Microtúbulos/metabolismo , Pessoa de Meia-Idade , Escalas de Graduação Psiquiátrica , AutorrelatoRESUMO
Major Depressive Disorder (MDD, ICD-10: F-33) is a prevalent illness in which the pathogenic mechanism remains elusive. Recently an important role has been attributed to neuro-inflammation, and specifically the NLRP3-inflammasome complex, in the pathogenesis of MDD. This suggests a key role for immunomodulation as a key pathway in the treatment of this disorder. This study evaluates the involvement of nine common antidepressants in the NLRP3-inflammasome complex (fluoxetine, paroxetine, mianserin, mirtazapine, venlafaxine, desvenlafaxine, amitriptyline, imipramine and agomelatine), both in in vitro THP-1 cells stimulated by ATP, and in a stress-induced depressive animal or MDD patients. Antidepressant treatment induced inflammasome inhibition was observed by decreased serum levels of IL-1ß and IL-18 and decrease of NLRP3 and IL-1ß (p17) protein expression. This was also observed under stress-induced depressive behaviour and inflammasome activation in C57Bl/6 mice in vivo. Deletion of key autophagy mediator Atg5 in embryonic fibroblasts (MEF cells) showed an autophagy dependent-NLRP3-inflammasome inhibition by antidepressant treatment. These results suggest the NLRP3-inflammasome could be a biomarker for antidepressant treatment response in MDD patients, and therefore the monitoring of NLRP3 expression levels and/or IL-1ß/IL-18 release may have clinical value in drug selection. Existing evidence suggests an anti-inflammatory effect of some antidepressants shown by IL-1ß, IL-6 and TNF-α. Our data have shown that antidepressant-mediated autophagy may have a role in restoration of certain metabolic and immunological pathways in MDD patients.
Assuntos
Antidepressivos/uso terapêutico , Autofagia/efeitos dos fármacos , Transtorno Depressivo Maior/tratamento farmacológico , Inflamassomos/antagonistas & inibidores , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Adulto , Animais , Antidepressivos/farmacologia , Linhagem Celular , Transtorno Depressivo Maior/sangue , Transtorno Depressivo Maior/imunologia , Feminino , Humanos , Inflamassomos/imunologia , Interleucina-1beta/sangue , Interleucina-1beta/imunologia , Masculino , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologiaRESUMO
Stress is a complex event that induces disturbances to physiological and psychological homeostasis, and it may have a detrimental impact on certain brain and physiological functions. In the last years, a dual role of the stress effect has been studied in order to elucidate the molecular mechanism by which can induce physiological symptoms after psychological stress exposition and vice versa. In this sense, inflammation has been proposed as an important starring. And in the same line, the inflammasome complex has emerged to give responses because of its role of stress sensor. The implication of the same complex, NLRP3 inflammasome, in different diseases such as cardiovascular, neurodegenerative, psychiatric, and metabolic diseases opens a door to develop new therapeutic perspectives.
Assuntos
Inflamassomos/imunologia , Inflamação/etiologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/imunologia , Estresse Fisiológico , Estresse Psicológico/complicações , Animais , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/imunologia , Humanos , Inflamação/imunologia , Transtornos Mentais/etiologia , Transtornos Mentais/imunologia , Neoplasias/etiologia , Neoplasias/imunologia , Doenças Neurodegenerativas/etiologia , Doenças Neurodegenerativas/imunologia , Estresse Psicológico/imunologiaAssuntos
Fibromialgia/tratamento farmacológico , Fibromialgia/psicologia , Psicotrópicos/uso terapêutico , Ubiquinona/análogos & derivados , Administração Oral , Adulto , Índice de Massa Corporal , Método Duplo-Cego , Fibromialgia/metabolismo , Humanos , Pessoa de Meia-Idade , Resultado do Tratamento , Ubiquinona/uso terapêuticoRESUMO
Conventional cardiovascular risk factors (CVRFs) are accepted to identify asymptomatic individuals with high risk of acute myocardial infarction (AMI). However, AMI affects many patients previously classified at low risk. New biomarkers are needed to improve risk prediction. We propose to evaluate the NLRP3-inflammasome complex as a potential conventional cardiovascular risk (CVR) indicator in healthy males and post-AMI patients and compare both groups by known CVRFs. We included 109 men with no history of cardiovascular disease (controls) and 150 AMI patients attending a cardiac rehabilitation program. AMI patients had higher mean of body mass index (BMI) and waist circumference than the controls. However, high percentages of the controls had a high BMI and a waist circumference >95 cm. The controls also had higher systolic blood pressure (p > 0.001), total and low-density lipoprotein cholesterol, dietary nutrient, and calorific intake. Fuster BEWAT score (FBS) correlated more closely than Framingham risk score (FRS) with most CVRF, groups. However, only the FBS showed a correlation with inflammasome cytokine interleukin 1ß (IL-1ß). Several CVRFs were significantly better in AMI patients; however, this group also had higher mRNA expression of the inflammasome gene NLRP3 and lower expression of the autophagy gene MAP-LC3. The controls had high levels of CVRF, probably reflecting unhealthy lifestyle. FBS reflects the efficiency of strategies to induce lifestyle changes such as cardiac rehabilitation programs, and could provide a sensitive evaluation CVR. These results lead to the hypothesis that NLRP3-inflammasome and associated IL-1ß release have potential as CVR biomarkers, particularly in post-AMI patients with otherwise low risk scores. Antioxid. Redox Signal. 27, 269-275.
Assuntos
Inflamassomos/metabolismo , Infarto do Miocárdio/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Adulto , Idoso , Biomarcadores , Pressão Sanguínea/fisiologia , Índice de Massa Corporal , Humanos , Inflamassomos/sangue , Interleucina-1beta/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Fatores de RiscoAssuntos
Transtorno Depressivo Maior/metabolismo , Expressão Gênica/fisiologia , Biogênese de Organelas , Superóxido Dismutase/metabolismo , Adulto , Citocinas/genética , Citocinas/metabolismo , Transtorno Depressivo Maior/genética , Feminino , Humanos , Interleucina-1beta/metabolismo , Análise em Microsséries , Pessoa de Meia-Idade , Proteínas Mitocondriais/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Escalas de Graduação PsiquiátricaRESUMO
AIMS: Impairment in adenosine monophosphate-activated protein kinase (AMPK) activity and NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation are associated with several metabolic and inflammatory diseases. In this study, we investigated the role of AMPK/NLRP3 inflammasome axis in the molecular mechanism underlying pain perception. RESULTS: Impairment in AMPK activation induced by compound C or sunitinib, two AMPK inhibitors, provoked hyperalgesia in mice (p<0.001) associated with marked NLRP3 inflammasome protein activation and increased serum levels of interleukin-1ß (IL-1ß) (24.56±0.82 pg/ml) and IL-18 (23.83±1.882 pg/ml) compared with vehicle groups (IL-1ß: 8.15±0.44; IL-18: 4.92±0.4). This effect was rescued by increasing AMPK phosphorylation via metformin treatment (p<0.001), caloric restriction diet (p<0.001), or NLRP3 inflammasome genetic inactivation using NLRP3 knockout (nlrp3(-/-)) mice (p<0.001). Deficient AMPK activation and overactivation of NLRP3 inflammasome axis were also observed in blood cells from patients with fibromyalgia (FM), a prevalent human chronic pain disease. In addition, metformin treatment (200 mg/daily), which increased AMPK activation, restored all biochemical alterations examined by us in blood cells and significantly improved clinical symptoms, such as, pain, fatigue, depression, disturbed sleep, and tender points, in patients with FM. INNOVATION AND CONCLUSIONS: These data suggest that AMPK/NLRP3 inflammasome axis participates in chronic pain and that NLRP3 inflammasome inhibition by AMPK modulation may be a novel therapeutic target to fight against chronic pain and inflammatory diseases as FM.