RESUMEN
KEAP1 is a cytoplasmic protein that functions as an adaptor for the Cullin-3-based ubiquitin E3 ligase system, which regulates the degradation of many proteins, including NFE2L2/NRF2 and p62/SQSTM1. Loss of KEAP1 leads to an accumulation of protein ubiquitin aggregates and defective autophagy. To better understand the role of KEAP1 in the degradation machinery, we investigated whether Keap1 deficiency affects the endosome-lysosomal pathway. We used KEAP1-deficient mouse embryonic fibroblasts (MEFs) and combined Western blot analysis and fluorescence microscopy with fluorometric and pulse chase assays to analyze the levels of lysosomal-endosomal proteins, lysosomal function, and autophagy activity. We found that the loss of keap1 downregulated the protein levels and activity of the cathepsin D enzyme. Moreover, KEAP1 deficiency caused lysosomal alterations accompanied by an accumulation of autophagosomes. Our study demonstrates that KEAP1 deficiency increases nondegradative lysosomes and identifies a new role for KEAP1 in lysosomal function that may have therapeutic implications.
RESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder with poorly understood etiology. Increasing evidence suggests that age-dependent compromise of the maintenance of mitochondrial function is a key risk factor. Several proteins encoded by PD-related genes are associated with mitochondria including PTEN-induced putative kinase 1 (PINK1), which was first identified as a gene that is upregulated by PTEN. Loss-of-function PINK1 mutations induce mitochondrial dysfunction and, ultimately, neuronal cell death. To mitigate the negative effects of altered cellular functions cells possess a degradation mechanism called autophagy for recycling damaged components; selective elimination of dysfunctional mitochondria by autophagy is termed mitophagy. Our study indicates that autophagy and mitophagy are upregulated in PINK1-deficient cells, and is the first report to demonstrate efficient fluxes by one-step analysis. We propose that autophagy is induced to maintain cellular homeostasis under conditions of non-regulated mitochondrial quality control.
RESUMEN
We characterized the dynamics of autophagy in vitro using four different cell systems and analyzing markers widely used in this field, i.e. LC3 (microtubule-associated protein 1 light chain 3; protein recruited from the cytosol (LC3-I) to the autophagosomal membrane where it is lipidated (LC3-II)) and p62/SQSTM1 (adaptor protein that serves as a link between LC3 and ubiquitinated substrates), (Klionsky et al., 2016) [1]. Data provided include analyses of protein levels of LC3 and p62 by Western-blotting and endogenous immunofluorescence experiments, but also p62 mRNA levels obtained by quantitative PCR (qPCR). To monitor the turnover of these autophagy markers and, thus, measure the flux of this pathway, cells were under starvation conditions and/or treated with bafilomycin A1 (Baf. A1) to block fusion of autophagosomes with lysosomes.
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Most laboratories interested in autophagy use different imaging software for managing and analyzing heterogeneous parameters in immunofluorescence experiments (e.g., LC3-puncta quantification and determination of the number and size of lysosomes). One solution would be software that works on a user's laptop or workstation that can access all image settings and provide quick and easy-to-use analysis of data. Thus, we have designed and implemented an application called IFDOTMETER, which can run on all major operating systems because it has been programmed using JAVA (Sun Microsystems). Briefly, IFDOTMETER software has been created to quantify a variety of biological hallmarks, including mitochondrial morphology and nuclear condensation. The program interface is intuitive and user-friendly, making it useful for users not familiar with computer handling. By setting previously defined parameters, the software can automatically analyze a large number of images without the supervision of the researcher. Once analysis is complete, the results are stored in a spreadsheet. Using software for high-throughput cell image analysis offers researchers the possibility of performing comprehensive and precise analysis of a high number of images in an automated manner, making this routine task easier.
Asunto(s)
Técnica del Anticuerpo Fluorescente/métodos , Procesamiento de Imagen Asistido por Computador/métodos , Programas Informáticos , Ensayos Analíticos de Alto RendimientoRESUMEN
The pathogenesis of neurodegenerative diseases involves altered activity of proteolytic systems and accumulation of protein aggregates. Autophagy is an intracellular process in which damaged organelles and long-lived proteins are degraded and recycled for maintaining normal cellular homeostasis. Disruption of autophagic activity in neurons leads to modify the cellular homeostasis, causing deficient elimination of abnormal and toxic protein aggregates that promotes cellular stress and death. Therefore, induction of autophagy has been proposed as a reasonable strategy to help neurons to clear abnormal protein aggregates and survive. This review aims to give an overview of some of the main modulators of autophagy that are currently being studied as possible alternatives in the search of therapies that slow the progression of neurodegenerative diseases, which are incurable to date.
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Autofagia/efectos de los fármacos , Autofagia/fisiología , Isotiocianatos/farmacología , Enfermedades Neurodegenerativas/tratamiento farmacológico , Estilbenos/farmacología , Trehalosa/farmacología , Animales , Modelos Animales de Enfermedad , Alimentos , Humanos , Litio/farmacología , Resveratrol , Sirolimus/farmacología , Espermidina/farmacología , Sulfóxidos , Ácido Valproico/farmacologíaRESUMEN
At present, the analysis of autophagic flux by Western blotting (WB), which measures two of the most important markers of autophagy, i.e., microtubule-associated protein 1 light chain 3 (LC3) and p62, is widely accepted in the scientific community. In this study, we addressed the possible disadvantages and limitations that this method presents for a correct interpretation of the results according to the lysis buffer used for extracting proteins. Here, we tested the LC3 and p62 protein levels by WB in four cell models (mouse embryonic and human fibroblasts (MEFs and HFs, respectively), N27 rat mesencephalic dopaminergic neurons and SH-SY5Y human neuroblastoma cells). The cells were exposed to the autophagy inhibitor bafilomycin A1 (Baf. A1) in combination (or not) with nutrient deprivation to induce autophagy, and they were lysed by using four different buffers (nonyl phenoxypolyethoxylethanol (NP-40), radioimmunoprecipitation assay (RIPA), Triton X-100, and sample buffer (SB) 1×). Based on our observations, we want to highlight that this technique is not always appropriate for analyzing and monitoring autophagy. In this report, we show conflicting data that hinder the correct interpretation of the results, especially in relation to p62 protein levels, at least in the models studied in this work.
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Autofagia , Western Blotting/métodos , Animales , Biomarcadores/metabolismo , Línea Celular , Humanos , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Unión al ARN/metabolismo , RatasRESUMEN
Parkinson's disease (PD) is a neurodegenerative disorder of unknown etiology. It is considered as a multifactorial disease dependent on environmental and genetic factors. Deregulation in cell degradation has been related with a significant increase in cell damage, becoming a target for studies on the PD etiology. In the present study, we have characterized the parkinsonian toxin 1-methyl-4-phenylpyridinium ion (MPP(+))-induced damage in fibroblasts from Parkinson's patients with the mutation G2019S in leucine-rich repeat kinase 2 protein (LRRK2) and control individuals without this mutation. The results reveal that MPP(+) induces mTOR-dependent autophagy in fibroblasts. Moreover, the effects of caspase-dependent cell death to MPP(+) were higher in cells with the G2019S LRRK2 mutation, which showed basal levels of autophagy due to the G2019S LRRK2 mutation (mTOR-independent). The inhibition of autophagy by 3-methyladenine (3-MA) treatment reduces these sensitivity differences between both cell types, however, the inhibition of autophagosome-lysosome fusion by bafilomycin A1 (Baf A1) increases these differences. This data confirm the importance of the combination of genetic and environmental factors in the PD etiology. Thereby, the sensitivity to the same damage may be different in function of a genetic predisposition, reason why individuals with certain mutations can develop some early-onset diseases, such as individuals with G2019S LRRK2 mutation and PD.
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1-Metil-4-fenilpiridinio/toxicidad , Autofagia/efectos de los fármacos , Fibroblastos/efectos de los fármacos , Mutación , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Proteínas Serina-Treonina Quinasas/genética , Adenina/análogos & derivados , Adenina/farmacología , Estudios de Casos y Controles , Caspasas/metabolismo , Células Cultivadas , Relación Dosis-Respuesta a Droga , Fibroblastos/enzimología , Fibroblastos/patología , Interacción Gen-Ambiente , Predisposición Genética a la Enfermedad , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Lisosomas/patología , Macrólidos/farmacología , Enfermedad de Parkinson/patología , Fenotipo , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Riesgo , Serina-Treonina Quinasas TOR/metabolismo , TransfecciónRESUMEN
Mutations of the PTEN-induced kinase 1 (PINK1) gene are a cause of autosomal recessive Parkinson's disease (PD). This gene encodes a mitochondrial serine/threonine kinase, which is partly localized to mitochondria, and has been shown to play a role in protecting neuronal cells from oxidative stress and cell death, perhaps related to its role in mitochondrial dynamics and mitophagy. In this study, we report that increased mitochondrial PINK1 levels observed in human neuroblastoma SH-SY5Y cells after carbonyl cyanide m-chlorophelyhydrazone (CCCP) treatment were due to de novo protein synthesis, and not just increased stabilization of full length PINK1 (FL-PINK1). PINK1 mRNA levels were significantly increased by 4-fold after 24h. FL-PINK1 protein levels at this time point were significantly higher than vehicle-treated, or cells treated with CCCP for 3h, despite mitochondrial content being decreased by 29%. We have also shown that CCCP dissipated the mitochondrial membrane potential (Δψm) and induced entry of extracellular calcium through L/N-type calcium channels. The calcium chelating agent BAPTA-AM impaired the CCCP-induced PINK1 mRNA and protein expression. Furthermore, CCCP treatment activated the transcription factor c-Fos in a calcium-dependent manner. These data indicate that PINK1 expression is significantly increased upon CCCP-induced mitophagy in a calcium-dependent manner. This increase in expression continues after peak Parkin mitochondrial translocation, suggesting a role for PINK1 in mitophagy that is downstream of ubiquitination of mitochondrial substrates. This sensitivity to intracellular calcium levels supports the hypothesis that PINK1 may also play a role in cellular calcium homeostasis and neuroprotection.
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Calcio/metabolismo , Expresión Génica , Mitocondrias/enzimología , Mitocondrias/metabolismo , Proteínas Quinasas/metabolismo , Autofagia/efectos de los fármacos , Carbonil Cianuro m-Clorofenil Hidrazona/toxicidad , Línea Celular Tumoral , Humanos , Mitocondrias/efectos de los fármacos , Mitofagia/efectos de los fármacos , Mitofagia/fisiología , Neuroblastoma/enzimología , Neuroblastoma/metabolismo , Proteínas Quinasas/genética , Proteínas Proto-Oncogénicas c-fos/metabolismo , Ionóforos de Protónes/toxicidadRESUMEN
The Centers for Medicare & Medicaid Services has implemented 3 prevention interventions programs to bring diabetes self-management education to vulnerable populations via Medicare's Quality Improvement Organizations. The programs and the lessons derived from a Federal initiative geared to closing the health disparities gap are described.
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Centers for Medicare and Medicaid Services, U.S. , Diabetes Mellitus/prevención & control , Disparidades en Atención de Salud/normas , Desarrollo de Programa , Disparidades en Atención de Salud/estadística & datos numéricos , Humanos , Mejoramiento de la Calidad , Estados UnidosRESUMEN
Mutations in leucine-rich repeat kinase 2 (LRRK2) are a major cause of familial Parkinsonism, and the G2019S mutation of LRRK2 is one of the most prevalent mutations. The deregulation of autophagic processes in nerve cells is thought to be a possible cause of Parkinson's disease (PD). In this study, we observed that G2019S mutant fibroblasts exhibited higher autophagic activity levels than control fibroblasts. Elevated levels of autophagic activity can trigger cell death, and in our study, G2019S mutant cells exhibited increased apoptosis hallmarks compared to control cells. LRRK2 is able to induce the phosphorylation of MAPK/ERK kinases (MEK). The use of 1,4-diamino-2,3-dicyano-1,4-bis[2-aminophenylthio]butadiene (U0126), a highly selective inhibitor of MEK1/2, reduced the enhanced autophagy and sensibility observed in G2019S LRRK2 mutation cells. These data suggest that the G2019S mutation induces autophagy via MEK/ERK pathway and that the inhibition of this exacerbated autophagy reduces the sensitivity observed in G2019S mutant cells.
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Autofagia/genética , Sistema de Señalización de MAP Quinasas , Proteínas Serina-Treonina Quinasas/genética , Anciano , Sustitución de Aminoácidos , Células Cultivadas , Inhibidores Enzimáticos/farmacología , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/enzimología , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Macrólidos/farmacología , Masculino , Persona de Mediana Edad , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Fosforilación , ATPasas de Translocación de Protón/antagonistas & inhibidoresRESUMEN
Huntington's disease (HD) is a neurodegenerative disorder caused by a mutation in the gene encoding the huntingtin protein. Although the precise mechanism by which neuronal degeneration occurs is still unclear, several elements are important to its development: (1) altered gene expression and protein synthesis, (2) mitochondrial damage and (3) improper regulation of the autophagy programme. In this issue of British Journal of Pharmacology, Galindo and co-workers provide the first evidence for a role of the mitochondrial permeability transition pore (mPTP) in mitochondrial fragmentation and autophagy activation. In a model of cell death induced by 3-nitropropionic acid (3-NP) in human neural cells, the authors describe clear functions for mPTP and Bax, but not the mitochondrial fusion/fission machinery, mitochondrial fragmentation and autophagy (mitophagy). This commentary summarises the significance of this relationship and suggests several points for future development.
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Autofagia/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas de Transporte de Membrana Mitocondrial/biosíntesis , Proteínas de Transporte de Membrana Mitocondrial/efectos de los fármacos , Nitrocompuestos/farmacología , Propionatos/farmacología , Animales , Humanos , Masculino , Poro de Transición de la Permeabilidad MitocondrialRESUMEN
Parkinson's disease is the second common neurodegenerative disorder, after Alzheimer's disease. It is a clinical syndrome characterized by loss of dopamine-generating cells in the substancia nigra, a region of the midbrain. The etiology of Parkinson's disease has long been through to involve both genetic and environmental factors. Mutations in the leucine-rich repeat kinase 2 gene cause late-onset Parkinson's disease with a clinical appearance indistinguishable from Parkinson's disease idiopathic. Autophagy is an intracellular catabolic mechanism whereby a cell recycles or degrades damage proteins and cytoplasmic organelles. This degradative process has been associated with cellular dysfunction in neurodegenerative processes including Parkinson's disease. We discuss the role of leucine-rich repeat kinase 2 in autophagy, and how the deregulations of this degradative mechanism in cells can be implicated in the Parkinson's disease etiology.
RESUMEN
PD (Parkinson's disease) is a neurodegenerative disorder caused by loss of dopamine-generating cells in the substantia nigra. The implication of genetic factors in the aetiology of PD has an essential importance in our understanding of the development of the disease. Mutations in the LRRK2 (leucine-rich repeat kinase 2) gene cause late-onset PD with a clinical appearance indistinguishable from idiopathic PD. Moreover, LRRK2 has been associated with the process of autophagy regulation. Autophagy is an intracellular catabolic mechanism whereby a cell recycles or degrades damaged proteins and cytoplasmic organelles. In the present paper, we discuss the role of LRRK2 in autophagy, and the importance of this relationship in the development of nigral degeneration in PD.
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Autofagia , Enfermedad de Parkinson/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mutación , Enfermedad de Parkinson/enzimología , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genéticaRESUMEN
The link between the deregulation of autophagy and cell death processes can be essential in the development of several neurodegenerative diseases, such as Parkinson disease (PD). However, the molecular mechanism of deregulation of this degradative process in PD patients is unknown. The leucine-rich repeat kinase 2 (LRRK2) gene is related to PD and its implication in autophagy regulation has been described. Our recent work shows that the presence of the G2019S LRRK2 mutation, one of the most prevalent in LRRK2, is accompanied by a deregulation of autophagy basal levels dependent on the MAPK1/3 (ERK2/1) pathway.
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Autofagia , Fibroblastos/enzimología , Sistema de Señalización de MAP Quinasas , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Mutación/genética , Proteínas Serina-Treonina Quinasas/genética , Sustitución de Aminoácidos , Fibroblastos/patología , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Modelos Biológicos , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Fipronil is a phenylpyrazole insecticide known to elicit neurotoxicity via an interaction with ionotropic receptors, namely GABA and glutamate receptors. Recently, we showed that fipronil and other phenylpyrazole compounds trigger cell death in Caco-2 cells. In this study, we investigated the mode of action and the type of cell death induced by fipronil in SH-SY5Y human neuroblastoma cells. Flow cytometric and western blot analyses demonstrated that fipronil induces cellular events belonging to the apoptosis process, such as mitochondrial potential collapse, cytochrome c release, caspase-3 activation, nuclear condensation and phosphatidylserine externalization. In addition, fipronil induces a rapid ATP depletion with concomitant activation of anaerobic glycolysis. This cellular response is characteristic of mitochondrial injury associated with a defect of the respiration process. Therefore, we also investigated the effect of fipronil on the oxygen consumption in isolated mitochondria. Interestingly, we show for the first time that fipronil is a strong uncoupler of oxidative phosphorylation at relative low concentrations. Thus in this study, we report a new mode of action by which the insecticide fipronil could triggers apoptosis.
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Apoptosis/efectos de los fármacos , Insecticidas/toxicidad , Neuronas/efectos de los fármacos , Fosforilación Oxidativa/efectos de los fármacos , Pirazoles/toxicidad , Desacopladores/toxicidad , Adenosina Trifosfato/metabolismo , Western Blotting , Caspasa 3/metabolismo , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Citocromos c/metabolismo , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Glucólisis/efectos de los fármacos , Humanos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Mitocondrias/patología , Neuronas/metabolismo , Neuronas/patología , Fosfatidilserinas/metabolismo , Factores de TiempoRESUMEN
O pênfigo vulgar é uma doença mucocutânea, imunomediada, caracterizada por lesões vesiculobolhosas, enquanto a infecção pelo vírus herpes simples (HSV) é comum na cavidade oral. A coexistência das duas doenças tem sido relatada por alguns autores. Este artigo relata o caso de um paciente com múltiplas lesões em várias áreas da mucosa oral, cujo procedimento foi raspagem e biópsia incisional, que resultou no diagnóstico de pênfigo vulgar associado à infecção pelo HSV. Destaca-se a inusitada associação das doenças e a identificação citopatológica de duas populações celulares com aspectos morfológicos distintos e característicos, capazes de determinar o correto diagnóstico, sendo fundamental para a conduta e terapêutica adequada.
Pemphigus vulgaris is an autoimmune mucocutaneous disease, characterized by vesiculobullous lesions. Herpes simplex virus (HSV) infection is common in the oral cavity and the coexistence of pemphigus vulgaris and HSV infection has been reported by some authors. In this work, we report a case of a patient with multiple lesions involving several areas of the oral mucous membrane. Based on scraping cytology and incisional biopsy findings, the diagnosis was pemphigus vulgaris associated with HSV infection. We call attention to the uncommon association of both diseases and the cytological identification of two cell populations with different and characteristic morphological aspects, able enough to establish the correct diagnosis and define an appropriate therapeutic approach.
RESUMEN
O Pênfigo vulgar é um tipo de doença mucocutânea de origem auto-imune. As lesões orais, na maioria dos casos, são as primeiras manifestações. O objetivo deste trabalho foi verificar a prevalência das lesões de pênfigo vulgar do serviço de anatomia Patológica do Hospital Universitário Antônio Pedro (HUAP) diagnosticadas no período entre os anos de 1993 e 2003, bem como sua distribuição quanto ao sexo, localização da biópsia e faixa etária. Ambos os sexos foram igualmente acometidos; pele, gengiva, mucosa labial, mucosa bucal e palato mole foram as regiões biopsiadas. A maioria dos pacientes estava na faixa etária dos cinquenta anos, conforme a literatura.