RESUMO
OBJECTIVE: Multiple acyl-coenzyme A dehydrogenase deficiency (MADD) is a disorder of fatty acid oxidation and considered an inborn error of metabolism. In recent years, we have diagnosed an increasing number of patients where, despite extensive investigation, no disease-causing mutations have been found. We therefore investigated a cohort of consecutive patients, with the objective to detect possible non-genetic causes. METHODS: We searched the patient records and the registry of muscle biopsies, for patients with MADD, diagnosed within the past 10 years. The patient records were reviewed regarding symptoms, clinical findings, comorbidities, drugs, diagnostic investigations, and response to treatment. In addition, complementary investigations of muscle tissue were performed. RESULTS: We identified 9 patients diagnosed with late-onset MADD. All presented with muscle weakness and elevated levels of creatine kinase. A lipid storage myopathy was evident in the muscle biopsies, as was elevated acylcarnitines in blood. Despite thorough genetic investigations, a probable genetic cause was found in only 2 patients. Remarkably, all 7 patients without disease-causing mutations were treated with sertraline. In some cases, a deterioration of symptoms closely followed dose increase, and discontinuation resulted in an improved acylcarnitine profile. All 9 patients responded to riboflavin treatment with normalization of creatine kinase and muscle biopsy findings, and in 8 patients the clinical symptoms clearly improved. INTERPRETATION: Our findings strongly suggest that sertraline may induce an acquired form of MADD in some patients. Importantly, riboflavin treatment seems to be similarly effective as in genetic MADD, but discontinuation of sertraline is reasonably warranted. ANN NEUROL 2024.
RESUMO
Alzheimer's disease (AD) is the most common form of dementia, affecting millions of people and currently lacking available disease-modifying treatments. Appropriate disease models are necessary to investigate disease mechanisms and potential treatments. Drosophila melanogaster models of AD include the Aß fly model and the AßPP-BACE1 fly model. In the Aß fly model, the Aß peptide is fused to a secretion sequence and directly overexpressed. In the AßPP-BACE1 model, human AßPP and human BACE1 are expressed in the fly, resulting in in vivo production of Aß peptides and other AßPP cleavage products. Although these two models have been used for almost two decades, the underlying mechanisms resulting in neurodegeneration are not yet clearly understood. In this study, we have characterized toxic mechanisms in these two AD fly models. We detected neuronal cell death and increased protein carbonylation (indicative of oxidative stress) in both AD fly models. In the Aß fly model, this correlates with high Aß1-42 levels and down-regulation of the levels of mRNA encoding lysosomal-associated membrane protein 1, lamp1 (a lysosomal marker), while in the AßPP-BACE1 fly model, neuronal cell death correlates with low Aß1-42 levels, up-regulation of lamp1 mRNA levels and increased levels of C-terminal fragments. In addition, a significant amount of AßPP/Aß antibody (4G8)-positive species, located close to the endosomal marker rab5, was detected in the AßPP-BACE1 model. Taken together, this study highlights the similarities and differences in the toxic mechanisms which result in neuronal death in two different AD fly models. Such information is important to consider when utilizing these models to study AD pathogenesis or screening for potential treatments.
Assuntos
Doença de Alzheimer/metabolismo , Doença de Alzheimer/fisiopatologia , Doença de Alzheimer/genética , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Peptídeos beta-Amiloides/genética , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Proteínas de Drosophila , Drosophila melanogaster , Humanos , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/fisiopatologia , Estresse OxidativoRESUMO
Invited for this month's cover picture is the group of Professor Peter Nilsson at the Department of Physics, Chemistry and Biology at Linköping University (Sweden). The cover picture shows a fluoro-glycoporphyrin that selectively targets cancer cells. The selectivity towards cancer cells are afforded due to proper functionalization of the porphyrin scaffold with specific carbohydrates, and the cancer cells can be visualized because of the intrinsic fluorescence from the porphyrin. The molecule also exhibits properties for photodynamic therapy and the incorporation of fluorine, in the form of 2-fluoro-2-deoxy glucose (FDG), offers the possibility to apply these compounds as 18F positron emission tomography (PET) tracers. Thus, fluoro-glycoporphyrins display multimodal properties and can be employed as theranostic agents targeting cancer cells. Read the full text of their Full Paper at https://doi.org/10.1002/open.201800020.
RESUMO
Small molecules with modalities for a variety of imaging techniques as well as therapeutic activity are essential, as such molecules render opportunities to simultaneously conduct diagnosis and targeted therapy, so called theranostics. In this regard, glycoporphyrins have proven useful as theranostic agents towards cancer, as well as noncancerous conditions. Herein, the synthesis and characterization of heterobifunctional glycoconjugated porphyrins with two different sugar moieties, a common monosaccharide at three sites, and a 2-fluoro-2-deoxy glucose (FDG) moiety at the fourth site are presented. The fluoro-glycoconjugated porphyrins exhibit properties for multimodal imaging and photodynamic therapy, as well as specificity towards cancer cells. We foresee that our findings might aid in the chemical design of heterobifunctional glycoconjugated porphyrins that could be utilized as theranostic agents.
RESUMO
The accumulation of protein aggregates is associated with many devastating neurodegenerative diseases and the development of molecular ligands able to detect these pathological hallmarks is essential. Here, the synthesis of thiophene based optical ligands, denoted bi-thiophene-vinyl-benzothiazoles (bTVBTs) that can be utilized for selective assignment of tau aggregates in brain tissue with Alzheimer's disease (AD) pathology is reported. The ability of the ligands to selectively distinguish tau deposits from the other AD associated pathological hallmark, senile plaques consisting of aggregated amyloid-ß (Aß) peptide, was reduced when the chemical composition of the ligands was altered, verifying that specific molecular interactions between the ligands and the aggregates are necessary for the selective detection of tau deposits. Our findings provide the structural and functional basis for the development of new fluorescent ligands that can distinguish between aggregated proteinaceous species consisting of different proteins. In addition, the bTVBT scaffold might be utilized to create powerful practical research tools for studying the underlying molecular events of tau aggregation and for creating novel agents for clinical imaging of tau pathology in AD.
Assuntos
Doença de Alzheimer/metabolismo , Benzotiazóis/química , Corantes Fluorescentes/química , Tiofenos/química , Proteínas tau/química , Doença de Alzheimer/diagnóstico , Peptídeos beta-Amiloides/química , Peptídeos beta-Amiloides/metabolismo , Benzotiazóis/síntese química , Encéfalo/metabolismo , Corantes Fluorescentes/síntese química , Humanos , Ligantes , Imagem Óptica/métodos , Placa Amiloide/química , Placa Amiloide/metabolismo , Agregados Proteicos , Tiofenos/síntese química , Proteínas tau/metabolismoRESUMO
The acidic environment of the lysosomal lumen provides an optimal milieu for the acid hydrolases and is also essential for fusion/fission of endo-lysosomal compartments and sorting of cargo. Evidence suggests that maintaining lysosomal acidity is essential to avoid disease. In this chapter, we describe a protocol for analyzing the lysosomal pH in cultured cells using the fluorescent probe fluorescein isothiocyanate (FITC)-dextran together with a dual-emission ratiometric technique suitable for flow cytometry. Fluorescence-labeled dextran is endocytosed and accumulated in the lysosomal compartment. FITC shows a pH-dependent variation in fluorescence when analyzed at maximum emission wavelength and no variation when analyzing at the isosbestic point, thereby the ratio can be used to determine the lysosomal pH. A standard curve is obtained by equilibrating intralysosomal pH with extracellular pH using the ionophore nigericin. The protocol also includes information regarding procedures to induce lysosomal alkalinization and lysosomal membrane permeabilization.
Assuntos
Dextranos/química , Fluoresceína-5-Isotiocianato/análogos & derivados , Lisossomos/metabolismo , Animais , Citometria de Fluxo , Fluoresceína-5-Isotiocianato/química , Humanos , Concentração de Íons de Hidrogênio , MacrolídeosRESUMO
Molecular tools for fluorescent imaging of specific compartments in cells are essential for understanding the function and activity of cells. Here, we report the synthesis of a series of pyridyl- and thienyl-substituted phospholes and the evaluation of these dyes for fluorescent imaging of cells. The thienyl-substituted phospholes proved to be successful for staining of cultured normal and malignant cells due to their fluorescent properties and low toxicity. Co-staining experiments demonstrated that these probes target lipid droplets, which are, lipid-storage organelles found in the cytosol of nearly all cell types. Our findings confirm that thienyl-substituted phospholes can be utilized as fluorescent tools for vital staining of cells, and we foresee that these fluorescent dyes might be used in studies to unravel the roles that lipid droplets play in cellular physiology and in diseases.
RESUMO
Altered lipid metabolism and extensive lipid storage in cells have been associated with various medical disorders, including cancer. The development of fluorescent probes that specifically accumulate in lipid deposits is therefore of great interest in order to study pathological processes that are linked to dysregulated lipogenesis. In the present study, we present a small fluorescent benzothiadiazole dye that specifically stains lipid droplets in living and fixated cells. The photophysical characterization of the probe revealed strong solvatochromic behavior, large Stokes shifts, and high fluorescent quantum yields in hydrophobic solvents. In addition, the fluorophore exhibits a nontoxic profile and a high signal-to-noise ratio in cells (i.e., lipid droplets vs cytosol), which make it an excellent candidate for studying lipid biology using confocal fluorescent microscopy.
Assuntos
Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador/métodos , Gotículas Lipídicas/química , Tiadiazóis/química , Humanos , Razão Sinal-Ruído , Espectrometria de FluorescênciaRESUMO
Deposits comprised of amyloid-ß (Aß) are one of the pathological hallmarks of Alzheimer's disease (AD) and small hydrophobic ligands targeting these aggregated species are used clinically for the diagnosis of AD. Herein, we observed that anionic oligothiophenes efficiently displaced X-34, a Congo Red analogue, but not Pittsburgh compoundâ B (PIB) from recombinant Aß amyloid fibrils and Alzheimer's disease brain-derived Aß. Overall, we foresee that the oligothiophene scaffold offers the possibility to develop novel high-affinity ligands for Aß pathology only found in human AD brain, targeting a different site than PIB.
Assuntos
Alcenos/química , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/química , Compostos de Anilina/química , Benzoatos/química , Encéfalo/patologia , Tiazóis/química , Tiofenos/metabolismo , Alcenos/metabolismo , Peptídeos beta-Amiloides/metabolismo , Compostos de Anilina/metabolismo , Benzoatos/metabolismo , Humanos , Tomografia por Emissão de Pósitrons , Tiazóis/metabolismo , Tiofenos/químicaRESUMO
Molecular tools for fluorescent imaging of cells and their components are vital for understanding the function and activity of cells. Here, we report an imidazole functionalized pentameric oligothiophene, p-HTIm, that can be utilized for fluorescent imaging of cells. p-HTIm fluorescence in normal cells appeared in a peripheral punctate pattern partially co-localized with lysosomes, whereas a one-sided perinuclear Golgi associated localization of the dye was observed in malignant cells. The uptake of p-HTIm was temperature dependent and the intracellular target was reached within 1 h after staining. The ability of p-HTIm to stain cells was reduced when the imidazole side chain was chemically altered, verifying that specific imidazole side-chain functionalities are necessary for achieving the observed cellular staining. Our findings confirm that properly functionalized oligothiophenes can be utilized as fluorescent tools for vital staining of cells and that the selectivity toward distinct intracellular targets are highly dependent on the side-chain functionalities along the conjugated thiophene backbone.
RESUMO
Molecular probes for imaging of live cells are of great interest for studying biological and pathological processes. The anionic luminescent conjugated polythiophene (LCP) polythiophene acetic acid (PTAA), has previously been used for vital staining of cultured fibroblasts as well as transformed cells with results indicating differential staining due to cell phenotype. Herein, we investigated the behavior of PTAA in two normal and five transformed cells lines. PTAA fluorescence in normal cells appeared in a peripheral punctated pattern whereas the probe was more concentrated in a one-sided perinuclear localization in the five transformed cell lines. In fibroblasts, PTAA fluorescence was initially associated with fibronectin and after 24 h partially localized to lysosomes. The uptake and intracellular target in malignant melanoma cells was more ambiguous and the intracellular target of PTAA in melanoma cells is still elusive. PTAA was well tolerated by both fibroblasts and melanoma cells, and microscopic analysis as well as viability assays showed no signs of negative influence on growth. Stained cells maintained their proliferation rate for at least 12 generations. Although the probe itself was nontoxic, photoinduced cellular toxicity was observed in both cell lines upon irradiation directly after staining. However, no cytotoxicity was detected when the cells were irradiated 24 h after staining, indicating that the photoinduced toxicity is dependent on the cellular location of the probe. Overall, these studies certified PTAA as a useful agent for vital staining of cells, and that PTAA can potentially be used to study cancer-related biological and pathological processes.
Assuntos
Ácido Acético/análise , Fibroblastos/química , Melanoma/patologia , Polímeros/análise , Coloração e Rotulagem/métodos , Tiofenos/análise , Linhagem Celular Transformada , Células HeLa , Humanos , Sondas Moleculares/análiseRESUMO
Lysosomes are acidic organelles essential for degradation, signalling and cell homoeostasis. In addition, they play a key role in cell death. Permeabilization of the lysosomal membrane and release of hydrolytic enzymes to the cytosol accompanies apoptosis signalling in several systems. The regulatory mechanism of lysosomal stability is, however, poorly understood. Lipophilic or amphiphilic compounds with a basic moiety will become protonated and trapped within lysosomes, and such lysosomotropic behaviour is also found in many pharmacological drugs. The natural sphingolipid sphingosine exhibits lysosomotropic detergent ability and is an endogenous candidate for controlling lysosomal membrane permeabilization. The lysosomotropic properties of certain detergents might be of use in lysosome-targeting anticancer drugs and drug delivery system in the future. The present review summarizes the current knowledge on the targeting and permeabilizing properties of lysosomotropic detergents from a cellular and physicochemical perspective.
Assuntos
Apoptose/efeitos dos fármacos , Membranas Intracelulares/efeitos dos fármacos , Lisossomos/efeitos dos fármacos , Moduladores de Transporte de Membrana/farmacologia , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Fenômenos Químicos , Detergentes/farmacologia , Sistemas de Liberação de Medicamentos , Humanos , Membranas Intracelulares/química , Bicamadas Lipídicas/química , Lisossomos/química , Permeabilidade/efeitos dos fármacos , Esfingosina/farmacologiaRESUMO
BACKGROUND & AIMS: Acid sphingomyelinase (ASMase) is activated in non-alcoholic steatohepatitis (NASH). However, the contribution of ASMase to NASH is poorly understood and limited to hepatic steatosis and glucose metabolism. Here we examined the role of ASMase in high fat diet (HFD)-induced NASH. METHODS: Autophagy, endoplasmic reticulum (ER) stress and lysosomal membrane permeabilization (LMP) were determined in ASMase(-/-) mice fed a HFD. The impact of pharmacological ASMase inhibition on NASH was analyzed in wild type mice fed a HFD. RESULTS: ASMase deficiency determined resistance to hepatic steatosis mediated by a HFD or methionine-choline deficient diet. ASMase(-/-) mice were resistant to HFD-induced hepatic ER stress, but sensitive to tunicamycin-mediated ER stress, indicating selectivity in the resistance of ASMase(-/-) mice to ER stress and steatosis. Autophagic flux, determined in the presence of rapamycin and/or chloroquine, was lower in primary mouse hepatocytes (PMH) from ASMase(-/-) mice and accompanied by increased p62 levels, suggesting autophagic impairment. Moreover, autophagy suppression by chloroquine and brefeldin A caused ER stress in PMH from ASMase(+/+) mice but not in ASMase(-/-) mice. ASMase(-/-) PMH exhibited increased lysosomal cholesterol loading, decreased LMP and apoptosis resistance induced by O-methyl-serine dodecylamide hydrochloride or palmitic acid, effects that were reversed by decreasing cholesterol levels by oxysterol 25-hydroxycholesterol. In vivo pharmacological ASMase inhibition by amitriptyline, a widely used tricyclic antidepressant, protected wild type mice against HFD-induced hepatic steatosis, fibrosis, and liver damage, effects indicative of early-stage NASH. CONCLUSIONS: These findings underscore a critical role for ASMase in diet-induced NASH and suggest the potential of amitriptyline as a treatment for patients with NASH.
Assuntos
Autofagia/fisiologia , Hepatopatia Gordurosa não Alcoólica/enzimologia , Hepatopatia Gordurosa não Alcoólica/prevenção & controle , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/metabolismo , Amitriptilina/farmacologia , Animais , Ceramidas/metabolismo , Colesterol/metabolismo , Deficiência de Colina/complicações , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Humanos , Fígado/metabolismo , Fígado/patologia , Lisossomos/metabolismo , Metionina/deficiência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Hepatopatia Gordurosa não Alcoólica/etiologia , Permeabilidade , Esfingomielina Fosfodiesterase/deficiência , Esfingomielinas/metabolismoRESUMO
The success of future intervention strategies for Alzheimer's disease (AD) will likely rely on the development of treatments starting early in the disease course, before irreversible brain damage occurs. The pre-symptomatic stage of AD occurs at least one decade before the clinical onset, highlighting the need for validated biomarkers that reflect this early period. Reliable biomarkers for AD are also needed in research and clinics for diagnosis, patient stratification, clinical trials, monitoring of disease progression and the development of new treatments. Changes in the lysosomal network, i.e., the endosomal, lysosomal and autophagy systems, are among the first alterations observed in an AD brain. In this study, we performed a targeted search for lysosomal network proteins in human cerebrospinal fluid (CSF). Thirty-four proteins were investigated, and six of them, early endosomal antigen 1 (EEA1), lysosomal-associated membrane proteins 1 and 2 (LAMP-1, LAMP-2), microtubule-associated protein 1 light chain 3 (LC3), Rab3 and Rab7, were significantly increased in the CSF from AD patients compared with neurological controls. These results were confirmed in a validation cohort of CSF samples, and patients with no neurochemical evidence of AD, apart from increased total-tau, were found to have EEA1 levels corresponding to the increased total-tau levels. These findings indicate that increased levels of LAMP-1, LAMP-2, LC3, Rab3 and Rab7 in the CSF might be specific for AD, and increased EEA1 levels may be a sign of general neurodegeneration. These six lysosomal network proteins are potential AD biomarkers and may be used to investigate lysosomal involvement in AD pathogenesis.
Assuntos
Doença de Alzheimer/líquido cefalorraquidiano , Proteínas do Líquido Cefalorraquidiano/líquido cefalorraquidiano , Proteínas de Membrana Lisossomal/líquido cefalorraquidiano , Proteína 2 de Membrana Associada ao Lisossomo/líquido cefalorraquidiano , Lisossomos/química , Proteínas Associadas aos Microtúbulos/líquido cefalorraquidiano , Proteínas de Transporte Vesicular/líquido cefalorraquidiano , Proteínas rab de Ligação ao GTP/líquido cefalorraquidiano , Proteínas rab3 de Ligação ao GTP/líquido cefalorraquidiano , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminas/líquido cefalorraquidiano , Peptídeos beta-Amiloides/líquido cefalorraquidiano , Autofagia , Biomarcadores/líquido cefalorraquidiano , Endossomos/química , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas do Tecido Nervoso/líquido cefalorraquidiano , Fragmentos de Peptídeos/líquido cefalorraquidiano , Fagossomos/química , proteínas de unión al GTP Rab7 , Proteínas tau/líquido cefalorraquidianoRESUMO
Ultraviolet (UV) irradiation is a major environmental carcinogen involved in the development of skin cancer. To elucidate the initial signaling during UV-induced damage in human keratinocytes, we investigated lysosomal exocytosis and apoptosis induction. UVA, but not UVB, induced plasma membrane damage, which was repaired by Ca(2+)-dependent lysosomal exocytosis. The lysosomal exocytosis resulted in extracellular release of cathepsin D and acid sphingomyelinase (aSMase). Two hours after UVA irradiation, we detected activation of caspase-8, which was reduced by addition of anti-aSMAse. Furthermore, caspase-8 activation and apoptosis was reduced by prevention of endocytosis and by the use of cathepsin inhibitors. We conclude that lysosomal exocytosis is part of the keratinocyte response to UVA and is followed by cathepsin-dependent activation of caspase-8. The findings have implications for the understanding of UV-induced skin damage and emphasize that UVA and UVB initiate apoptosis through different signaling pathways in keratinocytes.
Assuntos
Apoptose/efeitos da radiação , Caspase 8/metabolismo , Exocitose , Queratinócitos/enzimologia , Lisossomos/metabolismo , Raios Ultravioleta , Catepsina D/metabolismo , Células Cultivadas , Pré-Escolar , Ativação Enzimática/efeitos da radiação , Humanos , Lactente , Queratinócitos/fisiologia , Queratinócitos/efeitos da radiação , Proteínas de Membrana Lisossomal/metabolismo , Masculino , Estresse Oxidativo , Transporte Proteico , Transdução de SinaisRESUMO
Lysosomes are ubiquitous membrane-bound intracellular organelles with an acidic interior. They are central for degradation and recycling of macromolecules delivered by endocytosis, phagocytosis, and autophagy. In contrast to the rather simplified view of lysosomes as waste bags, nowadays lysosomes are recognized as advanced organelles involved in many cellular processes and are considered crucial regulators of cell homeostasis. The function of lysosomes is critically dependent on soluble lysosomal hydrolases (e.g. cathepsins) as well as lysosomal membrane proteins (e.g. lysosome-associated membrane proteins). This review focuses on lysosomal involvement in digestion of intra- and extracellular material, plasma membrane repair, cholesterol homeostasis, and cell death. Regulation of lysosomal biogenesis and function via the transcription factor EB (TFEB) will also be discussed. In addition, lysosomal contribution to diseases, including lysosomal storage disorders, neurodegenerative disorders, cancer, and cardiovascular diseases, is presented.
Assuntos
Lisossomos/fisiologia , Modelos Biológicos , Apoptose/fisiologia , Membrana Celular/metabolismo , Endocitose , Homeostase , Humanos , Doenças por Armazenamento dos Lisossomos/metabolismo , Lisossomos/metabolismo , Proteínas/metabolismo , Proteínas/fisiologiaRESUMO
Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-ß-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.
Assuntos
Morte Celular/fisiologia , Colesterol/metabolismo , Lisossomos/metabolismo , Neurônios/metabolismo , Androstenos , Western Blotting , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Fibroblastos/metabolismo , Citometria de Fluxo , Humanos , Hidroxicolesteróis , Imuno-Histoquímica , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana Lisossomal/metabolismo , Lisossomos/fisiologia , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Microscopia de Contraste de Fase , Mutação/genética , Proteína C1 de Niemann-Pick , Quinacrina , Estatísticas não Paramétricas , beta-CiclodextrinasRESUMO
Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Bax activator Bid, and translocation of Bax to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.
Assuntos
Apoptose , Proteína Agonista de Morte Celular de Domínio Interatuante com BH3/metabolismo , Catepsina D/metabolismo , Lisossomos/metabolismo , Fenilalanina/metabolismo , Processamento de Proteína Pós-Traducional , Triptofano/metabolismo , Proteínas 14-3-3/metabolismo , Animais , Apoptose/efeitos dos fármacos , Humanos , Membranas Intracelulares/efeitos dos fármacos , Membranas Intracelulares/metabolismo , Lisossomos/efeitos dos fármacos , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Permeabilidade/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Estaurosporina/farmacologia , Especificidade por Substrato/efeitos dos fármacos , Proteína X Associada a bcl-2/metabolismoRESUMO
In the past decade, lysosomal membrane permeabilization (LMP) has emerged as a significant component of cell death signaling. The mechanisms by which lysosomal stability is regulated are not yet fully understood, but changes in the lysosomal membrane lipid composition have been suggested to be involved. Our aim was to investigate the importance of cholesterol in the regulation of lysosomal membrane permeability and its potential impact on apoptosis. Treatment of normal human fibroblasts with U18666A, an amphiphilic drug that inhibits cholesterol transport and causes accumulation of cholesterol in lysosomes, rescued cells from lysosome-dependent cell death induced by the lysosomotropic detergent O-methyl-serine dodecylamide hydrochloride (MSDH), staurosporine (STS), or cisplatin. LMP was decreased by pretreating cells with U18666A, and there was a linear relationship between the cholesterol content of lysosomes and their resistance to permeabilization induced by MSDH. U18666A did not induce changes in expression or localization of 70-kDa heat shock proteins (Hsp70) or antiapoptotic Bcl-2 proteins known to protect the lysosomal membrane. Induction of autophagy also was excluded as a contributor to the protective mechanism. By using Chinese hamster ovary (CHO) cells with lysosomal cholesterol overload due to a mutation in the cholesterol transporting protein Niemann-Pick type C1 (NPC1), the relationship between lysosomal cholesterol accumulation and protection from lysosome-dependent cell death was confirmed. Cholesterol accumulation in lysosomes attenuates apoptosis by increasing lysosomal membrane stability.
Assuntos
Colesterol/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Amidas/farmacologia , Androstenos/farmacologia , Animais , Autofagia/efeitos dos fármacos , Células CHO , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Cisplatino/farmacologia , Cricetinae , Cricetulus , Citoproteção/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Lisossomos/efeitos dos fármacos , Glicoproteínas de Membrana/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Substâncias Protetoras/farmacologia , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Serina/análogos & derivados , Serina/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Proteína bcl-X/metabolismoRESUMO
The Niemann-Pick type C1 (NPC1) protein mediates the trafficking of cholesterol from lysosomes to other organelles. Mutations in the NPC1 gene lead to the retention of cholesterol and other lipids in the lysosomal compartment, and such defects are the basis of NPC disease. Several parallels exist between NPC disease and Alzheimer's disease (AD), including altered cholesterol homeostasis, changes in the lysosomal system, neurofibrillary tangles, and increased amyloid-beta generation. How the expression of NPC1 in the human brain is affected in AD has not been investigated so far. In the present study, we measured NPC1 mRNA and protein expression in three distinct regions of the human brain, and we revealed that NPC1 expression is upregulated at both mRNA and protein levels in the hippocampus and frontal cortex of AD patients compared to control individuals. In the cerebellum, a brain region that is relatively spared in AD, no difference in NPC1 expression was detected. Similarly, murine NPC1 mRNA levels were increased in the hippocampus of 12-month-old transgenic mice expressing a familial AD form of human amyloid-beta precursor protein (APP) and presenilin-1 (APP/PS1tg) compared to 12-month-old wild type mice, whereas no change in NPC1 was detected in mouse cerebellum. Immunohistochemical analysis of human hippocampus indicated that NPC1 expression was strongest in neurons. However, in vitro studies revealed that NPC1 expression was not induced by transfecting SK-N-SH neurons with human APP or by treating them with oligomeric amyloid-beta peptide. Total cholesterol levels were reduced in hippocampus from AD patients compared to control individuals, and it is therefore possible that the increased expression of NPC1 is linked to perturbed cholesterol homeostasis in AD.