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1.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638703

RESUMO

The peri-infarct region, which surrounds the irreversible ischemic stroke area is named ischemic penumbra. This term emphasizes the borderline conditions for neurons placed within such a critical region. Area penumbra separates the ischemic core, where frank cell loss occurs, from the surrounding healthy brain tissue. Within such a brain region, nervous matter, and mostly neurons are impaired concerning metabolic conditions. The classic biochemical marker, which reliably marks area penumbra is the over-expression of the heat shock protein 70 (HSP70). However, other proteins related to cell clearing pathways are modified within area penumbra. Among these, autophagy proteins like LC3 increase in a way, which recapitulates Hsp70. In contrast, components, such as P20S, markedly decrease. Despite apparent discrepancies, the present study indicates remarkable overlapping between LC3 and P20S redistribution within area penumbra. In fact, the amount of both proteins is markedly reduced within vacuoles. Specifically, a massive loss of LC3 + P20S immuno-positive vacuoles (autophagoproteasomes) is reported here. This represents the most relevant sub-cellular alteration here described in cell clearing pathways within area penumbra. The functional significance of these findings remains to be determined and it will take a novel experimental stream to decipher the fine-tuning of such a phenomenon.


Assuntos
Autofagossomos , Autofagia , Proteínas de Choque Térmico HSP70/metabolismo , AVC Isquêmico , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Biomarcadores/metabolismo , AVC Isquêmico/metabolismo , AVC Isquêmico/patologia , Masculino , Camundongos
2.
Nutrients ; 13(10)2021 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-34684361

RESUMO

Lactoferrin (LF) was used at first as a vehicle to deliver non-soluble active compounds to the body, including the central nervous system (CNS). Nonetheless, it soon became evident that, apart from acting as a vehicle, LF itself owns active effects in the CNS. In the present study, the effects of LF are assessed both in baseline conditions, as well as to counteract methamphetamine (METH)-induced neurodegeneration by assessing cell viability, cell phenotype, mitochondrial status, and specific autophagy steps. In detail, cell integrity in baseline conditions and following METH administration was carried out by using H&E staining, Trypan blue, Fluoro Jade B, and WST-1. Western blot and immuno-fluorescence were used to assess the expression of the neurofilament marker ßIII-tubulin. Mitochondria were stained using Mito Tracker Red and Green and were further detailed and quantified by using transmission electron microscopy. Autophagy markers were analyzed through immuno-fluorescence and electron microscopy. LF counteracts METH-induced degeneration. In detail, LF significantly attenuates the amount of cell loss and mitochondrial alterations produced by METH; and mitigates the dissipation of autophagy-related proteins from the autophagy compartment, which is massively induced by METH. These findings indicate a protective role of LF in the molecular mechanisms of neurodegeneration.


Assuntos
Autofagia , Lactoferrina/farmacologia , Metanfetamina/toxicidade , Mitocôndrias/metabolismo , Substâncias Protetoras/farmacologia , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Catepsina D/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lactoferrina/administração & dosagem , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Fusão de Membrana/efeitos dos fármacos , Metanfetamina/administração & dosagem , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Células PC12 , Fenótipo , Ratos , Fatores de Tempo , Tubulina (Proteína)/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
3.
Ecotoxicol Environ Saf ; 221: 112438, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34175825

RESUMO

Microcystin-leucine arginine (MCLR), a widespread environmental contaminant produced by cyanobacteria, poses a severe threat to the male reproductive system. However, the mechanisms of MCLR-induced testis injury accompanied by autophagy are still obscure. This study aimed to investigate the effects of MCLR on autophagy and apoptosis on the male reproductive system and its mechanism both in vitro and in vivo. MCLR caused damage to the testis of zebrafish, resulting in decreased hatching and growth retardation in the offspring. It also remarkably enhanced autophagic flux by elevating the expression of LC3BII, ATG5, and ATG12 proteins. The autophagic flux was also confirmed through the formation of autophagosomes in the ultrastructure of the zebrafish testis and the accumulation of LC3-positive puncta in zebrafish testis and mouse TM4 cells. Further evaluations revealed that inhibition of autophagy by 3-methyladenine (3-MA) significantly attenuated MCLR-induced apoptosis. This finding indicated that autophagy plays an essential role in cell death in the male reproductive system. Besides, inhibiting endoplasmic reticulum (ER) stress using 4-phenylbutyrate (4-PBA) remarkably blocked autophagy and partially suppressed apoptosis in TM4 cells induced by MCLR. This phenomenon suggested that ER stress-related autophagy was involved in MCLR-induced apoptosis. This study reveals crosstalk between ER stress and autophagy via the PERK/eIF2α/ATF4 signaling pathway. It further suggests that ER stress-related autophagy contributes to MCLR-induced apoptosis and injury in the male reproductive system. These findings provide a novel insight into MCLR-induced impairments of the testis.


Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Microcistinas/toxicidade , Testículo/efeitos dos fármacos , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular , Masculino , Camundongos , Fenilbutiratos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Testículo/ultraestrutura , Peixe-Zebra
4.
J Leukoc Biol ; 110(4): 629-649, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34085299

RESUMO

Despite the important function of neutrophils in the eradication of infections and induction of inflammation, the molecular mechanisms regulating the activation and termination of the neutrophil immune response is not well understood. Here, the function of the small GTPase from the RGK family, Gem, is characterized as a negative regulator of the NADPH oxidase through autophagy regulation. Gem knockout (Gem KO) neutrophils show increased NADPH oxidase activation and increased production of extracellular and intracellular reactive oxygen species (ROS). Enhanced ROS production in Gem KO neutrophils was associated with increased NADPH oxidase complex-assembly as determined by quantitative super-resolution microscopy, but normal exocytosis of gelatinase and azurophilic granules. Gem-deficiency was associated with increased basal autophagosomes and autolysosome numbers but decreased autophagic flux under phorbol ester-induced conditions. Neutrophil stimulation triggered the localization of the NADPH oxidase subunits p22phox and p47phox at LC3-positive structures suggesting that the assembled NADPH oxidase complex is recruited to autophagosomes, which was significantly increased in Gem KO neutrophils. Prevention of new autophagosome formation by treatment with SAR405 increased ROS production while induction of autophagy by Torin-1 decreased ROS production in Gem KO neutrophils, and also in wild-type neutrophils, suggesting that macroautophagy contributes to the termination of NADPH oxidase activity. Autophagy inhibition decreased NETs formation independently of enhanced ROS production. NETs production, which was significantly increased in Gem-deficient neutrophils, was decreased by inhibition of both autophagy and calmodulin, a known GEM interactor. Intracellular ROS production was increased in Gem KO neutrophils challenged with live Gram-negative bacteria Pseudomonas aeruginosa or Salmonella Typhimurium, but phagocytosis was not affected in Gem-deficient cells. In vivo analysis in a model of Salmonella Typhimurium infection indicates that Gem-deficiency provides a genetic advantage manifested as a moderate increased in survival to infections. Altogether, the data suggest that Gem-deficiency leads to the enhancement of the neutrophil innate immune response by increasing NADPH oxidase assembly and NETs production and that macroautophagy differentially regulates ROS and NETs in neutrophils.


Assuntos
Armadilhas Extracelulares/metabolismo , Macroautofagia , Proteínas Monoméricas de Ligação ao GTP/metabolismo , NADPH Oxidases/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Calmodulina/metabolismo , Modelos Animais de Doenças , Espaço Intracelular/metabolismo , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Monoméricas de Ligação ao GTP/deficiência , Ativação de Neutrófilo , Neutrófilos/metabolismo , Neutrófilos/ultraestrutura , Pseudomonas aeruginosa/fisiologia , Espécies Reativas de Oxigênio/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Salmonella typhimurium/fisiologia
5.
Nat Commun ; 12(1): 2587, 2021 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-33972537

RESUMO

Host cells use several anti-bacterial pathways to defend against pathogens. Here, using a uropathogenic Escherichia coli (UPEC) infection model, we demonstrate that bacterial infection upregulates RhoB, which subsequently promotes intracellular bacteria clearance by inducing LC3 lipidation and autophagosome formation. RhoB binds with Beclin 1 through its residues at 118 to 140 and the Beclin 1 CCD domain, with RhoB Arg133 being the key binding residue. Binding of RhoB to Beclin 1 enhances the Hsp90-Beclin 1 interaction, preventing Beclin 1 degradation. RhoB also directly interacts with Hsp90, maintaining RhoB levels. UPEC infections increase RhoB, Beclin 1 and LC3 levels in bladder epithelium in vivo, whereas Beclin 1 and LC3 levels as well as UPEC clearance are substantially reduced in RhoB+/- and RhoB-/- mice upon infection. We conclude that when stimulated by UPEC infections, host cells promote UPEC clearance through the RhoB-Beclin 1-HSP90 complex, indicating RhoB may be a useful target when developing UPEC treatment strategies.


Assuntos
Autofagossomos/metabolismo , Proteína Beclina-1/metabolismo , Infecções por Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP90/metabolismo , Infecções Urinárias/metabolismo , Escherichia coli Uropatogênica/crescimento & desenvolvimento , Proteína rhoB de Ligação ao GTP/metabolismo , Animais , Autofagossomos/genética , Autofagossomos/ultraestrutura , Proteína Beclina-1/genética , Linhagem Celular , Epitélio/metabolismo , Epitélio/microbiologia , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Técnicas de Silenciamento de Genes , Proteínas de Choque Térmico HSP90/genética , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica , Estabilidade Proteica , RNA Interferente Pequeno , Proteínas Recombinantes , Bexiga Urinária/metabolismo , Bexiga Urinária/microbiologia , Infecções Urinárias/genética , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/patogenicidade , Proteína rhoB de Ligação ao GTP/genética
6.
Nat Commun ; 12(1): 2522, 2021 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-33947846

RESUMO

Haematopoietic stem cells (HSCs) tightly regulate their quiescence, proliferation, and differentiation to generate blood cells during the entire lifetime. The mechanisms by which these critical activities are balanced are still unclear. Here, we report that Macrophage-Erythroblast Attacher (MAEA, also known as EMP), a receptor thus far only identified in erythroblastic island, is a membrane-associated E3 ubiquitin ligase subunit essential for HSC maintenance and lymphoid potential. Maea is highly expressed in HSCs and its deletion in mice severely impairs HSC quiescence and leads to a lethal myeloproliferative syndrome. Mechanistically, we have found that the surface expression of several haematopoietic cytokine receptors (e.g. MPL, FLT3) is stabilised in the absence of Maea, thereby prolonging their intracellular signalling. This is associated with impaired autophagy flux in HSCs but not in mature haematopoietic cells. Administration of receptor kinase inhibitor or autophagy-inducing compounds rescues the functional defects of Maea-deficient HSCs. Our results suggest that MAEA provides E3 ubiquitin ligase activity, guarding HSC function by restricting cytokine receptor signalling via autophagy.


Assuntos
Autofagossomos/genética , Autofagia/genética , Moléculas de Adesão Celular/metabolismo , Proteínas do Citoesqueleto/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Proteínas do Citoesqueleto/genética , Perfilação da Expressão Gênica , Hematopoese/efeitos dos fármacos , Hematopoese/genética , Células-Tronco Hematopoéticas/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia Eletrônica de Transmissão , Estabilidade Proteica , Receptores de Trombopoetina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitinação , Tirosina Quinase 3 Semelhante a fms/metabolismo
7.
J Histochem Cytochem ; 69(6): 407-414, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33884901

RESUMO

During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.


Assuntos
Autofagia , Estruturas da Membrana Celular/metabolismo , Proteína Sequestossoma-1/metabolismo , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Estruturas da Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Fosfolipídeos/metabolismo , Proteína Sequestossoma-1/análise
8.
Dev Cell ; 56(10): 1452-1468.e8, 2021 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-33878344

RESUMO

Niemann-Pick disease type C (NPC) is a neurodegenerative lysosomal storage disorder characterized by lipid accumulation in endolysosomes. An early pathologic hallmark is axonal dystrophy occurring at presymptomatic stages in NPC mice. However, the mechanisms underlying this pathologic change remain obscure. Here, we demonstrate that endocytic-autophagic organelles accumulate in NPC dystrophic axons. Using super-resolution and live-neuron imaging, we reveal that elevated cholesterol on NPC lysosome membranes sequesters kinesin-1 and Arl8 independent of SKIP and Arl8-GTPase activity, resulting in impaired lysosome transport into axons, contributing to axonal autophagosome accumulation. Pharmacologic reduction of lysosomal membrane cholesterol with 2-hydroxypropyl-ß-cyclodextrin (HPCD) or elevated Arl8b expression rescues lysosome transport, thereby reducing axonal autophagic stress and neuron death in NPC. These findings demonstrate a pathological mechanism by which altered membrane lipid composition impairs lysosome delivery into axons and provide biological insights into the translational application of HPCD in restoring axonal homeostasis at early stages of NPC disease.


Assuntos
Autofagia , Axônios/metabolismo , Lipídeos/química , Lisossomos/metabolismo , Distrofias Musculares/patologia , Doença de Niemann-Pick Tipo C/patologia , Estresse Fisiológico , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Transporte Biológico , Morte Celular , Colesterol/metabolismo , Vesículas Citoplasmáticas/metabolismo , Vesículas Citoplasmáticas/ultraestrutura , GTP Fosfo-Hidrolases/metabolismo , Membranas Intracelulares/metabolismo , Cinesina/metabolismo , Camundongos Endogâmicos BALB C , Distrofias Musculares/complicações , Proteína C1 de Niemann-Pick/deficiência , Proteína C1 de Niemann-Pick/metabolismo , Doença de Niemann-Pick Tipo C/complicações
9.
Int J Mol Sci ; 22(4)2021 Feb 12.
Artigo em Inglês | MEDLINE | ID: mdl-33673233

RESUMO

Autophagy is an intracellular self-devouring system that plays a central role in cellular recycling. The formation of functional autophagosomes depends on several autophagy-related proteins, including the microtubule-associated proteins 1A/1B light chain 3 (LC3) and the conserved autophagy-related gene 12 (Atg12). We have recently developed a novel scanning electron-assisted dielectric microscope (SE-ADM) for nanoscale observations of intact cells. Here, we used the SE-ADM system to observe LC3- and Atg12-containing autophagosomes in cells labelled in the culture medium with antibodies conjugated to colloidal gold particles. We observed that, during autophagosome formation, Atg12 localized along the actin meshwork structure, whereas LC3 formed arcuate or circular alignments. Our system also showed a difference in the distribution of LC3 and Atg12; Atg12 was broadly distributed while LC3 was more localized. The difference in the spatial distribution demonstrated by our system explains the difference in the size of fluorescent spots due to the fluorescently labelled antibodies observed using optical microscopy. The direct SE-ADM observation of cells should thus be effective in analyses of autophagosome formation.


Assuntos
Autofagossomos , Proteína 12 Relacionada à Autofagia/metabolismo , Microscopia Eletrônica de Varredura , Proteínas Associadas aos Microtúbulos/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular Tumoral , Camundongos , Ratos
10.
Mol Cell ; 81(9): 2013-2030.e9, 2021 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-33773106

RESUMO

The sequestration of damaged mitochondria within double-membrane structures termed autophagosomes is a key step of PINK1/Parkin mitophagy. The ATG4 family of proteases are thought to regulate autophagosome formation exclusively by processing the ubiquitin-like ATG8 family (LC3/GABARAPs). We discover that human ATG4s promote autophagosome formation independently of their protease activity and of ATG8 family processing. ATG4 proximity networks reveal a role for ATG4s and their proximity partners, including the immune-disease protein LRBA, in ATG9A vesicle trafficking to mitochondria. Artificial intelligence-directed 3D electron microscopy of phagophores shows that ATG4s promote phagophore-ER contacts during the lipid-transfer phase of autophagosome formation. We also show that ATG8 removal during autophagosome maturation does not depend on ATG4 activity. Instead, ATG4s can disassemble ATG8-protein conjugates, revealing a role for ATG4s as deubiquitinating-like enzymes. These findings establish non-canonical roles of the ATG4 family beyond the ATG8 lipidation axis and provide an AI-driven framework for rapid 3D electron microscopy.


Assuntos
Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Cisteína Endopeptidases/metabolismo , Metabolismo dos Lipídeos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/genética , Inteligência Artificial , Autofagossomos/genética , Autofagossomos/ultraestrutura , Família da Proteína 8 Relacionada à Autofagia/genética , Família da Proteína 8 Relacionada à Autofagia/metabolismo , Proteínas Relacionadas à Autofagia/genética , Cisteína Endopeptidases/genética , Células HEK293 , Células HeLa , Humanos , Imageamento Tridimensional , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Proteínas Associadas aos Microtúbulos/genética , Mitocôndrias/genética , Mitocôndrias/ultraestrutura , Mitofagia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Transporte Proteico , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Transporte Vesicular/genética , Proteínas de Transporte Vesicular/metabolismo
11.
Biochem Biophys Res Commun ; 549: 179-186, 2021 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-33677390

RESUMO

Intervertebral disc degeneration (IDD) is closely related to loss of the extracellular matrix (ECM), apoptosis and inflammation in nucleus pulposus cells (NPCs). It has been reported that Zinc finger protein A20/TNFAIP3 (A20) can inhibit the activity of the NF-κB pathway and promote autophagy. Therefore, we speculated that A20 can regulate inflammation and ameliorate IDD through autophagy mediated by NF-κB in human NPCs. Our results indicated that the expression of A20 and inflammatory factors in IDD tissues was increased. A20 is an essential negative regulator in the NF-κB pathway. Constructed adenoviral shRNA and overexpression vectors for A20 could effectively regulate the inflammation, autophagy, and activity of NF-κB, which in turn affected the progression of IDD. Inhibition of NF-κB on the basis of knocking down A20 results in increased autophagy, suggesting that A20-regulated autophagy was mediated by NF-κB. In vivo, A20 overexpression could ameliorate the progression of IDD and promote autophagy at the same time, while deletion of A20 leads to low levels of autophagy and severe degeneration. In summary, A20 plays an important role in inhibiting inflammation through autophagy mediated by NF-κB in NPCs and ameliorating IDD.


Assuntos
Autofagia , Inflamação/patologia , Degeneração do Disco Intervertebral/metabolismo , Degeneração do Disco Intervertebral/patologia , NF-kappa B/metabolismo , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Proteína 3 Induzida por Fator de Necrose Tumoral alfa/metabolismo , Adulto , Animais , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Coelhos , Transdução de Sinais
12.
Biomolecules ; 11(2)2021 02 05.
Artigo em Inglês | MEDLINE | ID: mdl-33562550

RESUMO

Cardiolipin (CL) is a hallmark phospholipid localized within the inner mitochondrial membrane. Upon several mitochondrial stress conditions, CL is translocated to specialized platforms, where it may play a role in signaling events to promote mitophagy and apoptosis. Recent studies characterized the molecular composition of MAM-associated lipid microdomains and their implications in regulating the autophagic process. In this study we analyzed the presence of CL within MAMs following autophagic stimulus and the possible implication of raft-like microdomains enriched in CL as a signaling platform in autophagosome formation. Human 2FTGH fibroblasts and SKNB-E-2 cells were stimulated under nutrient deprivation with HBSS. MAM fraction was obtained by an ultracentrifugation procedure and analyzed by HPTLC immunostaining. CL interactions with mitofusin2 (MFN2), calnexin (CANX) and AMBRA1 were analyzed by scanning confocal microscopy and coimmunoprecipitation. The analysis revealed that CL accumulates in MAMs fractions following autophagic stimulus, where it interacts with MFN2 and CANX. It associates with AMBRA1, which in turn interacts with BECN1 and WIPI1. This study demonstrates that CL is present in MAM fractions following autophagy triggering and interacts with the multimolecular complex (AMBRA1/BECN1/WIPI1) involved in autophagosome formation. It may have both structural and functional implications in the pathophysiology of neurodegenerative disease(s).


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagossomos/metabolismo , Calnexina/metabolismo , Cardiolipinas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Proteínas Relacionadas à Autofagia/genética , Proteínas Relacionadas à Autofagia/metabolismo , Proteína Beclina-1/genética , Proteína Beclina-1/metabolismo , Western Blotting , Calnexina/genética , Cardiolipinas/isolamento & purificação , Fracionamento Celular , Linhagem Celular , Fibroblastos/metabolismo , Fibroblastos/ultraestrutura , GTP Fosfo-Hidrolases/genética , Expressão Gênica , Humanos , Soluções Isotônicas/farmacologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Membranas Mitocondriais/ultraestrutura , Proteínas Mitocondriais/genética , Mitofagia/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/ultraestrutura , Ligação Proteica
13.
J Alzheimers Dis ; 79(3): 1171-1184, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33386800

RESUMO

BACKGROUND: Alzheimer's disease (AD) is characterized by amyloid-ß (Aß) deposition. The metabolism of Aß is critically affected by autophagy. Although rifampicin is known to mediate neuroinflammation, the underlying mechanism by which rifampicin regulates the cognitive sequelae remains unknown. OBJECTIVE: Based on our previous findings that rifampicin possesses neuroprotective effects on improving cognitive function after neuroinflammation, we aimed to examine in this study whether rifampicin can inhibit Aß accumulation by enhancing autophagy in a mouse model of lipopolysaccharide (LPS)-induced cognitive impairment. METHODS: Adult C57BL/6 mice were intraperitoneally injected with rifampicin, chloroquine, and/or LPS every day for 7 days. Pathological and biochemical assays and behavioral tests were performed to determine the therapeutic effect and mechanism of rifampicin on the hippocampus of LPS-induced mice. RESULTS: We found that rifampicin ameliorated cognitive impairments in the LPS-induced mice. In addition, rifampicin attenuated the inhibition of autophagosome formation, suppressed the accumulation of Aß1-42, and protected the hippocampal neurons against LPS-induced damage. Our results further demonstrated that rifampicin improved the neurological function by promoting autophagy through the inhibition of Akt/mTOR/p70S6K signaling pathway in the hippocampus of LPS-induced mice. CONCLUSION: Rifampicin ameliorates cognitive impairment by suppression of Aß1-42 accumulation through inhibition of Akt/mTOR/p70S6K signaling and enhancement of autophagy in the hippocampus of LPS-induced mice.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Autofagia/efeitos dos fármacos , Disfunção Cognitiva/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Rifampina/uso terapêutico , Peptídeos beta-Amiloides/antagonistas & inibidores , Animais , Autofagossomos/ultraestrutura , Western Blotting , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Imunofluorescência , Hipocampo/metabolismo , Hipocampo/patologia , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão , Teste do Labirinto Aquático de Morris/efeitos dos fármacos
14.
Nat Commun ; 12(1): 16, 2021 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-33397898

RESUMO

Autophagy contributes to the selective degradation of liquid droplets, including the P-Granule, Ape1-complex and p62/SQSTM1-body, although the molecular mechanisms and physiological relevance of selective degradation remain unclear. In this report, we describe the properties of endogenous p62-bodies, the effect of autophagosome biogenesis on these bodies, and the in vivo significance of their turnover. p62-bodies are low-liquidity gels containing ubiquitin and core autophagy-related proteins. Multiple autophagosomes form on the p62-gels, and the interaction of autophagosome-localizing Atg8-proteins with p62 directs autophagosome formation toward the p62-gel. Keap1 also reversibly translocates to the p62-gels in a p62-binding dependent fashion to activate the transcription factor Nrf2. Mice deficient for Atg8-interaction-dependent selective autophagy show that impaired turnover of p62-gels leads to Nrf2 hyperactivation in vivo. These results indicate that p62-gels are not simple substrates for autophagy but serve as platforms for both autophagosome formation and anti-oxidative stress.


Assuntos
Autofagossomos/metabolismo , Estresse Oxidativo , Proteína Sequestossoma-1/metabolismo , Animais , Proteínas Reguladoras de Apoptose/metabolismo , Autofagossomos/ultraestrutura , Autofagia , Linhagem Celular , Géis , Hepatócitos/metabolismo , Hepatócitos/ultraestrutura , Humanos , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fígado/lesões , Fígado/patologia , Camundongos Endogâmicos C57BL , Proteínas Associadas aos Microtúbulos/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Ligação Proteica , Lipossomas Unilamelares
15.
J Cell Biol ; 220(3)2021 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33439214

RESUMO

The mechanism of isolation membrane formation in autophagy is receiving intensive study. We recently found that Atg9 translocates phospholipids across liposomal membranes and proposed that this functionality plays an essential role in the expansion of isolation membranes. The distribution of phosphatidylinositol 3-phosphate in both leaflets of yeast autophagosomal membranes supports this proposal, but if Atg9-mediated lipid transport is crucial, symmetrical distribution in autophagosomes should be found broadly for other phospholipids. To test this idea, we analyzed the distributions of phosphatidylcholine, phosphatidylserine, and phosphatidylinositol 4-phosphate by freeze-fracture electron microscopy. We found that all these phospholipids are distributed with comparable densities in the two leaflets of autophagosomes and autophagic bodies. Moreover, de novo-synthesized phosphatidylcholine is incorporated into autophagosomes preferentially and shows symmetrical distribution in autophagosomes within 30 min after synthesis, whereas this symmetrical distribution is compromised in yeast expressing an Atg9 mutant. These results indicate that transbilayer phospholipid movement that is mediated by Atg9 is involved in the biogenesis of autophagosomes.


Assuntos
Autofagossomos/metabolismo , Proteínas Relacionadas à Autofagia/metabolismo , Membrana Celular/metabolismo , Proteínas de Membrana/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Autofagossomos/ultraestrutura , Membrana Celular/ultraestrutura , Técnica de Fratura por Congelamento , Humanos , Saccharomyces cerevisiae/ultraestrutura
16.
Drug Res (Stuttg) ; 71(1): 43-50, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33022720

RESUMO

Glucagon-like peptide-2 (GLP-2) is a peptide hormone that belongs to the glucagon-derived peptide family. We have previously shown that analogues of the sister hormone Glucagon-like peptide-1 (GLP-1) showed neuroprotective effects. Here we investigated the effect of a GLP-2 agonist in a cell model of Parkinson's disease (PD) created by treating SH-SY5Y or Neuro-2a cells with 1-Methyl-4-phenyl-pyridine ion (MPP+). Cell viability and cell cytotoxicity was detected by MTT and LDH assays, respectively. The protein expression levels of mitochondrial, autophagy and apoptotic biomarkers including PGC-1α, Mfn2, IRE1, ATG7, LC3B, Beclin1 and Bcl-2 were detected by western blot. Mitochondrial superoxide was detected by MitoSOX Red. In addition, mitochondrial morphology, autophagosome and apoptotic corpuscles were observed by transmission electron microscope (TEM). We found that the GLP-1 and the GLP-2 agonists both protect cells against mitochondrial damage, autophagy impairments and apoptosis induced by MPP+both in SH-SY5Y and Neuro-2a cells. Cell signaling for mitogenesis was enhanced, and oxidative stress levels much reduced by the drugs. This demonstrates for the first time the neuroprotective effects of a GLP-2 analogue in PD cellular models, in which oxidative stress, autophagy and apoptosis play crucial roles. The protective effects were comparable to those seen with the GLP-1 analogue liraglutide. The results suggest that not only GLP-1, but also GLP-2 has neuroprotective properties and may be useful as a novel treatment of PD.


Assuntos
Peptídeo 2 Semelhante ao Glucagon/agonistas , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson/tratamento farmacológico , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Linhagem Celular Tumoral , Avaliação Pré-Clínica de Medicamentos , Peptídeo 1 Semelhante ao Glucagon/agonistas , Humanos , Liraglutida/farmacologia , Liraglutida/uso terapêutico , Microscopia Eletrônica de Transmissão , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Fármacos Neuroprotetores/uso terapêutico , Estresse Oxidativo/efeitos dos fármacos , Doença de Parkinson/patologia , Transdução de Sinais/efeitos dos fármacos
17.
J Cell Physiol ; 236(5): 4050-4065, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33174204

RESUMO

Arsenic is an environmental toxicant. Its overdose can cause liver damage. Autophagy has been reported to be involved in arsenite (iAs3+ ) cytotoxicity and plays a dual role in cell proliferation and cell death. However, the effect and molecular regulative mechanisms of iAs3+ on autophagy in hepatocytes remains largely unknown. Here, we found that iAs3+ exposure lead to hepatotoxicity by inducing autophagosome and autolysosome accumulation. On the one hand, iAs3+ promoted autophagosome synthesis by inhibiting E2F1/mTOR pathway in L-02 human hepatocytes. On the other, iAs3+ blocked autophagosome degradation partially via suppressing the expression of INPP5E and Rab7 as well as impairing lysosomal activity. More importantly, autophagosome and autolysosome accumulation induced by iAs3+ increased the protein level of E2F7a, which could further inhibit cell viability and induce apoptosis of L-02 cells. The treatment of Ginkgo biloba extract (GBE) effectively reduced autophagosome and autolysosome accumulation and thus alleviated iAs3+ -induced hepatotoxicity. Moreover, GBE could also protect lysosomal activity, promote the phosphorylation level of E2F1 (Ser364 and Thr433) and Rb (Ser780) as well as suppress the protein level of E2F7a in iAs3+ -treated L-02 cells. Taken together, our data suggested that autophagosome and autophagolysosome accumulation play a critical role for iAs3+ -induced hepatotoxicity, and GBE is a promising candidate for intervening iAs3+ induced liver damage by regulating E2F1-autophagy-E2F7a pathway and restoring lysosomal activity.


Assuntos
Arsenitos/toxicidade , Autofagia , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F7/metabolismo , Fígado/patologia , Lisossomos/metabolismo , Extratos Vegetais/farmacologia , Transdução de Sinais , Apoptose/efeitos dos fármacos , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Humanos , Fígado/efeitos dos fármacos , Fígado/ultraestrutura , Lisossomos/efeitos dos fármacos , Lisossomos/ultraestrutura , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos
18.
Cell Mol Neurobiol ; 41(4): 813-826, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-32577848

RESUMO

As a widely known plant hormone, Abscisic acid plays an important role in the progress of planting cell and their stress response. Recently, we reported that ABA might play an anti-cancer role in glioma tissues. In the present study, the molecular mechanism of ABA anti-cancer was further explored in glioblastoma cells. By measuring LC3 puncta formation and conversion in glioblastoma cells, inhibiting the autophagic pathway, targeting the essential autophagic modulator beclin 1 with RNA interference, and analysing cellular morphology via transmission electron microscopy, we found that ABA-treated glioblastoma cells exhibited the features of autophagy. Specifically, ABA-induced autophagy in glioblastoma cells was mediated by the MAPK/JNK signalling pathway rather than the PI3K/AKT/mTOR axis. Moreover, the inhibition or knockdown of JNK specifically blocked ABA-induced autophagic cell death. ABA-induced autophagy was further confirmed in tumour-bearing mice and was accompanied by the inhibition of glioma growth in vivo. This report is the first to describe autophagy induced by ABA and mediated by the MAPK/JNK pathway in human cancer cells and tumour-bearing mice. These results may shed some light in new therapeutic strategies of glioma.


Assuntos
Ácido Abscísico/farmacologia , Autofagia , Glioblastoma/enzimologia , Glioblastoma/patologia , Sistema de Sinalização das MAP Quinases , Aloenxertos/efeitos dos fármacos , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Camundongos Nus , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Vacúolos/efeitos dos fármacos , Vacúolos/metabolismo , Vacúolos/ultraestrutura
19.
Breast Cancer ; 28(1): 60-66, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-32654094

RESUMO

LED red light has been reported to have many health benefits. The present study was conducted to characterise anti-proliferation properties of four LED red light wavelengths (615, 630, 660 and 730 nm) against non-triple negative (MCF-7) and triple negative (MDA-MB-231) breast cancer-origin cell lines. It has been shown by MTT assay that at 24 h post-exposure time point, only LED red light with wavelength 660 nm possessed anti-proliferative effects against both cell lines with 40% reduction of cell viability. The morphology of LED 660 nm irradiated cells was found flatten with enlarged cell size, typical characteristic of cell senescent. Indications of autophagy activities following the irradiation have been provided by acridine orange staining, showing high presence of acidic vesicle organelles (AVOs). In addition, high LC3-II/LC3-I to LC3 ratio has been observed qualitatively in Western blot analysis indicating an increase number of autophagosomes formation in LED 660 nm irradiated cells compared to control cells. Electron dense bodies observed in these cells under TEM micrographs provided additional support to the above observations, leading to the conclusion that LED 660 nm irradiation promoted anti-proliferative activities through autophagy in breast cancer-origin cells. These findings have suggested that LED 660 nm might be developed and be employed as an alternative cancer treatment method in future.


Assuntos
Autofagossomos/metabolismo , Autofagia/efeitos da radiação , Neoplasias da Mama/terapia , Fototerapia/métodos , Apoptose , Autofagossomos/efeitos da radiação , Autofagossomos/ultraestrutura , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos da radiação , Sobrevivência Celular/efeitos da radiação , Feminino , Humanos , Microscopia Eletrônica de Transmissão , Semicondutores
20.
Sci Rep ; 10(1): 20924, 2020 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-33262410

RESUMO

Resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) has become the main clinical challenge of advanced lung cancer. This research aimed to explore the role of PARP1-mediated autophagy in the progression of TKI therapy. PARP1-mediated autophagy was evaluated in vitro by CCK-8 assay, clonogenic assay, immunofluorescence, and western blot in the HCC-827, H1975, and H1299 cells treated with icotinib (Ico), rapamycin, and AZD2281 (olaparib) alone or in combination. Our results and GEO dataset analysis confirmed that PARP1 is expressed at lower levels in TKI-sensitive cells than in TKI-resistant cells. Low PARP1 expression and high p62 expression were associated with good outcomes among patients with NSCLC after TKI therapy. AZD2281 and a lysosomal inhibitor reversed resistance to Ico by decreasing PARP1 and LC3 in cells, but an mTOR inhibitor did not decrease Ico resistance. The combination of AZD2281 and Ico exerted a markedly enhanced antitumor effect by reducing PARP1 expression and autophagy in vivo. Knockdown of PARP1 expression reversed the resistance to TKI by the mTOR/Akt/autophagy pathway in HCC-827IR, H1975, and H1299 cells. PARP1-mediated autophagy is a key pathway for TKI resistance in NSCLC cells that participates in the resistance to TKIs. Olaparib may serve as a novel method to overcome the resistance to TKIs.


Assuntos
Autofagia , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/tratamento farmacológico , Poli(ADP-Ribose) Polimerase-1/metabolismo , Inibidores de Proteínas Quinases/uso terapêutico , Animais , Autofagossomos/efeitos dos fármacos , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Autofagia/efeitos dos fármacos , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Éteres de Coroa/farmacologia , Éteres de Coroa/uso terapêutico , Intervalo Livre de Doença , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , Camundongos Nus , Proteínas Associadas aos Microtúbulos/metabolismo , Ftalazinas/farmacologia , Ftalazinas/uso terapêutico , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Quinazolinas/farmacologia , Quinazolinas/uso terapêutico , Proteína Sequestossoma-1/metabolismo , Transdução de Sinais/efeitos dos fármacos
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