Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 48
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Curr Top Dev Biol ; 155: 1-37, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-38043949

RESUMO

Autophagy is a fundamental catabolic process whereby excessive or damaged cytoplasmic components are degraded through lysosomes to maintain cellular homeostasis. Studies of mTOR signaling have revealed that mTOR controls biomass generation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Primary cilia, the assembly of which depends on kinesin molecular motors, serve as sensory organelles and signaling platforms. Given these pathways' central role in maintaining cellular and physiological homeostasis, a connection between mTOR and primary cilia signaling is starting to emerge in a variety of diseases. In this review, we highlight recent advances in our understanding of the complex crosstalk between the mTOR pathway and cilia and discuss its function in the context of related diseases.


Assuntos
Cílios , Serina-Treonina Quinases TOR , Humanos , Cílios/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Transdução de Sinais/fisiologia , Autofagia/fisiologia , Homeostase
2.
Autophagy Rep ; 2(1)2023.
Artigo em Inglês | MEDLINE | ID: mdl-37637387

RESUMO

The Schlemm's canal (SC) is a circular, lymphatic-like vessel located at the limbus of the eye that participates in the regulation of aqueous humor drainage to control intraocular pressure (IOP). Circumferential flow of aqueous humor within the SC lumen generates shear stress, which regulates SC cell behaviour. Using biochemical analysis and real-time live cell imaging techniques, we have investigated the activation of autophagy in SC cells by shear stress. We report, for the first time, the primary cilium (PC)-dependent activation of autophagy in SC cells in response to shear stress. Moreover, we identified PC-dependent shear stress-induced autophagy to be positively regulated by phosphorylation of SMAD2 in its linker and C-terminal regions. Additionally, SMAD2/3 signaling was found to transcriptionally activate LC3B, ATG5 and ATG7 in SC cells. Intriguingly, concomitant to SMAD2-dependent activation of autophagy, we also report here the activation of mTOR pathway, a classical autophagy inhibitor, in SC cells by shear stress. mTOR activation was found to also be dependent on the PC. Moreover, pharmacological inhibition of class I PI3K increased phosphorylation of SMAD2 at the linker and activated autophagy. Together, our data indicates an interplay between PI3K and SMAD2/3 signaling pathways in the regulation of PC-dependent shear stress-induced autophagy in SC cells.

3.
Cell Death Dis ; 14(8): 554, 2023 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-37620383

RESUMO

Glaucoma is a group of diseases that leads to chronic degeneration of retinal ganglion cell (RGC) axons and progressive loss of RGCs, resulting in vision loss. While aging and elevated intraocular pressure (IOP) have been identified as the main contributing factors to glaucoma, the molecular mechanisms and signaling pathways triggering RGC death and axonal degeneration are not fully understood. Previous studies in our laboratory found that overactivation of autophagy in DBA/2J::GFP-LC3 mice led to RGC death and optic nerve degeneration with glaucomatous IOP elevation. We found similar findings in aging GFP-LC3 mice subjected to chronic IOP elevation. Here, we further investigated the impact of autophagy deficiency on autophagy-deficient DBA/2J-Atg4bko and DBA/2J-Atg4b+/- mice, generated in our laboratory via CRISPR/Cas9 technology; as well as in Atg4bko mice subjected to the experimental TGFß2 chronic ocular hypertensive model. Our data shows that, in contrast to DBA/2J and DBA/2J-Atg4b+/- littermates, DBA/2J-Atg4bko mice do not develop glaucomatous IOP elevation. Atg4b deficiency also protected against glaucomatous IOP elevation in the experimental TGFß2 chronic ocular hypertensive model. Atg4 deletion did not compromise RGC or optic nerve survival in Atg4bko mice. Moreover, our results indicate a protective role of autophagy deficiency against RGC death and ON atrophy in the hypertensive DBA/2J-Atg4b+/- mice. Together, our data suggests a pathogenic role of autophagy activation in ocular hypertension and glaucoma.


Assuntos
Glaucoma , Hipertensão Ocular , Animais , Camundongos , Camundongos Endogâmicos DBA , Glaucoma/complicações , Glaucoma/genética , Autofagia/genética , Modelos Animais de Doenças , Células Ganglionares da Retina
4.
Autophagy Rep ; 2(1)2023.
Artigo em Inglês | MEDLINE | ID: mdl-37034386

RESUMO

Autophagy is a catabolic self-degradative pathway that promotes the degradation and recycling of intracellular material through the lysosomal compartment. Although first believed to function in conditions of nutritional stress, autophagy is emerging as a critical cellular pathway, involved in a variety of physiological and pathophysiological processes. Autophagy dysregulation is associated with an increasing number of diseases, including ocular diseases. On one hand, mutations in autophagy-related genes have been linked to cataracts, glaucoma, and corneal dystrophy; on the other hand, alterations in autophagy and lysosomal pathways are a common finding in essentially all diseases of the eye. Moreover, LC3-associated phagocytosis, a form of non-canonical autophagy, is critical in promoting visual cycle function. This review collects the latest understanding of autophagy in the context of the eye. We will review and discuss the respective roles of autophagy in the physiology and/or pathophysiology of each of the ocular tissues, its diurnal/circadian variation, as well as its involvement in diseases of the eye.

5.
Autophagy ; 19(2): 388-400, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-35468037

RESUMO

Age-related macular degeneration (AMD) is the leading cause of visual impairment in the aging population with limited understanding of its pathogenesis and a lack of effective treatment. The progression of AMD is initially characterized by atrophic alterations in the retinal pigment epithelium, as well as the formation of lysosomal lipofuscin and extracellular drusen deposits. Damage caused by chronic oxidative stress, protein aggregation and inflammatory processes may lead to geographic atrophy and/or choroidal neovascularization and fibrosis. The role of macroautophagy/autophagy in AMD pathology is steadily emerging. This review describes selective and secretory autophagy and their role in drusen biogenesis, senescence-associated secretory phenotype, inflammation and epithelial-mesenchymal transition in the pathogenesis of AMD.Abbreviations: Aß: amyloid-beta; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ATF6: activating transcription factor 6; ATG: autophagy related; BACE1: beta-secretase 1; BHLHE40: basic helix-loop-helix family member e40; BNIP3: BCL2 interacting protein 3; BNIP3L/NIX: BCL2 interacting protein 3 like; C: complement; CALCOCO2/NDP52: calcium binding and coiled-coil domain 2; CARD: caspase recruitment domain; CDKN2A/p16: cyclin dependent kinase inhibitor 2A; CFB: complement factor B; DELEC1/Dec1; deleted in esophageal cancer 1; EIF2AK3/PERK: eukaryotic translation initiation factor 2 alpha kinase 3; EMT: epithelial-mesenchymal transition; ER: endoplasmic reticulum; ERN1/IRE1: endoplasmic reticulum to nucleus signaling 1; FUNDC1: FUN14 domain containing 1; GABARAP: GABA type A receptor-associated protein; HMGB1: high mobility group box 1; IL: interleukin; KEAP1: kelch like ECH associated protein 1; LAP: LC3-associated phagocytosis; LAMP2: lysosomal associated membrane protein 2; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NFE2L2: NFE2 like bZIP transcription factor 2; NLRP3; NLR family pyrin domain containing 3; NFKB/NFκB: nuclear factor kappa B; OPTN: optineurin; PARL: presenilin associated rhomboid like; PGAM5: PGAM family member 5, mitochondrial serine/threonine protein phosphatase; PINK1: PTEN induced kinase 1; POS: photoreceptor outer segment; PPARGC1A: PPARG coactivator 1 alpha; PRKN: parkin RBR E3 ubiquitin protein ligase; PYCARD/ASC: PYD and CARD domain containing; ROS: reactive oxygen species; RPE: retinal pigment epithelium; SA: secretory autophagy; SASP: senescence-associated secretory phenotype; SEC22B: SEC22 homolog B, vesicle trafficking protein; SNAP: synaptosome associated protein; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX: syntaxin; TGFB2: transforming growth factor beta 2; TRIM16: tripartite motif containing 16; TWIST: twist family bHLH transcription factor; Ub: ubiquitin; ULK: unc-51 like autophagy activating kinase; UPR: unfolded protein response; UPS: ubiquitin-proteasome system; V-ATPase: vacuolar-type H+-translocating ATPase; VIM: vimentin.


Assuntos
Autofagia , Degeneração Macular , Humanos , Autofagia/fisiologia , Proteína 1 Associada a ECH Semelhante a Kelch , Secretases da Proteína Precursora do Amiloide , Fator 2 Relacionado a NF-E2 , Ácido Aspártico Endopeptidases , Adenosina Trifosfatases , Ubiquitinas , Proteínas Proto-Oncogênicas c-bcl-2 , Proteínas com Motivo Tripartido , Ubiquitina-Proteína Ligases , Proteínas Adaptadoras de Transdução de Sinal
6.
Prog Retin Eye Res ; 90: 101064, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-35370083

RESUMO

During the last few years, the autophagy lysosomal system is emerging as a central cellular pathway with roles in survival, acting as a housekeeper and stress response mechanism. Studies by our and other labs suggest that autophagy might play an essential role in maintaining aqueous humor outflow homeostasis, and that malfunction of autophagy in outflow pathway cells might predispose to ocular hypertension and glaucoma pathogenesis. In this review, we will collect the current knowledge and discuss the molecular mechanisms by which autophagy does or might regulate normal outflow pathway tissue function, and its response to different types of stressors (oxidative stress and mechanical stress). We will also discuss novel roles of autophagy and lysosomal enzymes in modulation of TGFß signaling and ECM remodeling, and the link between dysregulated autophagy and cellular senescence. We will examine what we have learnt, using pre-clinical animal models about how dysregulated autophagy can contribute to disease and apply that to the current status of autophagy in human glaucoma. Finally, we will consider and discuss the challenges and the potential of autophagy as a therapeutic target for the treatment of ocular hypertension and glaucoma.


Assuntos
Humor Aquoso , Glaucoma , Animais , Humor Aquoso/metabolismo , Autofagia/fisiologia , Humanos , Pressão Intraocular , Lisossomos/metabolismo , Malha Trabecular/fisiologia
7.
Invest Ophthalmol Vis Sci ; 63(2): 12, 2022 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-35129590

RESUMO

Due to their similarities in anatomy, physiology, and pharmacology to humans, mice are a valuable model system to study the generation and mechanisms modulating conventional outflow resistance and thus intraocular pressure. In addition, mouse models are critical for understanding the complex nature of conventional outflow homeostasis and dysfunction that results in ocular hypertension. In this review, we describe a set of minimum acceptable standards for developing, characterizing, and utilizing mouse models of open-angle ocular hypertension. We expect that this set of standard practices will increase scientific rigor when using mouse models and will better enable researchers to replicate and build upon previous findings.


Assuntos
Humor Aquoso/fisiologia , Consenso , Glaucoma/metabolismo , Pressão Intraocular/fisiologia , Hipertensão Ocular/metabolismo , Malha Trabecular/metabolismo , Animais , Modelos Animais de Doenças , Glaucoma/fisiopatologia , Camundongos , Hipertensão Ocular/fisiopatologia , Tonometria Ocular
8.
Invest Ophthalmol Vis Sci ; 62(12): 15, 2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34533563

RESUMO

Purpose: The Slc4a11 knock out (KO) mouse model recapitulates the human disease phenotype associated with congenital hereditary endothelial dystrophy (CHED). Increased mitochondrial reactive oxygen species (ROS) in the Slc4a11 KO mouse model is a major cause of edema and endothelial cell loss. Here, we asked if autophagy was activated by ROS in the KO mice. Methods: Immortalized cell lines and mouse corneal endothelia were used to measure autophagy and lysosome associated protein expressions using Protein Simple Wes immunoassay. Autophagy and lysosome functions were examined in wild type (WT) and KO cells as well as animals treated with the mitochondrial ROS quencher MitoQ. Results: Even though autophagy activation was evident, autophagy flux was aberrant in Slc4a11 KO cells and corneal endothelium. Expression of lysosomal proteins and lysosomal mass were decreased along with reduced nuclear translocation of lysosomal master regulator, transcription factor EB (TFEB). MitoQ reversed aberrant lysosomal functions and TFEB nuclear localization in KO cells. MitoQ injections in KO animals reduced corneal edema and decreased the rate of endothelial cell loss. Conclusions: Mitochondrial ROS disrupts TFEB signaling causing lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction in an animal model of CHED, and to identify a potential therapeutic approach.


Assuntos
Autofagia/fisiologia , Distrofias Hereditárias da Córnea/metabolismo , Modelos Animais de Doenças , Lisossomos/metabolismo , Mitocôndrias/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Proteínas de Transporte de Ânions/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Western Blotting , Catepsina L/metabolismo , Células Cultivadas , Distrofias Hereditárias da Córnea/genética , Distrofias Hereditárias da Córnea/patologia , Endotélio Corneano/efeitos dos fármacos , Endotélio Corneano/metabolismo , Regulação da Expressão Gênica , Imuno-Histoquímica , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microscopia de Fluorescência , Compostos Organofosforados/farmacologia , Reação em Cadeia da Polimerase em Tempo Real , Simportadores/genética , Transfecção , Ubiquinona/análogos & derivados , Ubiquinona/farmacologia
9.
Proc Natl Acad Sci U S A ; 118(13)2021 03 30.
Artigo em Inglês | MEDLINE | ID: mdl-33753495

RESUMO

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.


Assuntos
Cílios/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Malha Trabecular/metabolismo , Autofagia , Células Cultivadas , Cílios/patologia , Técnicas de Silenciamento de Genes , Humanos , Pressão Intraocular/fisiologia , Microscopia Intravital , Mecanotransdução Celular/genética , Hipertensão Ocular/patologia , Hipertensão Ocular/fisiopatologia , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-akt/genética , Proteína Smad2/genética , Proteína Smad3/genética , Estresse Mecânico , Imagem com Lapso de Tempo , Malha Trabecular/citologia , Malha Trabecular/patologia
10.
J Clin Med ; 10(1)2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33379277

RESUMO

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFß/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

11.
Adv Biosyst ; 4(8): e2000059, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-33179869

RESUMO

Cell engineering relies heavily on viral vectors for the delivery of molecular cargo into cells due to their superior efficiency compared to nonviral ones. However, viruses are immunogenic and expensive to manufacture, and have limited delivery capacity. Nonviral delivery approaches avoid these limitations but are currently inefficient for clinical applications. This work demonstrates that the efficiency of nonviral delivery of plasmid DNA, mRNA, Sleeping Beauty transposon, and ribonucleoprotein can be significantly enhanced through pretreatment of cells with the nondegradable sugars (NDS), such as sucrose, trehalose, and raffinose. The enhancement is mediated by the incorporation of the NDS into cell membranes, causing enlargement of lysosomes and formation of large (>500 nm) amphisome-like bodies (ALBs). The changes in subcellular structures redirect transport of cargo to ALBs rather than to lysosomes, reducing cargo degradation in cells. The data indicate that pretreatment of cells with NDS is a promising approach to improve nonviral cargo delivery in biomedical applications.


Assuntos
Sistemas de Liberação de Medicamentos/métodos , Terapia Genética/métodos , Rafinose/farmacologia , Sacarose/farmacologia , Trealose/farmacologia , Transporte Biológico , Sistemas CRISPR-Cas , Elementos de DNA Transponíveis , Eletroporação , Células HEK293 , Humanos , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Plasmídeos/química , Plasmídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
12.
Invest Ophthalmol Vis Sci ; 61(10): 31, 2020 08 03.
Artigo em Inglês | MEDLINE | ID: mdl-32797200

RESUMO

Purpose: To investigate autophagy in the outflow pathway and ganglion cell layer in the aging and ocular hypertensive mouse. Methods: Both 4-month-old and 18-month-old C57BL/6J and GFP-LC3 mice were subjected to unilateral injection of hypertonic saline into a limbal vein, causing sclerosis of the outflow pathway and subsequent elevation of intraocular pressure (IOP). IOP was measured on a weekly basis using a rebound tonometer. Protein expression levels of LC3B, Lamp1, and p62 were evaluated by western blot and/or immunofluorescence. Retinal ganglion cell (RGC) count was performed in whole retinal flat mounts using an anti-Brn3a antibody. Optic nerves were fixed with 4% paraformaldehyde and resin-embedded for axon counts and electron microscopy. Results: In contrast to 18-month-old mice, which developed sustained elevated IOP with a single injection, 4-month-old mice were refractory to high elevations of IOP. Interestingly, both the percentage of animals that developed elevated IOP and the mean ∆IOP were significantly higher in the transgenic mice compared to C57BL/6J. Immunofluorescence and western blot analysis showed dysregulated autophagy in the iridocorneal and retina tissues from 18-month-old mice compared to 4-month-old ones. Moreover, the LC3-II/LC3-I ratio correlated with IOP. As expected, injected hypertensive eyes displayed axonal degeneration and RGC death. RGC and axon loss were significantly exacerbated with aging, especially when combined with GFP-LC3 expression. Autophagic structures were observed in the degenerating axons. Conclusions: Our results indicate dysregulation of autophagy in the trabecular meshwork and retinal tissues with aging and suggest that such dysregulation of autophagy contributes to neurodegeneration in glaucoma.


Assuntos
Envelhecimento , Autofagia , Hipertensão Ocular/metabolismo , Envelhecimento/metabolismo , Animais , Western Blotting , Modelos Animais de Doenças , Feminino , Imunofluorescência , Proteínas de Fluorescência Verde , Pressão Intraocular , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Hipertensão Ocular/patologia , Nervo Óptico/patologia , Células Ganglionares da Retina/metabolismo , Células Ganglionares da Retina/patologia , Malha Trabecular/metabolismo , Malha Trabecular/patologia
13.
Autophagy ; 16(7): 1248-1261, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-31476975

RESUMO

The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in turn leads to elevated IOP, a main risk factor of glaucomatous neurodegeneration. Due to variations in IOP, TM cells are continuously exposed to mechanical deformations. We previously reported activation of macroautophagy/autophagy, as one of the physiological responses elicited in TM cells following mechanical strain application. By using biochemical fractionation analysis and imaging techniques, we demonstrate here for the first time the nuclear accumulation of the autophagic marker MAP1LC3/LC3 (microtubule associated protein1 light chain 3)-II, endogenous and exogenously added (AdGFP-LC3, AdtfLC3), in response to cyclic mechanical stress (CMS). Wheat germ agglutinin (WGA) and leptomycin B treatment suggest LC3 to enter the nucleus by passive diffusion, but to exit in an XPO1/CRM1 (exportin 1)-dependent manner in human TM (hTM) cells. While blockage of nuclear export leads to accumulation of LC3 with promyelocytic leukemia (PML) bodies, nuclear LC3 localizes in the nucleolus in cells under CMS. Moreover, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for starvation-induced ribophagy. More interestingly, we further demonstrate that NUFIP1 translocates from the nucleus to LAMP2 (lysosomal associated membrane protein 2)-positive organelles in the stretched cells without triggering ribophagy, suggesting a more general role of NUFIP1 as a selective autophagy receptor for another yet-to-be-identified target in CMS and a surveillance role of nuclear LC3 against stretch-induced damage. ABBREVIATION: AdGFP: adenovirus encoding GFP; ATG: autophagy-related; BSA: bovine serum albumin; CMS: cyclic mechanical stretch; Co-IP: coimmunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; DFCs: dense fibrillar components; EM: electron microscopy; FCs: fibrillar centers; GCs: granular components; GFP: green fluorescent protein; hTM: human trabecular meshwork; HBSS: Hanks balanced salt solution; IOP: intraocular pressure; LAMP1/2: lysosomal associated membrane protein 1/2; LepB: leptomycin B; MTOR: mechanistic target of rapamacyin kinase; NES: nuclear export signals; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NLS: nuclear localization signal; NPCs: nuclear pore complexes; NUFIP1: nuclear FMR1 interacting protein 1; NS: non-stretched; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; pfu: plaque-forming units; PML: promyelocytic leukemia; RFP: red fluorescent protein; RPS15A: ribosomal protein S15a; RPL26: ribosomal protein L26; rRNA: ribosomal RNA; SIRT1: sirtuin 1; SQSTM1/p62: sequestosome 1; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3; TM: trabecular meshwork; WB: western blot; WDR36: WD repeat domain 36; WGA: wheat germ agglutinin; XPO1/CRM1: exportin 1.


Assuntos
Autofagia , Nucléolo Celular/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/metabolismo , Estresse Mecânico , Animais , Autofagossomos/metabolismo , Citosol/metabolismo , Humanos , Carioferinas/metabolismo , Camundongos , Modelos Biológicos , Células NIH 3T3 , Ligação Proteica , Transporte Proteico , Receptores Citoplasmáticos e Nucleares/metabolismo , Sirtuína 1/metabolismo , Malha Trabecular/metabolismo , Malha Trabecular/ultraestrutura , Proteína Exportina 1
14.
Sci Rep ; 9(1): 16092, 2019 11 06.
Artigo em Inglês | MEDLINE | ID: mdl-31695131

RESUMO

The trabecular meshwork (TM) is a specialized ocular tissue, which is responsible, together with the Schlemm's canal (SC), for maintaining appropriate levels of intraocular pressure. Dysfunction of these tissues leads to ocular hypertension and increases the risk for developing glaucoma. Previous work by our laboratory revealed dysregulated autophagy in aging and in glaucomatous TM cells. In order to gain more insight in the role of autophagy in the TM pathophysiology, we have conducted transcriptome and functional network analyses of TM primary cells with silenced expression of the autophagy genes Atg5 and Atg7. Atg5/7-deficient TM cells showed changes in transcript levels of several fibrotic genes, including TGFß2, BAMBI, and SMA. Furthermore, genetic and pharmacological inhibition of autophagy was associated with a parallel reduction in TGFß-induced fibrosis, caused by a BAMBI-mediated reduced activation of Smad2/3 signaling in autophagy-deficient cells. At the same time, TGFß treatment led to Smad2/3-dependent dysregulation of autophagy in TM cells, characterized by increased LC3-II levels and autophagic vacuoles content. Together, our results indicate a cross-talk between autophagy and TGFß signaling in TM cells.


Assuntos
Autofagia , Fibrose/metabolismo , Proteína Smad2/metabolismo , Proteína Smad3/metabolismo , Malha Trabecular/citologia , Fator de Crescimento Transformador beta2/metabolismo , Proteína 5 Relacionada à Autofagia/genética , Proteína 5 Relacionada à Autofagia/metabolismo , Proteína 7 Relacionada à Autofagia/genética , Proteína 7 Relacionada à Autofagia/metabolismo , Células Cultivadas , Fibrose/genética , Fibrose/fisiopatologia , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteína Smad2/genética , Proteína Smad3/genética , Malha Trabecular/metabolismo
15.
Cell Death Discov ; 4: 14, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30210817

RESUMO

Glaucoma is a progressive optic neuropathy characterized by axonal degeneration and retinal ganglion cells loss. Several factors have been postulated to play a role in glaucoma, elevated intraocular pressure (IOP) being the best well-known causative factor. The mechanisms leading to ocular hypertension and glaucoma are still not fully understood. An increasing number of evidence indicates a role of autophagy in the pathophysiological process of ocular hypertension and glaucoma. However, while all of the studies agree that autophagy is induced in RGCs in response to injury, autophagy was found to either protect or promote cell death depending on the experimental model used. In order to gain more insight into both, the role of autophagy in the pathogenesis of glaucoma and the effect of chronic IOP elevation in the autophagy pathway, we have investigated here for the first time autophagy in the iridocorneal angle region, retinal ganglion cell bodies, and ON axons in the spontaneous ocular hypertensive DBA/2J mouse glaucoma model and in the transgenic DBA/2J::GFP-LC3 mice, generated in our laboratory. Our results indicate decreased autophagic flux in the outflow pathway cells in the DBA/2J mice, characterized by increased levels of LC3-II and p62 together with a decrease in the lysosomal marker LAMP1, evaluated by western blot and immunofluorescence. Elevated presence of autophagic vacuoles in the DBA/2J and, in particular, in the DBA/2J::GFP-LC3 mice was also observed. Expression of the GFP-LC3 transgene was associated to higher cumulative IOP in the DBA/2J background. In addition to higher elevation in IOP, DBA/2J::GFP-LC3 were characterized by further RGCs and exacerbated axonal degeneration compared to DBA/2J. This was accompanied by the notable high presence of autophagic figures within degenerating axons. These results strongly suggest overactivation of autophagy as a potential cellular mechanism leading to ON degeneration in the chronic hypertensive DBA/2J mice.

16.
Exp Eye Res ; 171: 164-173, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29526795

RESUMO

Cultured trabecular meshwork (TM) cells are a valuable model system to study the cellular mechanisms involved in the regulation of conventional outflow resistance and thus intraocular pressure; and their dysfunction resulting in ocular hypertension. In this review, we describe the standard procedures used for the isolation of TM cells from several animal species including humans, and the methods used to validate their identity. Having a set of standard practices for TM cells will increase the scientific rigor when used as a model, and enable other researchers to replicate and build upon previous findings.


Assuntos
Técnicas de Cultura de Células , Separação Celular/métodos , Guias como Assunto , Malha Trabecular/citologia , Fatores Etários , Animais , Biomarcadores/metabolismo , Consenso , Feto , Humanos , Doadores de Tecidos , Preservação de Tecido , Coleta de Tecidos e Órgãos , Malha Trabecular/metabolismo
17.
Sci Rep ; 7(1): 6656, 2017 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-28751712

RESUMO

Human corneal endothelial cells (HCEnCs) are terminally differentiated cells that have limited regenerative potential. The large numbers of mitochondria in HCEnCs are critical for pump and barrier function required for corneal hydration and transparency. Fuchs Endothelial Corneal Dystrophy (FECD) is a highly prevalent late-onset oxidative stress disorder characterized by progressive loss of HCEnCs. We previously reported increased mitochondrial fragmentation and reduced ATP and mtDNA copy number in FECD. Herein, carbonyl cyanide m-chlorophenyl hydrazone (CCCP)-induced mitochondrial depolarization decreased mitochondrial mass and Mfn2 levels, which were rescued with mitophagy blocker, bafilomycin, in FECD. Moreover, electron transport chain complex (I, V) decrease in FECD indicated deficient mitochondrial bioenergetics. Transmission electron microscopy of FECD tissues displayed an increased number of autophagic vacuoles containing degenerated and swollen mitochondria with cristolysis. An elevation of LC3-II and LAMP1 and downregulation of Mfn2 in mitochondrial fractions suggested that loss of fusion capacity targets fragmented mitochondria to the pre-autophagic pool and upregulates mitophagy. CCCP-induced mitochondrial fragmentation leads to Mfn2 and LC3 co-localization without activation of proteosome, suggesting a novel Mfn2 degradation pathway via mitophagy. These data indicate constitutive activation of mitophagy results in reduction of mitochondrial mass and abrogates cellular bioenergetics during degeneration of post-mitotic cells of ocular tissue.


Assuntos
Distrofia Endotelial de Fuchs/fisiopatologia , GTP Fosfo-Hidrolases/genética , Proteínas Mitocondriais/genética , Mitofagia , Idoso , Carbonil Cianeto m-Clorofenil Hidrazona , Células Cultivadas , Regulação para Baixo , Endotélio Corneano/metabolismo , Endotélio Corneano/fisiopatologia , Feminino , Distrofia Endotelial de Fuchs/metabolismo , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade
18.
Virus Res ; 236: 30-43, 2017 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-28465158

RESUMO

Measles virus (MV) remains a leading cause of vaccine-preventable deaths in children. Protection against MV is associated with neutralizing antibodies that preferentially recognize the viral hemagglutinin (MV-H), and to a lesser extent, the fusion protein (MV-F). Although MV is serologically monotypic, 24 genotypes have been identified. Here we report three neutralization epitopes conserved in the more prevalent circulating MV genotypes, two located in the MV-H receptor binding site (RBS) (antigenic site III) and a third in MV-H/MV-F interphase (antigenic site Ia) which are essential for MV multiplication. In contrast, two MV-H neutralization epitopes, showed a genotype-specific neutralization escape due to a single amino acid change, that we mapped in the "noose" antigenic site, or an enhanced neutralization epitope (antigenic site IIa). The monoclonal antibody (mAb) neutralization potency correlated with its binding affinity and was mainly driven by kinetic dissociation rate (koff). We developed an immunoassay for mAb binding to MV-H in its native hetero-oligomeric structure with MV-F on the surface of a MV productive steady-state persistently infected (p.i.) human cell lines, and a competitive-binding assay with serum from individuals with past infection by different MV genotypes. Binding assays revealed that a broad neutralization epitope, in RBS antigenic site, a genotype specific neutralization epitopes, in noose and IIa sites, were immunogenic in natural infection and vaccination and may elicit long-lasting humoral immunity that might contribute to explain MV immunogenic stability. These results support the design of improved measles vaccines, broad-spectrum prophylactic or therapeutic antibodies and MV-used in oncolytic therapies.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Antivirais/imunologia , Hemaglutininas Virais/imunologia , Vírus do Sarampo/imunologia , Sarampo/imunologia , Anticorpos Neutralizantes/imunologia , Epitopos/administração & dosagem , Epitopos/imunologia , Genótipo , Hemaglutininas Virais/administração & dosagem , Hemaglutininas Virais/genética , Humanos , Sarampo/prevenção & controle , Sarampo/virologia , Vacina contra Sarampo/administração & dosagem , Vacina contra Sarampo/imunologia , Vírus do Sarampo/classificação , Vírus do Sarampo/genética , Vírus do Sarampo/isolamento & purificação , Testes de Neutralização , Vacinação
19.
Mol Ther ; 25(3): 803-815, 2017 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-28129959

RESUMO

Electrotransfection is a widely used method for delivering genes into cells with electric pulses. Although different hypotheses have been proposed, the mechanism of electrotransfection remains controversial. Previous studies have indicated that uptake and intracellular trafficking of plasmid DNA (pDNA) are mediated by endocytic pathways, but it is still unclear which pathways are directly involved in the delivery. To this end, the present study investigated the dependence of electrotransfection on macropinocytosis. Data from the study demonstrated that electric pulses induced cell membrane ruffling and actin cytoskeleton remodeling. Using fluorescently labeled pDNA and a macropinocytosis marker (i.e., dextran), the study showed that electrotransfected pDNA co-localized with dextran in intracellular vesicles. Furthermore, electrotransfection efficiency could be decreased significantly by reducing temperature or treatment of cells with a pharmacological inhibitor of Rac1 and could be altered by changing Rac1 activity. Taken together, the findings suggested that electrotransfection of pDNA involved Rac1-dependent macropinocytosis.


Assuntos
Eletroporação , Pinocitose , Plasmídeos/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Actinas/metabolismo , Animais , Células COS , Linhagem Celular , Chlorocebus aethiops , Endocitose , Expressão Gênica , Técnicas de Transferência de Genes , Humanos , Camundongos , Microscopia de Fluorescência , Plasmídeos/genética , Transfecção
20.
Exp Eye Res ; 158: 146-153, 2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-27373974

RESUMO

Because of elevations in IOP and other forces, cells in the trabecular meshwork (TM) are constantly subjected to mechanical strain. In order to preserve cellular function and regain homeostasis, cells must sense and adapt to these morphological changes. We and others have already shown that mechanical stress can trigger a broad range of responses in TM cells; however, very little is known about the strategies that TM cells use to respond to this stress, so they can adapt and survive. Autophagy, a lysosomal degradation pathway, has emerged as an important cellular homeostatic mechanism promoting cell survival and adaptation to a number of cytotoxic stresses. Our laboratory has reported the activation of autophagy in TM cells in response to static biaxial strain and high pressure. Moreover, our newest data also suggest the activation of chaperon-assisted selective autophagy, a recently identified tension-induced autophagy essential for mechanotransduction, in TM cells under cyclic mechanical stress. In this review manuscript we will discuss autophagy as part of an integrated response triggered in TM cells in response to strain, exerting a dual role in repair and mechanotransduction, and the potential effects of dysregulated in outflow pathway pathophysiology.


Assuntos
Humor Aquoso/metabolismo , Autofagia/fisiologia , Mecanotransdução Celular/fisiologia , Malha Trabecular/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Humanos , Pressão Intraocular , Estresse Mecânico
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...