RESUMO
Multiple regulatory mechanisms are in place to ensure the normal processes of bone metabolism, encompassing both bone formation and absorption. This study has identified chaperone-mediated autophagy (CMA) as a critical regulator that safeguards bone formation from the detrimental effects of excessive inflammation. By silencing LAMP2A or HSCA8, we observed a hindrance in the osteoblast differentiation of human bone marrow mesenchymal stem cells (hBMSCs) in vitro. To further elucidate the role of LAMP2A, we generated LAMP2A gene knockdown and overexpression of mouse BMSCs (mBMSCs) using adenovirus. Our results showed that LAMP2A knockdown led to a decrease in osteogenic-specific proteins, while LAMP2A overexpression favored the osteogenesis of mBMSCs. Notably, active-ß-catenin levels were upregulated by LAMP2A overexpression. Furthermore, we found that LAMP2A overexpression effectively protected the osteogenesis of mBMSCs from TNF-α, through the PI3K/AKT/GSK3ß/ß-catenin pathway. Additionally, LAMP2A overexpression significantly inhibited osteoclast hyperactivity induced by TNF-α. Finally, in a murine bone defect model, we demonstrated that controlled release of LAMP2A overexpression adenovirus by alginate sodium capsule efficiently protected bone healing from inflammation, as confirmed by imaging and histological analyses. Collectively, our findings suggest that enhancing CMA has the potential to safeguard bone formation while mitigating hyperactivity in bone absorption.
Assuntos
Autofagia Mediada por Chaperonas , Glicogênio Sintase Quinase 3 beta , Inflamação , Proteína 2 de Membrana Associada ao Lisossomo , Células-Tronco Mesenquimais , Osteogênese , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , beta Catenina , Animais , Osteogênese/fisiologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases/metabolismo , beta Catenina/metabolismo , Humanos , Células-Tronco Mesenquimais/metabolismo , Inflamação/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Transdução de Sinais , Masculino , Camundongos Endogâmicos C57BL , Osteoblastos/metabolismo , Diferenciação Celular , Osteoclastos/metabolismoRESUMO
Hematopoietic stem and progenitor cells (HSPCs) are successfully employed for hematological transplantations, and impaired HSPC function causes hematological diseases and aging. HSPCs maintain the lifelong homeostasis of blood and immune cells through continuous self-renewal and maintenance of the multilineage differentiation potential. TMEM106B is a transmembrane protein localized on lysosomal membranes and associated with neurodegenerative and cardiovascular diseases; however, its roles in HSPCs and hematopoiesis are unknown. Here, we established tmem106bb-/- knockout (KO) zebrafish and showed that tmem106bb KO reduced the proliferation of HSPCs during definitive hematopoiesis. The differentiation potential of HSPCs to lymphoid lineage was reduced, whereas the myeloid and erythroid differentiation potentials of HPSCs were increased in tmem106bb-/- zebrafish. Similar results were obtained with morpholino knockdown of tmem106bb. Mechanistically, TMEM106B interacted with LAMP2A, the lysosomal associated membrane protein 2A, impaired LAMP2A-Cathepsin A interaction, and enhanced LAMP2A stability; tmem106bb KO or TMEM106B knockdown caused LAMP2A degradation and impairment of chaperone-mediated autophagy (CMA). Knockdown of lamp2a caused similar phenotypes to that in tmem106bb-/- zebrafish, and overexpression of lamp2a rescued the impaired phenotypes of HSPCs in tmem106bb-/- embryos. These results uncover a novel molecular mechanism for the maintenance of HSPC proliferation and differentiation through stabilizing LAMP2A via TMEM106B-LAMP2A interaction.
Assuntos
Diferenciação Celular , Proliferação de Células , Células-Tronco Hematopoéticas , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas de Membrana , Peixe-Zebra , Animais , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana/genética , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Hematopoéticas/citologia , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Lisossomos/metabolismo , Humanos , Hematopoese/fisiologiaRESUMO
The purpose of this study was to evaluate the spatiotemporal immunoexpression pattern of microtubule-associated protein 1 light chain 3 beta (LC3B), glucose-regulated protein 78 (GRP78), heat shock protein 70 (HSP70), and lysosomal-associated membrane protein 2A (LAMP2A) in normal human fetal kidney development (CTRL) and kidneys affected with congenital anomalies of the kidney and urinary tract (CAKUT). Human fetal kidneys (control, horseshoe, dysplastic, duplex, and hypoplastic) from the 18th to the 38th developmental week underwent epifluorescence microscopy analysis after being stained with antibodies. Immunoreactivity was quantified in various kidney structures, and expression dynamics were examined using linear and nonlinear regression modeling. The punctate expression of LC3B was observed mainly in tubules and glomerular cells, with dysplastic kidneys displaying distinct staining patterns. In the control group's glomeruli, LAMP2A showed a sporadic, punctate signal; in contrast to other phenotypes, duplex kidneys showed significantly stronger expression in convoluted tubules. GRP78 had a weaker expression in CAKUT kidneys, especially hypoplastic ones, while normal kidneys exhibited punctate staining of convoluted tubules and glomeruli. HSP70 staining varied among phenotypes, with dysplastic and hypoplastic kidneys exhibiting stronger staining compared to controls. Expression dynamics varied among observed autophagy markers and phenotypes, indicating their potential roles in normal and dysfunctional kidney development.
Assuntos
Autofagia , Chaperona BiP do Retículo Endoplasmático , Proteínas de Choque Térmico HSP70 , Rim , Proteína 2 de Membrana Associada ao Lisossomo , Proteínas Associadas aos Microtúbulos , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Rim/metabolismo , Rim/anormalidades , Rim/patologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico/metabolismo , Anormalidades Urogenitais/metabolismo , Anormalidades Urogenitais/patologia , Sistema Urinário/metabolismo , Sistema Urinário/anormalidades , Refluxo Vesicoureteral/metabolismo , Refluxo Vesicoureteral/patologiaRESUMO
Chaperone-mediated autophagy (CMA) is the selective degradation process of intracellular components by lysosomes, which is required for the degradation of aggregate-prone proteins and contributes to proteostasis maintenance. Proteostasis is essential for normal cell function and survival, and it is determined by the balance of protein synthesis and degradation. Because postmitotic neurons are highly susceptible to proteostasis disruption, CMA is vital for the nervous system. Since Parkinson's disease (PD) was first linked to CMA dysfunction, an increasing number of studies have shown that CMA loss, as seen during aging, occurs in the pathogenetic process of neurodegenerative diseases. Here, we review the molecular mechanisms of CMA, as well as the physiological function and regulation of this autophagy pathway. Following, we highlight its potential role in neurodegenerative diseases, and the latest advances and challenges in targeting CMA in therapy of neurodegenerative diseases.
Assuntos
Autofagia Mediada por Chaperonas , Doenças Neurodegenerativas , Doença de Parkinson , Humanos , Doenças Neurodegenerativas/terapia , Doenças Neurodegenerativas/metabolismo , Chaperonas Moleculares/metabolismo , Autofagia/fisiologia , Doença de Parkinson/terapia , Doença de Parkinson/metabolismo , Lisossomos/metabolismoRESUMO
Chaperone-mediated autophagy (CMA) is a major pathway of lysosomal proteolysis recognized as a key player of the control of numerous cellular functions, and whose defects have been associated with several human pathologies. To date, this cellular function is presumed to be restricted to mammals and birds, due to the absence of an identifiable lysosome-associated membrane protein 2A (LAMP2A), a limiting and essential protein for CMA, in nontetrapod species. However, the recent identification of expressed sequences displaying high homology with mammalian LAMP2A in several fish species challenges that view and suggests that CMA likely appeared earlier during evolution than initially thought. In the present study, we provide a comprehensive picture of the evolutionary history of the LAMP2 gene in vertebrates and demonstrate that LAMP2 indeed appeared at the root of the vertebrate lineage. Using a fibroblast cell line from medaka fish (Oryzias latipes), we further show that the splice variant lamp2a controls, upon long-term starvation, the lysosomal accumulation of a fluorescent reporter commonly used to track CMA in mammalian cells. Finally, to address the physiological role of Lamp2a in fish, we generated knockout medaka for that specific splice variant, and found that these deficient fish exhibit severe alterations in carbohydrate and fat metabolisms, in consistency with existing data in mice deficient for CMA in liver. Altogether, our data provide the first evidence for a CMA-like pathway in fish and bring new perspectives on the use of complementary genetic models, such as zebrafish or medaka, for studying CMA in an evolutionary perspective.
Assuntos
Autofagia Mediada por Chaperonas , Evolução Molecular , Proteína 2 de Membrana Associada ao Lisossomo/genética , Oryzias/genética , Animais , Metabolismo dos Carboidratos , Linhagem Celular , Éxons , Fibroblastos/fisiologia , Humanos , Metabolismo dos Lipídeos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Oryzias/metabolismoRESUMO
Huntington's disease (HD) is caused by a mutant huntingtin (mHtt) protein that contains abnormally extended polyglutamine (polyQ) repeats. The process of autophagy has been implicated in clearing mHtt aggregates, and microRNAs (miRNAs) have been reported as new players to regulate autophagy. However, the autophagy-associated target molecule of let7b miRNA remains unclear in HD. The present study showed that extended polyQ in mouse striatal neurons increased lysosomal membrane-associated protein 2A (LAMP2A) levels and influenced the inflammatory conditions, and these augmented levels correlated to the let7b miRNA expression level. The upregulated let7b increased LAMP2A and reduced the extended polyQ in mouse striatal cells. The let7b level was highly expressed in the striatum of pre-onset HD mice, whereas it was significantly reduced in the post-onset HD striatum. Considering the level changing pattern of let7b, LAMP2A protein levels were increased in the striatum of pre-onset HD mice, but decreased in the striatum of post-onset HD mice. These results suggest that LAMP2A related to chaperone-mediated autophagy (CMA) capacity might play an important role in HD symptom onset and progression.
Assuntos
Doença de Huntington/etiologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Animais , Autofagia/genética , Autofagia/fisiologia , Linhagem Celular , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/genética , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Peptídeos/genética , Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismoRESUMO
Chaperone Mediated Autophagy (CMA) is a selective autophagy pathway deregulated in many cancers. In this study, we were aiming at understanding the importance of CMA in breast cancer. To this end, we examined the expression of the CMA markers HSP8 and LAMP2A in different breast cancer cell lines and found a wide range of LAMP2A expression levels across the cell lines analyzed. Next, we applied a specific immunohistochemical staining protocol to a tissue microarray derived from a cohort of 365 breast cancer patients. Therefore, we were able to find a correlation of high LAMP2A but not HSPA8 (HSC70) with worse disease free survival in patients with HER2 negative tumors (p = 0.026) which was independent prognostic parameter from pT category, pN category and grading in a multivariate model (HR = 1.889; 95% CI = 1.039-3.421; p = 0.037). In line, low LAMP2A levels decrease proliferation of the breast cancer cell lines T47D and MCF-7 in vitro. Our data suggest that LAMP2A supports a more severe breast cancer cell phenotype.
Assuntos
Neoplasias da Mama/metabolismo , Técnicas de Cultura de Células/métodos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Receptor ErbB-2/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Western Blotting , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Linhagem Celular Tumoral , Proliferação de Células/genética , Sobrevivência Celular/genética , Autofagia Mediada por Chaperonas/genética , Intervalo Livre de Doença , Feminino , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Células MCF-7 , Pessoa de Meia-Idade , Interferência de RNARESUMO
Naturally-occurring autoantibodies to certain components of autophagy processes have been described in a few autoimmune diseases, but their fine specificity, their relationships with clinical phenotypes, and their potential pathogenic functions remain elusive. Here, we explored IgG autoantibodies reacting with a panel of cytoplasmic endosomal/lysosomal antigens and individual heat-shock proteins, all of which share links to autophagy. Sera from autoimmune patients and from MRL/lpr and NZB/W lupus-prone mice reacted with the C-terminal residues of lysosome-associated membrane glycoprotein (LAMP)2A. No cross-reaction was observed with LAMP2B or LAMP2C variants, with dsDNA or mononucleosomes, or with heat-shock protein A8. Moreover, administering chromatography-purified LAMP2A autoantibodies to MRL/lpr mice accelerated mortality. Furthermore, flow cytometry revealed elevated cell-surface expression of LAMP2A on MRL/lpr B cells. These findings reveal the involvement of a new class of autoantibodies targeting the C-terminus of LAMP2A, a receptor for cytosolic proteins targeted for degradation via chaperone-mediated autophagy. These autoantibodies could affect the autophagy process, which is abnormally upregulated in lupus. The data presented support a novel connection between autophagy dysregulation, autoimmune processes and pathophysiology in lupus.
Assuntos
Antígenos/imunologia , Suscetibilidade a Doenças/imunologia , Lúpus Eritematoso Sistêmico/etiologia , Lúpus Eritematoso Sistêmico/metabolismo , Lisossomos/imunologia , Animais , Autoanticorpos/imunologia , Autoantígenos/imunologia , Autoimunidade , Autofagia/imunologia , Biomarcadores , Estudos de Casos e Controles , Modelos Animais de Doenças , Endossomos/imunologia , Endossomos/metabolismo , Ensaio de Imunoadsorção Enzimática , Proteínas de Choque Térmico/imunologia , Humanos , Imunoglobulina G/imunologia , Lúpus Eritematoso Sistêmico/patologia , Proteína 2 de Membrana Associada ao Lisossomo/imunologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos MRL lpr , Peptídeos/imunologiaRESUMO
AIMS: Chaperone-mediated autophagy (CMA) is a pathway involved in the autophagy lysosome protein degradation system. CMA has attracted attention as a contributing factor to neurodegenerative diseases since it participates in the degradation of disease-causing proteins. We previously showed that CMA is generally impaired in cells expressing the proteins causing spinocerebellar ataxias (SCAs). Therefore, we investigated the effect of CMA impairment on motor function and the neural survival of cerebellar neurons using the micro RNA (miRNA)-mediated knockdown of lysosome-associated protein 2A (LAMP2A), a CMA-related protein. METHODS: We injected adeno-associated virus serotype 9 vectors, which express green fluorescent protein (GFP) and miRNA (negative control miRNA or LAMP2A miRNA) under neuron-specific synapsin I promoter, into cerebellar parenchyma of 4-week-old ICR mice. Motor function of mice was evaluated by beam walking and footprint tests. Immunofluorescence experiments of cerebellar slices were conducted to evaluate histological changes in cerebella. RESULTS: GFP and miRNA were expressed in interneurons (satellite cells and basket cells) in molecular layers and granule cells in the cerebellar cortices, but not in cerebellar Purkinje cells. LAMP2A knockdown in cerebellar neurons triggered progressive motor impairment, prominent loss of cerebellar Purkinje cells, interneurons, granule cells at the late stage, and astrogliosis and microgliosis from the early stage. CONCLUSIONS: CMA impairment in cerebellar interneurons and granule cells triggers the progressive ataxic phenotype, gliosis and the subsequent degeneration of cerebellar neurons, including Purkinje cells. Our present findings strongly suggest that CMA impairment is related to the pathogenesis of various SCAs.
Assuntos
Ataxia Cerebelar/patologia , Cerebelo/patologia , Autofagia Mediada por Chaperonas/fisiologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Degeneração Neural/patologia , Neurônios/patologia , Animais , Ataxia Cerebelar/metabolismo , Cerebelo/metabolismo , Camundongos Endogâmicos ICR , Degeneração Neural/metabolismo , Neurônios/metabolismo , FenótipoRESUMO
The cyanobacterial non-protein amino acid α-amino-ß-methylaminopropionic acid, more commonly known as BMAA, was first discovered in the seeds of the ancient gymnosperm Cycad circinalis (now Cycas micronesica Hill). BMAA was linked to the high incidence of neurological disorders on the island of Guam first reported in the 1950s. BMAA still attracts interest as a possible causative factor in amyotrophic lateral sclerosis (ALS) following the identification of ALS disease clusters associated with living in proximity to lakes with regular cyanobacterial blooms. Since its discovery, BMAA toxicity has been the subject of many in vivo and in vitro studies. A number of mechanisms of toxicity have been proposed including an agonist effect at glutamate receptors, competition with cysteine for transport system xc_ and other mechanisms capable of generating cellular oxidative stress. In addition, a wide range of studies have reported effects related to disturbances in proteostasis including endoplasmic reticulum stress and activation of the unfolded protein response. In the present studies we examine the effects of BMAA on the ubiquitin-proteasome system (UPS) and on chaperone-mediated autophagy (CMA) by measuring levels of ubiquitinated proteins and lamp2a protein levels in a differentiated neuronal cell line exposed to BMAA. The BMAA induced increases in oxidised proteins and the increase in CMA activity reported could be prevented by co-administration of L-serine but not by the two antioxidants examined. These data provide further evidence of a protective role for L-serine against the deleterious effects of BMAA.
Assuntos
Diamino Aminoácidos/efeitos adversos , Autofagia Mediada por Chaperonas , Toxinas de Cianobactérias/efeitos adversos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Neuroblastoma/tratamento farmacológico , Agregados Proteicos/efeitos dos fármacos , Serina/farmacologia , Ubiquitina/metabolismo , Antioxidantes/farmacologia , Diferenciação Celular , Agonistas de Aminoácidos Excitatórios/efeitos adversos , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/genética , Neuroblastoma/metabolismo , Neuroblastoma/patologia , Estresse Oxidativo , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Células Tumorais CultivadasRESUMO
BACKGROUND: Lysosome-associated membrane protein type 2A (LAMP-2A) is the key component of chaperone-mediated autophagy (CMA), a cargo-selective lysosomal degradation pathway. Aberrant LAMP-2A expression and CMA activation have been demonstrated in various human malignancies. The study focusing on the intrinsic role of LAMP-2A and CMA in glioblastoma (GBM), and downstream mechanism could provide valuable insight into the pathogenesis and novel therapeutic modality of GBM. METHODS: The levels of LAMP-2A, nuclear receptor co-repressor (N-CoR), unfolded protein response (UPR) and apoptosis were examined in clinical samples. LAMP-2A siRNA and shRNA were constructed to manipulate CMA activation. The role of CMA and downstream mechanism through degradation of N-CoR and arresting UPR mediated apoptosis were explored in GBM cells and nude mouse xenograft model. RESULTS: Elevated LAMP-2A and associated decreased N-CoR expression were observed in GBM as compared with peritumoral region and low-grade glioma. Inhibited UPR and apoptosis were observed in GBM with high LAMP-2A expression. In vitro study demonstrated co-localization and interaction between LAMP-2A and N-CoR. LAMP-2A silencing up-regulated N-CoR and aroused UPR pathway, leading to apoptosis, while N-CoR silencing led to an opposite result. In vivo study further confirmed that LAMP-2A inhibition arrested tumor growth by promoting apoptosis. CONCLUSIONS: Our results demonstrated the central role of CMA in mediating N-CoR degradation and protecting GBM cells against UPR and apoptosis, and provided evidence of LAMP-2A as potential biomarker. Further research focusing on CMA with other tumorigenic process is needed and selective modulators of LAMP-2A remain to be investigated to provide a novel therapeutic strategy for GBM. Video Abstract.
Assuntos
Apoptose , Biomarcadores Tumorais/metabolismo , Glioblastoma/metabolismo , Glioblastoma/patologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Proteólise , Animais , Apoptose/genética , Caspases/metabolismo , Linhagem Celular Tumoral , Autofagia Mediada por Chaperonas/genética , Regulação Neoplásica da Expressão Gênica , Glioblastoma/genética , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Gradação de Tumores , RNA Interferente Pequeno/metabolismo , Transdução de Sinais , Transcrição Gênica , Resposta a Proteínas não Dobradas/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The aryl hydrocarbon receptor (AHR) is a ligand-activated signaling molecule expressed in many cell types, including triple-negative and non-triple-negative breast cancer cells. It affects breast cancer growth and crosstalk with estrogen receptor signaling. Normally, this receptor is degraded shortly after ligand activation via the 26S proteasome. Here, we report that AHR undergoes chaperone-mediated autophagy in MDA-MB-468 triple-negative breast cancer cells. This lysosomal degradation of AHR exhibits the following characteristics: (1) it is triggered by 6 amino-nicotinamide, starvation, and piperazinylpyrimidine compound Q18; (2) it is not observed in non-triple-negative breast cancer cells (MCF-7, T47D, and MDA-MB-361); (3) it can be inhibited by progesterone receptor B but not estrogen receptor alpha; (4) it can be reversed by chloroquine but not MG132; (5) it requires LAMP2A; and (6) it involves AHR-HSC70 and AHR-LAMP2A interactions. The NEKFF sequence localized at amino acid 558 of human AHR appears to be a KFERQ-like motif of chaperone-mediated autophagy, responsible for the LAMP2A-mediated AHR protein degradation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Autofagia Mediada por Chaperonas/fisiologia , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteólise , Receptores de Hidrocarboneto Arílico/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Sequência de Aminoácidos , Linhagem Celular Tumoral , Cloroquina/farmacologia , Citocromo P-450 CYP1A1/genética , Citocromo P-450 CYP1A1/metabolismo , Receptor alfa de Estrogênio/metabolismo , Humanos , Leupeptinas/farmacologia , Lisossomos/metabolismo , Células MCF-7 , Interferência de RNA , Receptores de Progesterona/genética , Receptores de Progesterona/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Chaperone-mediated autophagy (CMA) is a lysosomal degradation pathway of selective soluble proteins. Lysosome-associated membrane protein type 2a (LAMP2A) is the key receptor protein of CMA; downregulation of LAMP2A leads to CMA blockade. Although CMA activation has been involved in cancer growth, CMA status and functions in non-small cell lung cancer (NSCLC) by focusing on the roles in regulating chemosensitivity remain to be clarified. In this study, we found that LAMP2A expression is elevated in NSCLC cell lines and patient's tumors, conferring poor survival and platinum resistance in NSCLC patients. LAMP2A knockdown in NSCLC cells suppressed cell proliferation and colony formation and increased the sensitivity to chemotherapeutic drugs in vitro. Furthermore, we found that intrinsic apoptosis signaling is the mechanism of cell death involved with CMA blockade. Remarkably, LAMP2A knockdown repressed tumorigenicity and sensitized the tumors to cisplatin treatment in NSCLC-bearing mice. Our discoveries suggest that LAMP2A is involved in the regulation of cancer malignant phenotypes and represents a promising new target against chemoresistant NSCLC.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Resistencia a Medicamentos Antineoplásicos , Neoplasias Pulmonares/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Lisossomos/metabolismo , Transdução de Sinais , Animais , Apoptose/genética , Biomarcadores Tumorais , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/mortalidade , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Proliferação de Células/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Imuno-Histoquímica , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/mortalidade , Neoplasias Pulmonares/patologia , Proteína 2 de Membrana Associada ao Lisossomo/genética , Prognóstico , ProteóliseRESUMO
Chaperone-mediated autophagy (CMA) is a substrate-specific mode of lysosomal proteolysis, with multiple lines of evidence connecting its dysfunction to both ageing and disease. We have recently shown that CMA impairment through knock-down of the lysosomal receptor LAMP2A is detrimental to neuronal viability in vivo; however, it is not clear which subset of proteins regulated by the CMA pathway mediate such changes. In this study, we have manipulated CMA function through alterations of LAMP2A abundance in primary rat cortical neurons, to identify potential changes to the neuronal proteome occurring prior to neurotoxic effects. We have identified a list of proteins with significant, >2-fold change in abundance following our manipulations, of which PARK7/DJ-1 - an anti-oxidant implicated in hereditary forms of Parkinson's Disease (PD), and DPYSL2/CRMP-2 - a microtubule-binding phosphoprotein involved in schizophrenia pathogenesis - were both found to have measurable effects on neuronal homeostasis and phenotype. Taken together, this study describes alterations in the abundance of neuronal proteins involved in neuropsychiatric disorders upon CMA manipulation, and suggests that such alterations may in part be responsible for the neurodegeneration observed upon CMA impairment in vivo.
Assuntos
Autofagia , Homeostase , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteína Desglicase DJ-1/metabolismo , Animais , Células Cultivadas , Feminino , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Proteínas do Tecido Nervoso/genética , Proteína Desglicase DJ-1/genética , Ratos , Ratos WistarRESUMO
Impaired cardiac metabolism in the obese and diabetic heart leads to glucolipotoxicity and ensuing cardiomyopathy. Glucolipotoxicity causes cardiomyocyte injury by increasing energy insufficiency, impairing proteasomal-mediated protein degradation and inducing apoptosis. Proteasome-evading proteins are degraded by autophagy in the lysosome, whose metabolism and function are regulated by master regulator transcription factor EB (TFEB). Limited studies have examined the impact of glucolipotoxicity on intra-lysosomal signaling proteins and their regulators. By utilizing a mouse model of diet-induced obesity, type-1 diabetes (Akita) and ex-vivo model of glucolipotoxicity (H9C2 cells and NRCM, neonatal rat cardiomyocyte), we examined whether glucolipotoxicity negatively targets TFEB and lysosomal proteins to dysregulate autophagy and cause cardiac injury. Despite differential effects of obesity and diabetes on LC3B-II, expression of proteins facilitating autophagosomal clearance such as TFEB, LAMP-2A, Hsc70 and Hsp90 were decreased in the obese and diabetic heart. In-vivo data was recapitulated in H9C2 and NRCM cells, which exhibited impaired autophagic flux and reduced TFEB content when exposed to a glucolipotoxic milieu. Notably, overloading myocytes with a saturated fatty acid (palmitate) but not an unsaturated fatty acid (oleate) depleted cellular TFEB and suppressed autophagy, suggesting a fatty acid specific regulation of TFEB and autophagy in the cardiomyocyte. The effect of glucolipotoxicity to reduce TFEB content was also confirmed in heart tissue from patients with Class-I obesity. Therefore, during glucolipotoxicity, suppression of lysosomal autophagy was associated with reduced lysosomal content, decreased cathepsin-B activity and diminished cellular TFEB content likely rendering myocytes susceptible to cardiac injury.
Assuntos
Autofagia/fisiologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Diabetes Mellitus/metabolismo , Lisossomos/metabolismo , Miócitos Cardíacos/metabolismo , Obesidade/metabolismo , Animais , Apoptose/fisiologia , Autofagossomos/metabolismo , Linhagem Celular , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ácido Oleico/metabolismo , Palmitatos/metabolismo , Proteínas/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/fisiologiaRESUMO
Antiretroviral therapy has increased the life span of HIV+ individuals; however, HIV-associated neurocognitive disorder (HAND) occurrence is increasing in aging HIV patients. Previous studies suggest HIV infection alters autophagy function in the aging CNS and HIV-1 proteins affect autophagy in monocyte-derived cells. Despite these findings, the mechanisms leading to dysregulated autophagy in the CNS remain unclear. Here we sought to determine how HIV Tat dysregulates autophagy in neurons. Tat caused a dose-dependent decrease in autophagosome markers, microtubule-associated protein-1 light chain ß II (LC3II), and sequestosome 1(SQSTM1), in a membrane-enriched fraction, suggesting Tat increases autophagic degradation. Bafilomycin A1 increased autophagosome number, LC3II, and SQSTM1 accumulation; Tat cotreatment diminished this effect. Tat had no effect when 3-methyladenine or knockdown of beclin 1 blocked early stages of autophagy. Tat increased numbers of LC3 puncta and resulted in the formation of abnormal autophagosomes in vitro. Likewise, in vivo studies in GFAP-Tat tg mice showed increased autophagosome accumulation in neurons, altered LC3II levels, and neurodegeneration. These effects were reversed by rapamycin treatment. Tat colocalized with autophagosome and lysosomal markers and enhanced the colocalization of autophagosome with lysosome markers. Furthermore, co-IP studies showed that Tat interacts with lysosomal-associated membrane protein 2A (LAMP2A) in vitro and in vivo, and LAMP2A overexpression reduces Tat-induced neurotoxicity. Hence, Tat protein may induce autophagosome and lysosome fusion through interaction with LAMP2A leading to abnormal neuronal autophagy function and dysregulated degradation of critical intracellular components. Therapies targeting Tat-mediated autophagy alterations may decrease neurodegeneration in aging patients with HAND.
Assuntos
Autofagia , Lisossomos/metabolismo , Neurônios/metabolismo , Fagossomos/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo , Complexo AIDS Demência/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , HIV-1/genética , Proteína 2 de Membrana Associada ao Lisossomo/genética , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Ligação Proteica , Ratos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genética , Produtos do Gene tat do Vírus da Imunodeficiência Humana/toxicidadeRESUMO
Vacuolar protein sorting-35 (VPS35) is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with autosomal dominant PD. However, it remains poorly understood if and how VPS35 deficiency or mutation contributes to PD pathogenesis. Here we provide evidence that links VPS35 deficiency to PD-like neuropathology. VPS35 was expressed in mouse dopamine (DA) neurons in substantia nigra pars compacta (SNpc) and STR (striatum)--regions that are PD vulnerable. VPS35-deficient mice exhibited PD-relevant deficits including accumulation of α-synuclein in SNpc-DA neurons, loss of DA transmitter and DA neurons in SNpc and STR, and impairment of locomotor behavior. Further mechanical studies showed that VPS35-deficient DA neurons or DA neurons expressing PD-linked VPS35 mutant (D620N) had impaired endosome-to-Golgi retrieval of lysosome-associated membrane glycoprotein 2a (Lamp2a) and accelerated Lamp2a degradation. Expression of Lamp2a in VPS35-deficient DA neurons reduced α-synuclein, supporting the view for Lamp2a as a receptor of chaperone-mediated autophagy to be critical for α-synuclein degradation. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis. Significance statement: VPS35 is a key component of the retromer complex that is essential for endosome-to-Golgi retrieval of membrane proteins. Mutations in the VPS35 gene have been identified in patients with PD. However, if and how VPS35 deficiency or mutation contributes to PD pathogenesis remains unclear. We demonstrated that VPS35 deficiency or mutation (D620N) in mice leads to α-synuclein accumulation and aggregation in the substantia nigra, accompanied with DA neurodegeneration. VPS35-deficient DA neurons exhibit impaired endosome-to-Golgi retrieval of Lamp2a, which may contribute to the reduced α-synuclein degradation through chaperone-mediated autophagy. These results suggest that VPS35 deficiency or mutation promotes PD pathogenesis, and reveals a crucial pathway, VPS35-Lamp2a-α-synuclein, to prevent PD pathogenesis.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Transtornos Parkinsonianos/metabolismo , Transporte Proteico/fisiologia , Proteínas de Transporte Vesicular/metabolismo , Animais , Autofagia/fisiologia , Western Blotting , Células Cultivadas , Neurônios Dopaminérgicos/patologia , Endossomos/metabolismo , Complexo de Golgi/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Confocal , Mutagênese Sítio-Dirigida , Transtornos Parkinsonianos/patologia , Reação em Cadeia da Polimerase em Tempo Real , Transfecção , alfa-Sinucleína/metabolismoRESUMO
One of the main pathways of lysosomal proteolysis is chaperone-mediated autophagy (CMA), which represents a selective mechanism for the degradation of specific soluble proteins within lysosomes. Along with the other two lysosomal pathways, macro- and micro-autophagy, CMA contributes to cellular quality control through the removal of damaged or malfunctioning proteins. The two intrinsic characteristics of CMA are the selective targeting and the direct translocation of substrate proteins into the lysosomal lumen, in a fine-tuned manner through the orchestrated action of a chaperone/co-chaperone complex localized both at the cytosol and the lysosomes. Even though CMA was originally identified as a stress-induced pathway, basal CMA activity is detectable in most cell types analyzed so far, including neurons. Additionally, CMA activity declines with age and this may become a major aggravating factor contributing to neurodegeneration. More specifically, it has been suggested that CMA impairment may underlie the accumulation of misfolded/aggregated proteins, such as alpha-synuclein or LRRK2, whose levels or conformations are critical to Parkinson's disease pathogenesis. On the other hand, CMA induction might accelerate clearance of pathogenic proteins and promote cell survival, suggesting that CMA represents a viable therapeutic target for the treatment of various proteinopathies. In the current review, we provide an overview of the current state of knowledge regarding the role of CMA under physiological and pathological conditions of the nervous system and discuss the implications of these findings for therapeutic interventions for Parkinson's disease and other neurodegenerative disorders. This article is part of Special Issue entitled "Neuronal Protein".
Assuntos
Autofagia/fisiologia , Chaperonas Moleculares/metabolismo , Terapia de Alvo Molecular/métodos , Doenças Neurodegenerativas/terapia , Animais , Autofagia/genética , Humanos , Chaperonas Moleculares/genética , Doenças Neurodegenerativas/genéticaRESUMO
INTRODUCTION: Radiotherapy is an equivalent alternative or complement to radical prostatectomy, with high therapeutic efficacy. High risk patients, however, experience high relapse rates, so that research on radio-sensitization is the most evident route to improve curability of this common disease. MATERIALS AND METHODS: In the current study we investigated the autophagic activity in a series of patients with localized prostate tumors treated with radical radiotherapy, using the LC3A and the LAMP2a proteins as markers of autophagosome and lysosome cellular content, respectively. The role of autophagy on prostate cancer cell line resistance to radiation was also examined. RESULTS: Using confocal microscopy on tissue biopsies, we showed that prostate cancer cells have, overall, high levels of LC3A and low levels of LAMP2a compared to normal prostate glands. Tumors with a 'highLC3A/lowLAMP2a' phenotype, suggestive of intensified lysosomal consumption, had a significantly poorer biochemical relapse free survival. The PC3 radioresistant cell line sustained remarkably its autophagic flux ability after radiation, while the DU145 radiosensitive one experiences a prolonged blockage of the autophagic process. This was assessed with aggresome accumulation detection and LC3A/LAMP2a double immunofluorescence, as well as with sequestrosome/p62 protein detection. By silencing the LC3A or LAMP2a expression, both cell lines became more sensitive to escalated doses of radiation. CONCLUSIONS: High base line autophagy activity and cell ability to sustain functional autophagy define resistance of prostate cancer cells to radiotherapy. This can be reversed by blocking up-regulated components of the autophagy pathway, which may prove of importance in the field of clinical radiotherapy.
Assuntos
Próstata/patologia , Próstata/efeitos da radiação , Neoplasias da Próstata/radioterapia , Idoso , Autofagia , Linhagem Celular Tumoral , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/análise , Masculino , Proteínas Associadas aos Microtúbulos/análise , Pessoa de Meia-Idade , Recidiva Local de Neoplasia/patologia , Neoplasias da Próstata/patologiaRESUMO
Autophagy is an intracellular degradative system that is believed to be involved in the aging process. The contribution of autophagy to age-related changes in the human skin is unclear. In this study, we examined the relationship between autophagy and skin aging. Transmission electron microscopy and immunofluorescence microscopy analyses of skin tissue and cultured dermal fibroblasts derived from women of different ages revealed an increase in the number of nascent double-membrane autophagosomes with age. Western blot analysis showed that the amount of LC3-II, a form associated with autophagic vacuolar membranes, was significantly increased in aged dermal fibroblasts compared with that in young dermal fibroblasts. Aged dermal fibroblasts were minimally affected by inhibition of autophagic activity. Although lipofuscin autofluorescence was elevated in aged dermal fibroblasts, the expression of Beclin-1 and Atg5-genes essential for autophagosome formation-was similar between young and aged dermal fibroblasts, suggesting that the increase of autophagosomes in aged dermal fibroblasts was due to impaired autophagic flux rather than an increase in autophagosome formation. Treatment of young dermal fibroblasts with lysosomal protease inhibitors, which mimic the condition of aged dermal fibroblasts with reduced autophagic activity, altered the fibroblast content of type I procollagen, hyaluronan and elastin, and caused a breakdown of collagen fibrils. Collectively, these findings suggest that the autophagy pathway is impaired in aged dermal fibroblasts, which leads to deterioration of dermal integrity and skin fragility.