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1.
Int J Mol Sci ; 22(19)2021 Sep 27.
Artigo em Inglês | MEDLINE | ID: mdl-34638755

RESUMO

Cholesterol and fatty acids are essential lipids that are critical for membrane biosynthesis and fetal organ development. Cholesteryl esters (CE) are degraded by hormone-sensitive lipase (HSL) in the cytosol and by lysosomal acid lipase (LAL) in the lysosome. Impaired LAL or HSL activity causes rare pathologies in humans, with HSL deficiency presenting less severe clinical manifestations. The infantile form of LAL deficiency, a lysosomal lipid storage disorder, leads to premature death. However, the importance of defective lysosomal CE degradation and its consequences during early life are incompletely understood. We therefore investigated how defective CE catabolism affects fetus and infant maturation using Lal and Hsl knockout (-/-) mouse models. This study demonstrates that defective lysosomal but not neutral lipolysis alters placental and fetal cholesterol homeostasis and exhibits an initial disease pathology already in utero as Lal-/- fetuses accumulate hepatic lysosomal lipids. Immediately after birth, LAL deficiency exacerbates with massive hepatic lysosomal lipid accumulation, which continues to worsen into young adulthood. Our data highlight the crucial role of LAL during early development, with the first weeks after birth being critical for aggravating LAL deficiency.


Assuntos
Lipólise , Fígado , Lisossomos , Esterol Esterase/deficiência , Doença de Wolman , Animais , Animais Recém-Nascidos , Modelos Animais de Doenças , Humanos , Fígado/metabolismo , Fígado/patologia , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos , Camundongos Knockout , Doença de Wolman/genética , Doença de Wolman/metabolismo , Doença de Wolman/patologia
2.
Cell Physiol Biochem ; 55(S4): 68-95, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34523304

RESUMO

Protein homeostasis strongly depends on the targeted and selective removal of unneeded or flawed proteins, of protein aggregates, and of damaged or excess organelles by the two main intracellular degradative systems, namely the ubiquitin proteasomal system (UPS) and the autophagosomal lysosomal system. Despite representing completely distinct mechanisms of degradation, which underlie differing regulatory mechanisms, growing evidence suggests that the UPS and autophagy strongly interact especially in situations of overwhelming and impairment, and that both are involved in podocyte proteostasis and in the pathogenesis of podocyte injury. The differential impact of autophagy and the UPS on podocyte biology and on podocyte disease development and progression is not understood. Recent advances in understanding the role of the UPS and autophagy in podocyte biology are reviewed here.


Assuntos
Autofagia , Nefropatias , Podócitos , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Animais , Autofagossomos/metabolismo , Autofagossomos/patologia , Humanos , Nefropatias/metabolismo , Nefropatias/patologia , Nefropatias/fisiopatologia , Lisossomos/metabolismo , Lisossomos/patologia , Podócitos/metabolismo , Podócitos/patologia
3.
Cells ; 10(9)2021 09 02.
Artigo em Inglês | MEDLINE | ID: mdl-34571934

RESUMO

Substrate reduction therapy (SRT) in clinic adequately manages the visceral manifestations in Gaucher disease (GD) but has no direct effect on brain disease. To understand the molecular basis of SRT in GD treatment, we evaluated the efficacy and underlying mechanism of SRT in an immortalized neuronal cell line derived from a Gba knockout (Gba-/-) mouse model. Gba-/- neurons accumulated substrates, glucosylceramide, and glucosylsphingosine. Reduced cell proliferation was associated with altered lysosomes and autophagy, decreased mitochondrial function, and activation of the mTORC1 pathway. Treatment of the Gba-/- neurons with venglustat analogue GZ452, a central nervous system-accessible SRT, normalized glucosylceramide levels in these neurons and their isolated mitochondria. Enlarged lysosomes were reduced in the treated Gba-/- neurons, accompanied by decreased autophagic vacuoles. GZ452 treatment improved mitochondrial membrane potential and oxygen consumption rate. Furthermore, GZ452 diminished hyperactivity of selected proteins in the mTORC1 pathway and improved cell proliferation of Gba-/- neurons. These findings reinforce the detrimental effects of substrate accumulation on mitochondria, autophagy, and mTOR in neurons. A novel rescuing mechanism of SRT was revealed on the function of mitochondrial and autophagy-lysosomal pathways in GD. These results point to mitochondria and the mTORC1 complex as potential therapeutic targets for treatment of GD.


Assuntos
Autofagia , Doença de Gaucher/tratamento farmacológico , Glucosilceramidase/antagonistas & inibidores , Inibidores de Glicosídeo Hidrolases/farmacologia , Mitocôndrias/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Animais , Doença de Gaucher/metabolismo , Doença de Gaucher/patologia , Glucosilceramidase/fisiologia , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Lisossomos/patologia , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Neurônios/metabolismo , Neurônios/patologia , Serina-Treonina Quinases TOR/genética
4.
Elife ; 102021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34585663

RESUMO

Removal of damaged organelles via the process of selective autophagy constitutes a major form of cellular quality control. Damaged organelles are recognized by a dedicated surveillance machinery, leading to the assembly of an autophagosome around the damaged organelle, prior to fusion with the degradative lysosomal compartment. Lysosomes themselves are also prone to damage and are degraded through the process of lysophagy. While early steps involve recognition of ruptured lysosomal membranes by glycan-binding galectins and ubiquitylation of transmembrane lysosomal proteins, many steps in the process, and their interrelationships, remain poorly understood, including the role and identity of cargo receptors required for completion of lysophagy. Here, we employ quantitative organelle capture and proximity biotinylation proteomics of autophagy adaptors, cargo receptors, and galectins in response to acute lysosomal damage, thereby revealing the landscape of lysosome-associated proteome remodeling during lysophagy. Among the proteins dynamically recruited to damaged lysosomes were ubiquitin-binding autophagic cargo receptors. Using newly developed lysophagic flux reporters including Lyso-Keima, we demonstrate that TAX1BP1, together with its associated kinase TBK1, are both necessary and sufficient to promote lysophagic flux in both HeLa cells and induced neurons (iNeurons). While the related receptor Optineurin (OPTN) can drive damage-dependent lysophagy when overexpressed, cells lacking either OPTN or CALCOCO2 still maintain significant lysophagic flux in HeLa cells. Mechanistically, TAX1BP1-driven lysophagy requires its N-terminal SKICH domain, which binds both TBK1 and the autophagy regulatory factor RB1CC1, and requires upstream ubiquitylation events for efficient recruitment and lysophagic flux. These results identify TAX1BP1 as a central component in the lysophagy pathway and provide a proteomic resource for future studies of the lysophagy process.


Assuntos
Autofagia/genética , Lisossomos/patologia , Macroautofagia/fisiologia , Ubiquitina/metabolismo , Humanos , Ligação Proteica , Proteômica
5.
Leukemia ; 35(10): 2759-2770, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34462526

RESUMO

Lysosomes, since their discovery, have been primarily known for degrading cellular macromolecules. However, in recent studies, they have begun to emerge as crucial regulators of cell homeostasis. They are at the crossroads of catabolic and anabolic pathways and are intricately involved in cellular trafficking, nutrient signaling, energy metabolism, and immune regulation. Their involvement in such essential cellular functions has renewed clinical interest in targeting the lysosome as a novel way to treat disease, particularly cancer. Acute myeloid leukemia (AML) is an aggressive blood cancer with a low survival probability, particularly in older patients. The genomic landscape of AML has been extensively characterized but few targeted therapies (with the exception of differentiation therapy) can achieve a long-term cure. Therefore, there is an unmet need for less intensive and more tolerable therapeutic interventions. In this review, we will give an overview on the myriad of functions performed by lysosomes and their importance in malignant disease. Furthermore, we will discuss their relevance in hematopoietic cells and different ways to potentially target them in AML.


Assuntos
Leucemia Mieloide Aguda/patologia , Lisossomos/patologia , Animais , Humanos , Terapia de Alvo Molecular/métodos , Transdução de Sinais/fisiologia
6.
Am J Physiol Lung Cell Mol Physiol ; 321(3): L555-L565, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34261337

RESUMO

Patients with cystic fibrosis (CF) have defective macrophage phagocytosis and efferocytosis. Several reports demonstrate that neutrophil elastase (NE), a major inflammatory protease in the CF airway, impairs macrophage phagocytic function. To date, NE-impaired macrophage phagocytic function has been attributed to cleavage of cell surface receptors or opsonins. We applied an unbiased proteomic approach to identify other potential macrophage targets of NE protease activity that may regulate phagocytic function. Using the murine macrophage cell line, RAW 264.7, human blood monocyte-derived macrophages, and primary alveolar macrophages from Cftr-null and wild-type littermate mice, we demonstrated that NE exposure blocked phagocytosis of Escherichia coli bio-particles. We performed liquid chromatography-tandem mass spectroscopy (LC-MS/MS) proteomic analysis of the conditioned media from RAW264.7 treated either with active NE or inactive (boiled) NE as a control. Out of 840 proteins identified in the conditioned media, active NE upregulated 142 proteins and downregulated 211 proteins. NE released not only cell surface proteins into the media but also cytoskeletal, mitochondrial, cytosolic, and nuclear proteins that were detected in the conditioned media. At least 32 proteins were associated with the process of phagocytosis including 11 phagocytic receptors [including lipoprotein receptor-related protein 1 (LRP1)], 7 proteins associated with phagocytic cup formation, and 14 proteins involved in phagocytic maturation (including calpain-2) and phagolysosome formation. NE had a broad effect on the proteome required for regulation of all stages of phagocytosis and phagolysosome formation. Furthermore, the NE sheddome/secretome included proteins from other macrophage cellular domains, suggesting that NE may globally regulate macrophage structure and function.


Assuntos
Elastase de Leucócito/metabolismo , Lisossomos/metabolismo , Macrófagos/metabolismo , Fagocitose , Fagossomos/metabolismo , Adolescente , Adulto , Animais , Criança , Pré-Escolar , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Feminino , Humanos , Elastase de Leucócito/genética , Lisossomos/genética , Lisossomos/patologia , Macrófagos/fisiologia , Masculino , Camundongos , Camundongos Mutantes , Fagossomos/genética , Fagossomos/patologia , Células RAW 264.7
7.
Cell Death Dis ; 12(7): 673, 2021 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-34218252

RESUMO

The STING pathway and its induction of autophagy initiate a potent immune defense response upon the recognition of pathogenic DNA. However, this protective response is minimal, as STING activation worsens organ damage, and abnormal autophagy is observed during progressive sepsis. Whether and how the STING pathway affects autophagic flux during sepsis-induced acute lung injury (sALI) are currently unknown. Here, we demonstrate that the level of circulating mtDNA and degree of STING activation are increased in sALI patients. Furthermore, STING activation was found to play a pivotal role in mtDNA-mediated lung injury by evoking an inflammatory storm and disturbing autophagy. Mechanistically, STING activation interferes with lysosomal acidification in an interferon (IFN)-dependent manner without affecting autophagosome biogenesis or fusion, aggravating sepsis. Induction of autophagy or STING deficiency alleviated lung injury. These findings provide new insights into the role of STING in the regulatory mechanisms behind extrapulmonary sALI.


Assuntos
Lesão Pulmonar Aguda/metabolismo , Autofagia , Ácidos Nucleicos Livres/sangue , DNA Mitocondrial/sangue , Pulmão/metabolismo , Proteínas de Membrana/metabolismo , Sepse/complicações , Lesão Pulmonar Aguda/etiologia , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/patologia , Animais , Proteínas Relacionadas à Autofagia/metabolismo , Ácidos Nucleicos Livres/genética , DNA Mitocondrial/genética , Modelos Animais de Doenças , Humanos , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/metabolismo , Interferons/metabolismo , Pulmão/patologia , Lisossomos/genética , Lisossomos/metabolismo , Lisossomos/patologia , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , Sepse/microbiologia
8.
Life Sci ; 279: 119665, 2021 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-34087281

RESUMO

AIMS: Although it is well established that skeletal muscle contains oxytocin (OT) receptors and OT-knockout mice show premature development of sarcopenia, the role of OT in controlling skeletal muscle mass is still unknown. Therefore, the present work aimed to determine OT's effects on skeletal muscle protein metabolism. MAIN METHODS: Total proteolysis, proteolytic system activities and protein synthesis were assessed in isolated soleus muscle from prepubertal female rats. Through in vivo experiments, rats received 3-day OT treatment (3UI.kg-1.day-1, i.p.) or saline, and muscles were harvested for mass-gain assessment. KEY FINDINGS: In vitro OT receptor stimulation reduced total proteolysis, specifically through attenuation of the lysosomal and proteasomal proteolytic systems, and in parallel activated the Akt/FoxO1 signaling and suppressed atrogenes (e.g., MuRF-1 and atrogin-1) expression induced by motor denervation. On the other hand, the protein synthesis was not altered by in vitro treatment with the OT receptor-selective agonist. Although short-term OT treatment did not change the atrogene mRNA levels, the protein synthesis was stimulated, resulting in soleus mass gain, probably through an indirect effect. SIGNIFICANCE: Taken together, these data show for the first time that OT directly inhibits the proteolytic activities of the lysosomal and proteasomal systems in rat oxidative skeletal muscle by suppressing atrogene expression via stimulation of Akt/FoxO signaling. Moreover, the data obtained from in vivo experiments suggest OT's ability to control rat oxidative skeletal muscle mass.


Assuntos
Anabolizantes/farmacologia , Lisossomos/metabolismo , Músculo Esquelético/metabolismo , Ocitocina/farmacologia , Biossíntese de Proteínas , Proteólise , Animais , Feminino , Lisossomos/efeitos dos fármacos , Lisossomos/patologia , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Estresse Oxidativo , Ocitócicos/farmacologia , Ratos , Ratos Wistar , Transdução de Sinais
9.
PLoS Genet ; 17(6): e1009603, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34143769

RESUMO

The inability to maintain a strictly regulated endo(lyso)somal acidic pH through the proton-pumping action of the vacuolar-ATPases (v-ATPases) has been associated with various human diseases including heritable connective tissue disorders. Autosomal recessive (AR) cutis laxa (CL) type 2C syndrome is associated with genetic defects in the ATP6V1E1 gene and is characterized by skin wrinkles or loose redundant skin folds with pleiotropic systemic manifestations. The underlying pathological mechanisms leading to the clinical presentations remain largely unknown. Here, we show that loss of atp6v1e1b in zebrafish leads to early mortality, associated with craniofacial dysmorphisms, vascular anomalies, cardiac dysfunction, N-glycosylation defects, hypotonia, and epidermal structural defects. These features are reminiscent of the phenotypic manifestations in ARCL type 2C patients. Our data demonstrates that loss of atp6v1e1b alters endo(lyso)somal protein levels, and interferes with non-canonical v-ATPase pathways in vivo. In order to gain further insights into the processes affected by loss of atp6v1e1b, we performed an untargeted analysis of the transcriptome, metabolome, and lipidome in early atp6v1e1b-deficient larvae. We report multiple affected pathways including but not limited to oxidative phosphorylation, sphingolipid, fatty acid, and energy metabolism together with profound defects on mitochondrial respiration. Taken together, our results identify complex pathobiological effects due to loss of atp6v1e1b in vivo.


Assuntos
Anormalidades Múltiplas/genética , Cútis Laxa/genética , Células Epiteliais/metabolismo , Pele/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Proteínas de Peixe-Zebra/genética , Anormalidades Múltiplas/metabolismo , Anormalidades Múltiplas/patologia , Animais , Cútis Laxa/metabolismo , Cútis Laxa/patologia , Modelos Animais de Doenças , Endossomos/metabolismo , Endossomos/patologia , Células Epiteliais/patologia , Regulação da Expressão Gênica , Humanos , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Lipidômica , Longevidade/genética , Lisossomos/metabolismo , Lisossomos/patologia , Metaboloma/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Fosforilação Oxidativa , Isoformas de Proteínas/deficiência , Isoformas de Proteínas/genética , Pele/patologia , Síndrome , Transcriptoma , ATPases Vacuolares Próton-Translocadoras/deficiência , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/deficiência
10.
Cells ; 10(5)2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-34067971

RESUMO

Many invasive cancers emerge through a years-long process of somatic evolution, characterized by an accumulation of heritable genetic and epigenetic changes and the emergence of increasingly aggressive clonal populations. In solid tumors, such as breast ductal carcinoma, the extracellular environment for cells within the nascent tumor is harsh and imposes different types of stress on cells, such as hypoxia, nutrient deprivation, and cytokine inflammation. Acidosis is a constant stressor of most cancer cells due to its production through fermentation of glucose to lactic acid in hypoxic or normoxic regions (Warburg effect). Over a short period of time, acid stress can have a profound effect on the function of lysosomes within the cells exposed to this environment, and after long term exposure, lysosomal function of the cancer cells can become completely dysregulated. Whether this dysregulation is due to an epigenetic change or evolutionary selection has yet to be determined, but understanding the mechanisms behind this dysregulation could identify therapeutic opportunities.


Assuntos
Acidose/metabolismo , Neoplasias da Mama/metabolismo , Carcinoma Ductal de Mama/metabolismo , Lisossomos/metabolismo , Microambiente Tumoral , Acidose/tratamento farmacológico , Acidose/genética , Acidose/patologia , Animais , Antineoplásicos/uso terapêutico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/tratamento farmacológico , Carcinoma Ductal de Mama/genética , Carcinoma Ductal de Mama/patologia , Metabolismo Energético , Feminino , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/efeitos dos fármacos , Lisossomos/patologia , Terapia de Alvo Molecular , Efeito Warburg em Oncologia
11.
Cell Physiol Biochem ; 55(3): 277-300, 2021 May 22.
Artigo em Inglês | MEDLINE | ID: mdl-34019755

RESUMO

The lysosome is a single ubiquitous membrane-enclosed intracellular organelle with an acidic pH present in all eukaryotic cells, which contains large numbers of hydrolytic enzymes with their maximal enzymatic activity at a low pH (pH ≤ 5) such as proteases, nucleases, and phosphatases that are able to degrade extracellular and intracellular components. It is well known that lysosomes act as a center for degradation and recycling of large numbers of macromolecules delivered by endocytosis, phagocytosis, and autophagy. Lysosomes are recognized as key organelles for cellular clearance and are involved in many cellular processes and maintain cellular homeostasis. Recently, it has been shown that lysosome function and its related pathways are of particular importance in vascular regulation and related diseases. In this review, we highlighted studies that have improved our understanding of the connection between lysosome function and vascular physiological and pathophysiological activities in arterial smooth muscle cells (SMCs) and endothelial cells (ECs). Sphingolipids-metabolizingenzymes in lysosomes play critical roles in intracellular signaling events that influence cellular behavior and function in SMCs and ECs. The focus of this review will be to define the mechanism by which the lysosome contributes to cardiovascular regulation and diseases. It is believed that exploring the role of lysosomal function and its sphingolipid metabolism in the initiation and progression of vascular disease and regulation may provide novel insights into the understanding of vascular pathobiology and helps develop more effective therapeutic strategies for vascular diseases.


Assuntos
Doenças Cardiovasculares/metabolismo , Células Endoteliais/metabolismo , Lisossomos/metabolismo , Miócitos de Músculo Liso/metabolismo , Esfingolipídeos/metabolismo , Animais , Doenças Cardiovasculares/patologia , Células Endoteliais/patologia , Humanos , Lisossomos/patologia , Miócitos de Músculo Liso/patologia
12.
Phytomedicine ; 87: 153587, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34044254

RESUMO

BACKGROUND: The survival rate and therapeutic options for patients with bladder cancer have improved little in recent decades. Guggulsterone (GS), a phytoestrogen, has been investigated as an anticancer drug in various malignancies. PURPOSE: The present study aimed to evaluate the anticancer effects of E-isomer and Z-isomer GS in the human bladder cancer cell lines TSGH8301 (low-grade) and T24 (high-grade) and their underlying mechanisms. METHODS: The cell survival effect of GS was investigated by the MTT and colony formation assays in bladder cancer cell lines. Flow cytometry was used to analyze the cell cycle and cell death. Migration ability was measured by wound healing and transwell assays. Protein expression was determined by Western blot after GS treatment. The potency of GS on subcutaneous TSGH8301 bladder tumors was evaluated using an in vivo imaging system. RESULTS: E-isomer GS reduced the survival rate of both low- and high-grade human bladder cancer cells. GS caused cell cycle arrest, accompanied by the decrease and increase in cyclin A and p21 levels, respectively. Additionally, caspase-dependent apoptosis was observed following GS treatment. Furthermore, GS treatment downregulated mTOR-Akt signaling and induced autophagy with p62 and LC3ß-II expression. Moreover, the farnesoid X receptor was involved in GS-inhibited cell growth. In addition, GS reduced the migration ability with a decrease in integrin-focal adhesion kinase and myosin light chain. Interestingly, the suppression of GS-mediated migration was prevented by the lysosomal inhibitor ammonium chloride (NH4Cl). GS also reduced TSGH8301 bladder cancer cell progression by increasing the level of p21, cleaved caspase 3, cleaved poly (ADP-ribose) polymerase (PARP), and LC3ß-II in vivo. CONCLUSIONS: The current findings suggest that GS treatment may serve as a potential anticancer therapy for different grades of urothelial carcinoma.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Lisossomos/efeitos dos fármacos , Pregnenodionas/farmacologia , Neoplasias da Bexiga Urinária/tratamento farmacológico , Neoplasias da Bexiga Urinária/patologia , Animais , Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Caspase 3/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Camundongos Endogâmicos BALB C , Serina-Treonina Quinases TOR/metabolismo , Neoplasias da Bexiga Urinária/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
13.
J Cell Mol Med ; 25(12): 5729-5743, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33949118

RESUMO

Cyclosporine A (CsA) is an immunosuppressor widely used for the prevention of acute rejection during solid organ transplantation. However, severe nephrotoxicity has substantially limited its long-term usage. Recently, an impaired autophagy pathway was suggested to be involved in the pathogenesis of chronic CsA nephrotoxicity. However, the underlying mechanisms of CsA-induced autophagy blockade in tubular cells remain unclear. In the present study, we observed that CsA suppressed the activation and expression of transcription factor EB (TFEB) by increasing the activation of mTOR, in turn promoting lysosomal dysfunction and autophagy flux blockade in tubular epithelial cells (TECs) in vivo and in vitro. Restoration of TFEB activation by Torin1-mediated mTOR inhibition significantly improved lysosomal function and rescued autophagy pathway activity, suppressing TEC injury. In summary, targeting TFEB-mediated autophagy flux represents a potential therapeutic strategy for CsA-induced nephrotoxicity.


Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Ciclosporina/toxicidade , Células Epiteliais/patologia , Túbulos Renais/patologia , Lisossomos/patologia , Serina-Treonina Quinases TOR/metabolismo , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Imunossupressores/toxicidade , Túbulos Renais/efeitos dos fármacos , Túbulos Renais/metabolismo , Lisossomos/efeitos dos fármacos , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Serina-Treonina Quinases TOR/genética
14.
J Cell Mol Med ; 25(12): 5769-5781, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33973365

RESUMO

Abnormal accumulation of lipids and massive deposition of foam cells is a primary event in the pathogenesis of atherosclerosis. Recent studies have demonstrated that autophagy and lysosomal function of atherosclerotic macrophages are impaired, which exacerbates the accumulation of lipid in macrophages and formation of foam cells. Gastrodin, a major active component of Gastrodia elata Bl., has exerted a protective effect on nervous system, but the effect of gastrodin on atherosclerotic vascular disease remains unknown. We aimed to evaluate the effect of gastrodin on autophagy and lysosomal function of foam cells and explored the mechanism underlying gastrodin's effect on the formation of foam cells. In an in vitro foam cell model constructed by incubating macrophages with oxygenized low-density lipoproteins (ox-LDL), our results showed that lysosomal function and autophagy of foam cells were compromised. Gastrodin restored lysosomal function and autophagic activity via the induction of lysosomal biogenesis and autophagy. The restoration of lysosomal function and autophagic activity enhanced cholesterol efflux from macrophages, therefore, reducing lipid accumulation and preventing formation of foam cells. AMP-activated protein kinase (AMPK) was activated by gastrodin to promote phosphorylation and nuclear translocation of forkhead box O1 (FoxO1), subsequently resulting in increased transcription factor EB (TFEB) expression. TFEB was activated by gastrodin to promote lysosomal biogenesis and autophagy. Our study revealed that the effect of gastrodin on foam cell formation and that induction of lysosomal biogenesis and autophagy of foam cells through AMPK-FoxO1-TFEB signalling axis may be a novel therapeutic target of atherosclerosis.


Assuntos
Aterosclerose/prevenção & controle , Autofagia , Álcoois Benzílicos/farmacologia , Células Espumosas/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Glucosídeos/farmacologia , Lisossomos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Células Espumosas/metabolismo , Células Espumosas/patologia , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Lisossomos/metabolismo , Lisossomos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos
15.
Nat Neurosci ; 24(7): 1020-1034, 2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34031600

RESUMO

Single-cell transcriptomics provide a systematic map of gene expression in different human cell types. The next challenge is to systematically understand cell-type-specific gene function. The integration of CRISPR-based functional genomics and stem cell technology enables the scalable interrogation of gene function in differentiated human cells. Here we present the first genome-wide CRISPR interference and CRISPR activation screens in human neurons. We uncover pathways controlling neuronal response to chronic oxidative stress, which is implicated in neurodegenerative diseases. Unexpectedly, knockdown of the lysosomal protein prosaposin strongly sensitizes neurons, but not other cell types, to oxidative stress by triggering the formation of lipofuscin, a hallmark of aging, which traps iron, generating reactive oxygen species and triggering ferroptosis. We also determine transcriptomic changes in neurons after perturbation of genes linked to neurodegenerative diseases. To enable the systematic comparison of gene function across different human cell types, we establish a data commons named CRISPRbrain.


Assuntos
Ferroptose/fisiologia , Perfilação da Expressão Gênica/métodos , Lisossomos/metabolismo , Neurônios/metabolismo , Saposinas/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Humanos , Lisossomos/patologia , Neurônios/patologia , Estresse Oxidativo/fisiologia
16.
Cells ; 10(3)2021 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-33800736

RESUMO

Parkinson's disease (PD) is a complex neurodegenerative disorder that is currently incurable. As a consequence of an incomplete understanding of the etiology of the disease, therapeutic strategies mainly focus on symptomatic treatment. Even though the majority of PD cases remain idiopathic (~90%), several genes have been identified to be causative for PD, facilitating the generation of animal models that are a good alternative to study disease pathways and to increase our understanding of the underlying mechanisms of PD. Drosophila melanogaster has proven to be an excellent model in these studies. In this review, we will discuss the different PD models in flies and key findings identified in flies in different affected pathways in PD. Several molecular changes have been identified, of which mitochondrial dysfunction and a defective endo-lysosomal pathway emerge to be the most relevant for PD pathogenesis. Studies in flies have significantly contributed to our knowledge of how disease genes affect and interact in these pathways enabling a better understanding of the disease etiology and providing possible therapeutic targets for the treatment of PD, some of which have already resulted in clinical trials.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Mitocôndrias/genética , Doença de Parkinson/genética , Proteínas Serina-Treonina Quinases/genética , Ubiquitina-Proteína Ligases/genética , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Modelos Animais de Doenças , Proteínas de Drosophila/deficiência , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Endossomos/patologia , Regulação da Expressão Gênica , Humanos , Lisossomos/metabolismo , Lisossomos/patologia , Redes e Vias Metabólicas/genética , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/genética , Estresse Oxidativo , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Proteínas Serina-Treonina Quinases/deficiência , Transdução de Sinais , Ubiquitina-Proteína Ligases/deficiência
17.
PLoS Genet ; 17(4): e1009406, 2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33830999

RESUMO

Phospholipase D3 (PLD3) is a protein of unclear function that structurally resembles other members of the phospholipase D superfamily. A coding variant in this gene confers increased risk for the development of Alzheimer's disease (AD), although the magnitude of this effect has been controversial. Because of the potential significance of this obscure protein, we undertook a study to observe its distribution in normal human brain and AD-affected brain, determine whether PLD3 is relevant to memory and cognition in sporadic AD, and to evaluate its molecular function. In human neuropathological samples, PLD3 was primarily found within neurons and colocalized with lysosome markers (LAMP2, progranulin, and cathepsins D and B). This colocalization was also present in AD brain with prominent enrichment on lysosomal accumulations within dystrophic neurites surrounding ß-amyloid plaques. This pattern of protein distribution was conserved in mouse brain in wild type and the 5xFAD mouse model of cerebral ß-amyloidosis. We discovered PLD3 has phospholipase D activity in lysosomes. A coding variant in PLD3 reported to confer AD risk significantly reduced enzymatic activity compared to wild-type PLD3. PLD3 mRNA levels in the human pre-frontal cortex inversely correlated with ß-amyloid pathology severity and rate of cognitive decline in 531 participants enrolled in the Religious Orders Study and Rush Memory and Aging Project. PLD3 levels across genetically diverse BXD mouse strains and strains crossed with 5xFAD mice correlated strongly with learning and memory performance in a fear conditioning task. In summary, this study identified a new functional mammalian phospholipase D isoform which is lysosomal and closely associated with both ß-amyloid pathology and cognition.


Assuntos
Doença de Alzheimer/genética , Disfunção Cognitiva/genética , Predisposição Genética para Doença , Fosfolipase D/genética , Doença de Alzheimer/enzimologia , Doença de Alzheimer/patologia , Animais , Autopsia , Disfunção Cognitiva/enzimologia , Disfunção Cognitiva/patologia , Modelos Animais de Doenças , Células HeLa , Humanos , Lisossomos/enzimologia , Lisossomos/patologia , Camundongos , Neurônios/enzimologia , Neurônios/patologia , Placa Amiloide/enzimologia , Placa Amiloide/genética , Placa Amiloide/patologia
18.
Acta Neuropathol Commun ; 9(1): 70, 2021 04 14.
Artigo em Inglês | MEDLINE | ID: mdl-33853668

RESUMO

Huntington's disease (HD) is a late onset, inherited neurodegenerative disorder for which early pathogenic events remain poorly understood. Here we show that mutant exon 1 HTT proteins are recruited to a subset of cytoplasmic aggregates in the cell bodies of neurons in brain sections from presymptomatic HD, but not wild-type, mice. This occurred in a disease stage and polyglutamine-length dependent manner. We successfully adapted a high-resolution correlative light and electron microscopy methodology, originally developed for mammalian and yeast cells, to allow us to correlate light microscopy and electron microscopy images on the same brain section within an accuracy of 100 nm. Using this approach, we identified these recruitment sites as single membrane bound, vesicle-rich endolysosomal organelles, specifically as (1) multivesicular bodies (MVBs), or amphisomes and (2) autolysosomes or residual bodies. The organelles were often found in close-proximity to phagophore-like structures. Immunogold labeling localized mutant HTT to non-fibrillar, electron lucent structures within the lumen of these organelles. In presymptomatic HD, the recruitment organelles were predominantly MVBs/amphisomes, whereas in late-stage HD, there were more autolysosomes or residual bodies. Electron tomograms indicated the fusion of small vesicles with the vacuole within the lumen, suggesting that MVBs develop into residual bodies. We found that markers of MVB-related exocytosis were depleted in presymptomatic mice and throughout the disease course. This suggests that endolysosomal homeostasis has moved away from exocytosis toward lysosome fusion and degradation, in response to the need to clear the chronically aggregating mutant HTT protein, and that this occurs at an early stage in HD pathogenesis.


Assuntos
Endossomos/patologia , Doença de Huntington/patologia , Corpos de Inclusão/ultraestrutura , Lisossomos/patologia , Neurônios/patologia , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Encéfalo/ultraestrutura , Endossomos/metabolismo , Endossomos/ultraestrutura , Técnicas de Introdução de Genes , Humanos , Proteína Huntingtina/genética , Doença de Huntington/genética , Doença de Huntington/metabolismo , Corpos de Inclusão/metabolismo , Corpos de Inclusão/patologia , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Camundongos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mutação , Neurônios/metabolismo , Neurônios/ultraestrutura
19.
Nat Commun ; 12(1): 2107, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33833240

RESUMO

Vacuolar H+-ATPases (V-ATPases) transport protons across cellular membranes to acidify various organelles. ATP6V0A1 encodes the a1-subunit of the V0 domain of V-ATPases, which is strongly expressed in neurons. However, its role in brain development is unknown. Here we report four individuals with developmental and epileptic encephalopathy with ATP6V0A1 variants: two individuals with a de novo missense variant (R741Q) and the other two individuals with biallelic variants comprising one almost complete loss-of-function variant and one missense variant (A512P and N534D). Lysosomal acidification is significantly impaired in cell lines expressing three missense ATP6V0A1 mutants. Homozygous mutant mice harboring human R741Q (Atp6v0a1R741Q) and A512P (Atp6v0a1A512P) variants show embryonic lethality and early postnatal mortality, respectively, suggesting that R741Q affects V-ATPase function more severely. Lysosomal dysfunction resulting in cell death, accumulated autophagosomes and lysosomes, reduced mTORC1 signaling and synaptic connectivity, and lowered neurotransmitter contents of synaptic vesicles are observed in the brains of Atp6v0a1A512P/A512P mice. These findings demonstrate the essential roles of ATP6V0A1/Atp6v0a1 in neuronal development in terms of integrity and connectivity of neurons in both humans and mice.


Assuntos
Encefalopatias/genética , Encéfalo/crescimento & desenvolvimento , Neurônios/fisiologia , Neurotransmissores/metabolismo , ATPases Vacuolares Próton-Translocadoras/genética , Animais , Autofagossomos/patologia , Mapeamento Encefálico/métodos , Catepsina D/metabolismo , Linhagem Celular , Células HEK293 , Humanos , Mutação com Perda de Função/genética , Lisossomos/patologia , Imageamento por Ressonância Magnética/métodos , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Camundongos , Mutação de Sentido Incorreto/genética , Neurônios/citologia , Vesículas Sinápticas/patologia
20.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-33804256

RESUMO

Lysosomal acid phosphatase 2 (Acp2) mutant mice (naked-ataxia, nax) have a severe cerebellar cortex defect with a striking reduction in the number of granule cells. Using a combination of in vivo and in vitro immunohistochemistry, Western blotting, BrdU assays, and RT-qPCR, we show downregulation of MYCN and dysregulation of the SHH signaling pathway in the nax cerebellum. MYCN protein expression is significantly reduced at P10, but not at the peak of proliferation at around P6 when the number of granule cells is strikingly reduced in the nax cerebellum. Despite the significant role of the SHH-MycN pathway in granule cell proliferation, our study suggests that a broader molecular pathway and additional mechanisms regulating granule cell development during the clonal expansion period are impaired in the nax cerebellum. In particular, our results indicate that downregulation of the protein synthesis machinery may contribute to the reduced number of granule cells in the nax cerebellum.


Assuntos
Fosfatase Ácida/genética , Ataxia Cerebelar/genética , Córtex Cerebelar/metabolismo , Proteínas Hedgehog/genética , Proteína Proto-Oncogênica N-Myc/genética , Animais , Diferenciação Celular/genética , Proliferação de Células/genética , Ataxia Cerebelar/metabolismo , Ataxia Cerebelar/patologia , Córtex Cerebelar/anormalidades , Córtex Cerebelar/patologia , Grânulos Citoplasmáticos/genética , Grânulos Citoplasmáticos/patologia , Modelos Animais de Doenças , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lisossomos/genética , Lisossomos/patologia , Camundongos , Mutação , Neurônios/metabolismo , Neurônios/patologia , Células de Purkinje/metabolismo , Células de Purkinje/patologia , Transdução de Sinais/genética
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