RESUMEN
Alternative lengthening of telomeres (ALT) is a homology-directed repair (HDR) mechanism of telomere elongation that controls proliferation in aggressive cancers. We show that the disruption of RAD51-associated protein 1 (RAD51AP1) in ALT+ cancer cells leads to generational telomere shortening. This is due to RAD51AP1's involvement in RAD51-dependent homologous recombination (HR) and RAD52-POLD3-dependent break induced DNA synthesis. RAD51AP1 KO ALT+ cells exhibit telomere dysfunction and cytosolic telomeric DNA fragments that are sensed by cGAS. Intriguingly, they activate ULK1-ATG7-dependent autophagy as a survival mechanism to mitigate DNA damage and apoptosis. Importantly, RAD51AP1 protein levels are elevated in ALT+ cells due to MMS21 associated SUMOylation. Mutation of a single SUMO-targeted lysine residue perturbs telomere dynamics. These findings indicate that RAD51AP1 is an essential mediator of the ALT mechanism and is co-opted by post-translational mechanisms to maintain telomere length and ensure proliferation of ALT+ cancer cells.
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Proteínas de Unión al ADN/metabolismo , Neoplasias/metabolismo , Proteínas de Unión al ARN/metabolismo , Homeostasis del Telómero , Telómero/metabolismo , Autofagia , Proteína 7 Relacionada con la Autofagia/genética , Proteína 7 Relacionada con la Autofagia/metabolismo , Homólogo de la Proteína 1 Relacionada con la Autofagia/genética , Homólogo de la Proteína 1 Relacionada con la Autofagia/metabolismo , Proliferación Celular , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Proteínas de Unión al ADN/genética , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Células HeLa , Recombinación Homóloga , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Ligasas/genética , Ligasas/metabolismo , Lisina , Neoplasias/genética , Neoplasias/patología , Nucleotidiltransferasas/genética , Nucleotidiltransferasas/metabolismo , Estabilidad Proteica , Proteínas de Unión al ARN/genética , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Transducción de Señal , Sumoilación , Telómero/genética , Telómero/patologíaRESUMEN
The recently identified ferroptotic cell death is characterized by excessive accumulation of hydroperoxy-arachidonoyl (C20:4)- or adrenoyl (C22:4)- phosphatidylethanolamine (Hp-PE). The selenium-dependent glutathione peroxidase 4 (GPX4) inhibits ferroptosis, converting unstable ferroptotic lipid hydroperoxides to nontoxic lipid alcohols in a tissue-specific manner. While placental oxidative stress and lipotoxicity are hallmarks of placental dysfunction, the possible role of ferroptosis in placental dysfunction is largely unknown. We found that spontaneous preterm birth is associated with ferroptosis and that inhibition of GPX4 causes ferroptotic injury in primary human trophoblasts and during mouse pregnancy. Importantly, we uncovered a role for the phospholipase PLA2G6 (PNPLA9, iPLA2beta), known to metabolize Hp-PE to lyso-PE and oxidized fatty acid, in mitigating ferroptosis induced by GPX4 inhibition in vitro or by hypoxia/reoxygenation injury in vivo. Together, we identified ferroptosis signaling in the human and mouse placenta, established a role for PLA2G6 in attenuating trophoblastic ferroptosis, and provided mechanistic insights into the ill-defined placental lipotoxicity that may inspire PLA2G6-targeted therapeutic strategies.
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Ferroptosis/fisiología , Fosfolipasas A2 Grupo VI/metabolismo , Trofoblastos/metabolismo , Animales , Femenino , Glutatión Peroxidasa/metabolismo , Fosfolipasas A2 Grupo VI/genética , Fosfolipasas A2 Grupo VI/fisiología , Humanos , Hierro/metabolismo , Peróxidos Lipídicos/metabolismo , Ratones , Ratones Noqueados , Fosfatidiletanolaminas/metabolismo , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Placenta/metabolismo , Embarazo , Nacimiento Prematuro/metabolismo , Transducción de SeñalRESUMEN
BACKGROUND: The epithelium is increasingly recognized as a pathologic contributor to asthma and its phenotypes. Although delayed wound closure by asthmatic epithelial cells is consistently observed, underlying mechanisms remain poorly understood, partly due to difficulties in studying dynamic physiologic processes involving polarized multilayered cell systems. Although type-2 immunity has been suggested to play a role, the mechanisms by which repair is diminished are unclear. OBJECTIVES: This study sought to develop and utilize primary multilayered polarized epithelial cell systems, derived from patients with asthma, to evaluate cell migration in response to wounding under type-2 and untreated conditions. METHODS: A novel wounding device for multilayered polarized cells, along with time-lapse live cell/real-time confocal imaging were evaluated under IL-13 and untreated conditions. The influence of inhibition of 15 lipoxygenase (15LO1), a type-2 enzyme, on the process was also addressed. Cell migration patterns were analyzed by high-dimensional frequency modulated Möbius for statistical comparisons. RESULTS: IL-13 stimulation negatively impacts wound healing by altering the total speed, directionality, and acceleration of individual cells. Inhibition 15LO1 partially improved the wound repair through improving total speed. CONCLUSIONS: Migration abnormalities contributed to markedly slower wound closure of IL-13 treated cells, which was modestly reversed by 15LO1 inhibition, suggesting its potential as an asthma therapeutic target. These novel methodologies offer new ways to dynamically study cell movements and identify contributing pathologic processes.
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Asma/etiología , Araquidonato 15-Lipooxigenasa/fisiología , Asma/diagnóstico por imagen , Asma/tratamiento farmacológico , Asma/inmunología , Movimiento Celular , Células Cultivadas , Células Epiteliales/fisiología , Humanos , Interleucina-13/farmacología , Inhibidores de la Lipooxigenasa/farmacología , Cicatrización de Heridas/efectos de los fármacosRESUMEN
Dopamine transporter (DAT) mediates the reuptake of synaptically released dopamine, and thus controls the duration and intensity of dopamine neurotransmission. Mammalian DAT has been observed to form oligomers, although the mechanisms of oligomerization and its role in DAT activity and trafficking remain largely unknown. We discovered a series of small molecule compounds that stabilize trimers and induce high-order oligomers of DAT and concomitantly promote its clathrin-independent endocytosis. Using a combination of chemical cross-linking, fluorescence resonance energy transfer microscopy, antibody-uptake endocytosis assay, live-cell lattice light sheet microscopy, ligand binding and substrate transport kinetics analyses, and molecular modeling and simulations, we investigated molecular basis of DAT oligomerization and endocytosis induced by these compounds. Our study showed that small molecule-induced DAT oligomerization and endocytosis are favored by the inward-facing DAT conformation and involve interactions of four hydrophobic residues at the interface between transmembrane (TM) helices TM4 and TM9. Surprisingly, a corresponding quadruple DAT mutant displays altered dopamine transport kinetics and increased cocaine-analog binding. The latter is shown to originate from an increased preference for outward-facing conformation and inward-to-outward transition. Taken together, our results demonstrate a direct coupling between conformational dynamics of DAT, functional activity of the transporter, and its oligomerization leading to endocytosis. The high specificity of such coupling for DAT makes the TM4-9 hub a new target for pharmacological modulation of DAT activity and subcellular localization.
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Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/efectos de los fármacos , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Dopamina/metabolismo , Animales , Línea Celular , Clatrina/metabolismo , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/fisiología , Endocitosis/efectos de los fármacos , Endocitosis/fisiología , Células Endoteliales/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Humanos , Modelos Moleculares , Unión Proteica , Conformación Proteica , Bibliotecas de Moléculas Pequeñas/farmacología , PorcinosRESUMEN
Nuclear factor κB (NF-κB) is a transcription factor important for regulating innate and adaptive immunity, cellular proliferation, apoptosis, and senescence. Dysregulation of NF-κB and its upstream regulator IκB kinase (IKK) contributes to the pathogenesis of multiple inflammatory and degenerative diseases as well as cancer. An 11-amino acid peptide containing the NF-κB essential modulator (NEMO)-binding domain (NBD) derived from the C-terminus of ß subunit of IKK, functions as a highly selective inhibitor of the IKK complex by disrupting the association of IKKß and the IKKγ subunit NEMO. A structure-based pharmacophore model was developed to identify NBD mimetics by in silico screening. Two optimized lead NBD mimetics, SR12343 and SR12460, inhibited tumor necrosis factor α (TNF-α)- and lipopolysaccharide (LPS)-induced NF-κB activation by blocking the interaction between IKKß and NEMO and suppressed LPS-induced acute pulmonary inflammation in mice. Chronic treatment of a mouse model of Duchenne muscular dystrophy (DMD) with SR12343 and SR12460 attenuated inflammatory infiltration, necrosis and muscle degeneration, demonstrating that these small-molecule NBD mimetics are potential therapeutics for inflammatory and degenerative diseases.
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Materiales Biomiméticos/farmacología , Quinasa I-kappa B/antagonistas & inhibidores , Distrofia Muscular de Duchenne/tratamiento farmacológico , Neumonía/tratamiento farmacológico , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , Animales , Materiales Biomiméticos/química , Línea Celular , Femenino , Células HEK293 , Humanos , Quinasa I-kappa B/química , Quinasa I-kappa B/metabolismo , Inflamación/tratamiento farmacológico , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Necrosis/tratamiento farmacológico , Dominios Proteicos , Células RAW 264.7RESUMEN
Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. In this study, we discovered that the sodium-dependent lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain containing 2A (MFSD2A) is upregulated on activated CD8+ T cells and is required for memory T cell maintenance. MFSD2A deficiency in mice resulted in decreased import of LPC esterified to long chain fatty acids into activated CD8+ T cells, and MFSD2A-deficient cells are at a competitive disadvantage resulting in reduced memory T cell formation and maintenance and reduced response to secondary infection. Mechanistically, import of LPCs was required to maintain T cell homeostatic turnover, which when lost resulted in a decreased memory T cell pool and thus a reduced secondary response to repeat infection.
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Linfocitos T CD8-positivos/inmunología , Listeria/fisiología , Listeriosis/inmunología , Simportadores/metabolismo , Animales , Células Cultivadas , Homeostasis , Memoria Inmunológica , Listeria/genética , Activación de Linfocitos , Lisofosfatidilcolinas/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ovalbúmina/inmunología , Simportadores/genética , Regulación hacia ArribaRESUMEN
Cancer cells thrive when challenged with proteotoxic stress by inducing components of the protein folding, proteasome, autophagy and unfolded protein response (UPR) pathways. Consequently, specific molecular chaperones have been validated as targets for anti-cancer therapies. For example, inhibition of Hsp70 family proteins (hereafter Hsp70) in rhabdomyosarcoma triggers UPR induction and apoptosis. To define how these cancer cells respond to compromised proteostasis, we compared rhabdomyosarcoma cells that were sensitive (RMS13) or resistant (RMS13-R) to the Hsp70 inhibitor MAL3-101. We discovered that endoplasmic reticulum-associated degradation (ERAD) and autophagy were activated in RMS13-R cells, suggesting that resistant cells overcome Hsp70 ablation by increasing misfolded protein degradation. Indeed, RMS13-R cells degraded ERAD substrates more rapidly than RMS cells and induced the autophagy pathway. Surprisingly, inhibition of the proteasome or ERAD had no effect on RMS13-R cell survival, but silencing of select autophagy components or treatment with autophagy inhibitors restored MAL3-101 sensitivity and led to apoptosis. These data indicate a route through which cancer cells overcome a chaperone-based therapy, define how cells can adapt to Hsp70 inhibition, and demonstrate the value of combined chaperone and autophagy-based therapies.This article has an associated First Person interview with the first author of the paper.
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Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteostasis , Rabdomiosarcoma/fisiopatología , Apoptosis , Autofagia , Línea Celular Tumoral , Degradación Asociada con el Retículo Endoplásmico , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Rabdomiosarcoma/genética , Rabdomiosarcoma/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Respuesta de Proteína DesplegadaRESUMEN
Lysosomes receive and degrade cargo from endocytosis, phagocytosis and autophagy. They also play an important role in sensing and instructing cells on their metabolic state. The lipid kinase PIKfyve generates phosphatidylinositol-3,5-bisphosphate to modulate lysosome function. PIKfyve inhibition leads to impaired degradative capacity, ion dysregulation, abated autophagic flux and a massive enlargement of lysosomes. Collectively, this leads to various physiological defects, including embryonic lethality, neurodegeneration and overt inflammation. The reasons for such drastic lysosome enlargement remain unclear. Here, we examined whether biosynthesis and/or fusion-fission dynamics contribute to swelling. First, we show that PIKfyve inhibition activates TFEB, TFE3 and MITF, enhancing lysosome gene expression. However, this did not augment lysosomal protein levels during acute PIKfyve inhibition, and deletion of TFEB and/or related proteins did not impair lysosome swelling. Instead, PIKfyve inhibition led to fewer but enlarged lysosomes, suggesting that an imbalance favouring lysosome fusion over fission causes lysosome enlargement. Indeed, conditions that abated fusion curtailed lysosome swelling in PIKfyve-inhibited cells.
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Lisosomas/química , Lisosomas/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Animales , Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Células HeLa , Humanos , Iones/metabolismo , Lisosomas/genética , Ratones , Ratones Noqueados , Fosfatidilinositol 3-Quinasas/genética , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
BACKGROUND: The hypercoagulable state associated with pancreatic adenocarcinoma (PDA) results in increased risk of venous thromboembolism, leading to substantial morbidity and mortality. Recently, neutrophil extracellular traps (NETs), whereby activated neutrophils release their intracellular contents containing DNA, histones, tissue factor, high mobility group box 1 (HMGB1) and other components have been implicated in PDA and in cancer-associated thrombosis. METHODS: Utilizing an orthotopic murine PDA model in C57/Bl6 mice and patient correlative samples, we studied the role of NETs in PDA hypercoagulability and targeted this pathway through treatment with the NET inhibitor chloroquine. PAD4 and RAGE knockout mice, deficient in NET formation, were used to study the role of NETs in platelet aggregation, release of tissue factor and hypercoagulability. Platelet aggregation was assessed using collagen-activated impedance aggregometry. Levels of circulating tissue factor, the initiator of extrinsic coagulation, were measured using ELISA. Thromboelastograms (TEGs) were performed to assess hypercoagulability and changes associated with treatment. Correlative data and samples from a randomized clinical trial of preoperative gemcitabine/nab-paclitaxel with and without hydroxychloroquine were studied and the impact of treatment on venous thromboembolism (VTE) rate was evaluated. RESULTS: The addition of NETs to whole blood stimulated platelet activation and aggregation. DNA and the receptor for advanced glycation end products (RAGE) were necessary for induction of NET associated platelet aggregation. PAD4 knockout tumor-burdened mice, unable to form NETs, had decreased aggregation and decreased circulating tissue factor. The NET inhibitor chloroquine reduces platelet aggregation, reduces circulating tissue factor and decreases hypercoagulability on TEG. Review of correlative data from patients treated on a randomized protocol of preoperative chemotherapy with and without hydroxychloroquine demonstrated a reduction in peri-operative VTE rate from 30 to 9.1% with hydroxychloroquine that neared statistical significance (p = 0.053) despite the trial not being designed to study VTE. CONCLUSION: NETs promote hypercoagulability in murine PDA through stimulation of platelets and release of tissue factor. Chloroquine inhibits NETs and diminishes hypercoagulability. These findings support clinical study of chloroquine to lower rates of venous thromboembolism in patients with cancer. TRIAL REGISTRATION: This study reports correlative data from two clinical trials that registered with clinicaltrials.gov, NCT01128296 (May 21, 2010) and NCT01978184 (November 7, 2013).
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Adenocarcinoma/complicaciones , Cloroquina/uso terapéutico , Trampas Extracelulares/efectos de los fármacos , Neoplasias Pancreáticas/complicaciones , Trombofilia/tratamiento farmacológico , Animales , ADN/fisiología , Femenino , Humanos , Hidrolasas/fisiología , Hidroxicloroquina/farmacología , Ratones , Ratones Endogámicos C57BL , Agregación Plaquetaria/efectos de los fármacos , Arginina Deiminasa Proteína-Tipo 4 , Receptor para Productos Finales de Glicación Avanzada/fisiología , Tromboelastografía , Tromboplastina/metabolismo , Tromboembolia Venosa/prevención & controlRESUMEN
Activation of the sympathetic nervous system (e.g., due to stress) has been implicated in cancer progression and recurrence, but its cancer-promoting effects have been variable between different studies. Here, we report that although catecholamines, mediators of systemic sympathetic activity, display only weak immunosuppressive impact on their own, their combination with inflammatory signals leads to the induction of COX-2 and multiple COX-2-dependent suppressive factors in human myeloid cells and cancer tissues. Human macrophages exposed to epinephrine and TNFα, or macrophages generated in 6day cultures in the presence of epinephrine, expressed high levels of COX-2, IDO and IL-10, and strongly suppressed both the proliferation and IFNγ production of CD8+ T cells. These suppressive effects of epinephrine were counteracted by celecoxib, a selective inhibitor of COX-2 activity, which inhibited the induction of immunosuppressive factors (including the elevated expression of COX-2 itself) and the ability of epinephrine-exposed macrophages to suppress CD8+ T cell responses. The activation of the COX-2/PGE2 system and COX-2-dependent suppressive events were also observed in ex vivo human breast and colon cancer explant cultures and were similarly counteracted by celecoxib. Our preliminary data also indicate elevated COX-2 expression in mammary tumors of chronic stress-exposed mice. The current demonstration of the interplay between inflammation and the induction of immunosuppressive factors by catecholamines suggest a contextual impact of stress, helping to explain variable results of epidemiologic studies of the link between sympathetic activity and cancer progression, and implicating COX-2 blockade as a potential means to mitigate stress-related immune suppression.
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Ciclooxigenasa 2/metabolismo , Epinefrina/farmacología , Neoplasias Mamarias Experimentales/inmunología , Células Mieloides/efectos de los fármacos , Animales , Celecoxib/farmacología , Proliferación Celular/efectos de los fármacos , Inhibidores de la Ciclooxigenasa 2/farmacología , Femenino , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/metabolismo , Interleucina-10/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Células Mieloides/inmunología , Células Mieloides/metabolismoRESUMEN
Advancements in microscopy and imaging have pushed the boundaries of what was once thought possible in many fields of research. New techniques, coupled with the application of new technologies, allow researchers to answer increasingly complex questions by probing deeper and with greater accuracy. While, these new techniques provide far greater specificity and increased sensitivity in regards to both resolution and frequency, the amount of data generated is swelling to a point where conventional data-management systems struggle to keep pace; this is especially true for large microscopy-and-imaging shared-user facilities. Sub-optimal data management can severely hinder the ability of a researcher to determine experimental results accurately or efficiently, and will inevitably limit the functionality of the research facility itself. This review discusses the source of the problem: how data are produced by systems available today, and the information's specificity and relative importance; techniques for management of these data to maximize functionality of the facility; and practices that can be detrimental in the research core environment.
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Curaduría de Datos/métodos , Sistemas de Administración de Bases de Datos , Microscopía/métodos , HumanosRESUMEN
Background: Human brain organoids are 3-dimensional cellular models that mimic architectural features of a developing brain. Generated from human induced pluripotent stem cells, these organoids offer an unparalleled physiologically relevant in vitro system for disease modeling and drug screening. In the current study, we sought to establish a foundation for a magnetic resonance imaging (MRI)-based, label-free imaging system that offers high-resolution capabilities for deep tissue imaging of whole organoids. Methods: An 11.7T Bruker/89 mm microimaging system was used to collect high-resolution multishell 3-dimensional diffusion images of 2 induced pluripotent stem cell-derived human hippocampal brain organoids. The MRI features identified in the study were interpreted on the basis of similarities with immunofluorescence microscopy. Results: MRI microscopy at ≤40 µm isotropic resolution provided a 3-dimensional view of organoid microstructure. T2-weighted contrast showed a rosette-like internal structure and a protruding spherical structure that correlated with immunofluorescence staining for the choroid plexus. Diffusion tractography methods can be used to model tissue microstructural features and possibly map neuronal organization. This approach complements traditional immunohistochemistry imaging methods without the need for tissue clearing. Conclusions: This proof-of-concept study shows, for the first time, the application of high-resolution diffusion MRI microscopy to image 2-mm diameter spherical human brain organoids. Application of ultrahigh-field MRI and diffusion tractography is a powerful modality for whole organoid imaging and has the potential to make a significant impact for probing microstructural changes in brain organoids used to model psychiatric disorders, neurodegenerative diseases, and viral infections of the human brain, as well as for assessing neurotoxicity in drug screening.
Versace et al. present a groundbreaking approach using ultrahigh-resolution MRI (11.7T) for deep tissue imaging of whole human brain organoids. These 3D miniature brains mimic the developing brain's architecture and hold promise for disease modeling and drug discovery. This label-free MRI approach offers the potential to characterize microstructural features in human brain organoids modeling psychiatric disorders, neurodegenerative diseases, viral infections, and/or drug-induced neurotoxicity.
RESUMEN
Non-neovascular or dry age-related macular degeneration (AMD) is a multi-factorial disease with degeneration of the aging retinal-pigmented epithelium (RPE). Lysosomes play a crucial role in RPE health via phagocytosis and autophagy, which are regulated by transcription factor EB/E3 (TFEB/E3). Here, we find that increased AKT2 inhibits PGC-1α to downregulate SIRT5, which we identify as an AKT2 binding partner. Crosstalk between SIRT5 and AKT2 facilitates TFEB-dependent lysosomal function in the RPE. AKT2/SIRT5/TFEB pathway inhibition in the RPE induced lysosome/autophagy signaling abnormalities, disrupted mitochondrial function and induced release of debris contributing to drusen. Accordingly, AKT2 overexpression in the RPE caused a dry AMD-like phenotype in aging Akt2 KI mice, as evident from decline in retinal function. Importantly, we show that induced pluripotent stem cell-derived RPE encoding the major risk variant associated with AMD (complement factor H; CFH Y402H) express increased AKT2, impairing TFEB/TFE3-dependent lysosomal function. Collectively, these findings suggest that targeting the AKT2/SIRT5/TFEB pathway may be an effective therapy to delay the progression of dry AMD.
Asunto(s)
Autofagia , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice , Lisosomas , Degeneración Macular , Proteínas Proto-Oncogénicas c-akt , Epitelio Pigmentado de la Retina , Transducción de Señal , Sirtuinas , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Animales , Proteínas Proto-Oncogénicas c-akt/metabolismo , Sirtuinas/metabolismo , Sirtuinas/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Degeneración Macular/genética , Humanos , Ratones , Epitelio Pigmentado de la Retina/metabolismo , Epitelio Pigmentado de la Retina/patología , Lisosomas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas/metabolismo , MasculinoRESUMEN
In dry age-related macular degeneration (AMD), LCN2 (lipocalin 2) is upregulated. Whereas LCN2 has been implicated in AMD pathogenesis, the mechanism remains unknown. Here, we report that in retinal pigmented epithelial (RPE) cells, LCN2 regulates macroautophagy/autophagy, in addition to maintaining iron homeostasis. LCN2 binds to ATG4B to form an LCN2-ATG4B-LC3-II complex, thereby regulating ATG4B activity and LC3-II lipidation. Thus, increased LCN2 reduced autophagy flux. Moreover, RPE cells from cryba1 KO, as well as sting1 KO and Sting1Gt mutant mice (models with abnormal iron chelation), showed decreased autophagy flux and increased LCN2, indicative of CGAS- and STING1-mediated inflammasome activation. Live cell imaging of RPE cells with elevated LCN2 also showed a correlation between inflammasome activation and increased fluorescence intensity of the Liperfluo dye, indicative of oxidative stress-induced ferroptosis. Interestingly, both in human AMD patients and in mouse models with a dry AMD-like phenotype (cryba1 cKO and KO), the LCN2 homodimer variant is increased significantly compared to the monomer. Sub-retinal injection of the LCN2 homodimer secreted by RPE cells into NOD-SCID mice leads to retinal degeneration. In addition, we generated an LCN2 monoclonal antibody that neutralizes both the monomer and homodimer variants and rescued autophagy and ferroptosis activities in cryba1 cKO mice. Furthermore, the antibody rescued retinal function in cryba1 cKO mice as assessed by electroretinography. Here, we identify a molecular pathway whereby increased LCN2 elicits pathophysiology in the RPE, cells known to drive dry AMD pathology, thus providing a possible therapeutic strategy for a disease with no current treatment options.Abbreviations: ACTB: actin, beta; Ad-GFP: adenovirus-green fluorescent protein; Ad-LCN2: adenovirus-lipocalin 2; Ad-LCN2-GFP: adenovirus-LCN2-green fluorescent protein; LCN2AKT2: AKT serine/threonine kinase 2; AMBRA1: autophagy and beclin 1 regulator 1; AMD: age-related macular degeneration; ARPE19: adult retinal pigment epithelial cell line-19; Asp278: aspartate 278; ATG4B: autophagy related 4B cysteine peptidase; ATG4C: autophagy related 4C cysteine peptidase; ATG7: autophagy related 7; ATG9B: autophagy related 9B; BLOC-1: biogenesis of lysosomal organelles complex 1; BLOC1S1: biogenesis of lysosomal organelles complex 1 subunit 1; C57BL/6J: C57 black 6J; CGAS: cyclic GMP-AMP synthase; ChQ: chloroquine; cKO: conditional knockout; Cys74: cysteine 74; Dab2: DAB adaptor protein 2; Def: deferoxamine; DHE: dihydroethidium; DMSO: dimethyl sulfoxide; ERG: electroretinography; FAC: ferric ammonium citrate; Fe2+: ferrous; FTH1: ferritin heavy chain 1; GPX: glutathione peroxidase; GST: glutathione S-transferase; H2O2: hydrogen peroxide; His280: histidine 280; IFNL/IFNλ: interferon lambda; IL1B/IL-1ß: interleukin 1 beta; IS: Inner segment; ITGB1/integrin ß1: integrin subunit beta 1; KO: knockout; LC3-GST: microtubule associated protein 1 light chain 3-GST; C-terminal fusion; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; LCN2: lipocalin 2; mAb: monoclonal antibody; MDA: malondialdehyde; MMP9: matrix metallopeptidase 9; NLRP3: NLR family pyrin domain containing 3; NOD-SCID: nonobese diabetic-severe combined immunodeficiency; OS: outer segment; PBS: phosphate-buffered saline; PMEL/PMEL17: premelanosome protein; RFP: red fluorescent protein; rLCN2: recombinant LCN2; ROS: reactive oxygen species; RPE SM: retinal pigmented epithelium spent medium; RPE: retinal pigment epithelium; RSL3: RAS-selective lethal; scRNAseq: single-cell ribonucleic acid sequencing; SD-OCT: spectral domain optical coherence tomography; shRNA: small hairpin ribonucleic acid; SM: spent medium; SOD1: superoxide dismutase 1; SQSTM1/p62: sequestosome 1; STAT1: signal transducer and activator of transcription 1; STING1: stimulator of interferon response cGAMP interactor 1; TYR: tyrosinase; VCL: vinculin; WT: wild type.
Asunto(s)
Ferroptosis , Degeneración Macular , Animales , Humanos , Ratones , Anticuerpos Monoclonales , Autofagia/fisiología , Inflamasomas/metabolismo , Lipocalina 2/genética , Degeneración Macular/genética , Degeneración Macular/metabolismo , Degeneración Macular/patología , Ratones Endogámicos NOD , Ratones SCID , Nucleotidiltransferasas/metabolismoRESUMEN
Age-related macular degeneration (AMD), the leading cause of geriatric blindness, is a multi-factorial disease with retinal-pigmented epithelial (RPE) cell dysfunction as a central pathogenic driver. With RPE degeneration, lysosomal function is a core process that is disrupted. Transcription factors EB/E3 (TFEB/E3) tightly control lysosomal function; their disruption can cause aging disorders, such as AMD. Here, we show that induced pluripotent stem cells (iPSC)-derived RPE cells with the complement factor H variant [ CFH (Y402H)] have increased AKT2, which impairs TFEB/TFE3 nuclear translocation and lysosomal function. Increased AKT2 can inhibit PGC1α, which downregulates SIRT5, an AKT2 binding partner. SIRT5 and AKT2 co-regulate each other, thereby modulating TFEB-dependent lysosomal function in the RPE. Failure of the AKT2/SIRT5/TFEB pathway in the RPE induced abnormalities in the autophagy-lysosome cellular axis by upregulating secretory autophagy, thereby releasing a plethora of factors that likely contribute to drusen formation, a hallmark of AMD. Finally, overexpressing AKT2 in RPE cells in mice led to an AMD-like phenotype. Thus, targeting the AKT2/SIRT5/TFEB pathway could be a potential therapy for atrophic AMD.
RESUMEN
Ligand binding to the EGF receptor (EGFR) triggers multiple signal-transduction processes and promotes endocytosis of the receptor. The mechanisms of EGFR endocytosis and its cross-talk with signaling are poorly understood. Here, we combine peroxidase-catalyzed proximity labeling, isobaric peptide tagging, and quantitative mass spectrometry to define the dynamics of the proximity proteome of ligand-activated EGFR. Using this approach, we identify a network of signaling proteins, which remain associated with the receptor during its internalization and trafficking through the endosomal system. We show that Trk-fused gene (TFG), a protein known to function at the endoplasmic reticulum exit sites, is enriched in the proximity proteome of EGFR in early/sorting endosomes and localized in these endosomes and demonstrate that TFG regulates endosomal sorting of EGFR. This study provides a comprehensive resource of time-dependent nanoscale environment of EGFR, thus opening avenues to discovering new regulatory mechanisms of signaling and intracellular trafficking of receptor tyrosine kinases.
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Receptores ErbB , Proteoma , Endocitosis/fisiología , Endosomas/metabolismo , Factor de Crecimiento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Ligandos , Transporte de Proteínas , Proteoma/metabolismoRESUMEN
Intrauterine infections during pregnancy by herpes simplex virus (HSV) can cause significant neurodevelopmental deficits in the unborn/newborn, but clinical studies of pathogenesis are challenging, and while animal models can model some aspects of disease, in vitro studies of human neural cells provide a critical platform for more mechanistic studies. We utilized a reductionist approach to model neurodevelopmental outcomes of HSV-1 infection of neural rosettes, which represent the in vitro equivalent of differentiating neural tubes. Specifically, we employed early-stage brain organoids (ES-organoids) composed of human induced pluripotent stem cells (hiPSCs)-derived neural rosettes to investigate aspects of the potential neuropathological effects induced by the HSV-1 infections on neurodevelopment. To allow for the long-term differentiation of ES-organoids, viral infections were performed in the presence of the antiviral drug acyclovir (ACV). Despite the antiviral treatment, HSV-1 infection caused organizational changes in neural rosettes, loss of structural integrity of infected ES-organoids, and neuronal alterations. The inability of ACV to prevent neurodegeneration was associated with the generation of ACV-resistant mutants during the interaction of HSV-1 with differentiating neural precursor cells (NPCs). This study models the effects of HSV-1 infection on the neuronal differentiation of NPCs and suggests that this environment may allow for accelerated development of ACV-resistance.