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Cold and ischemia/reperfusion (IR)-associated injuries are seemingly inevitable during liver transplantation and hepatectomy. Because Syrian hamsters demonstrate intrinsic tolerance to transplantation-like stimuli, cross-species comparative metabolomic analyses were conducted with hamster, rat, and donor liver samples to seek hepatic cold and IR-adaptive mechanisms. Lower hepatic phosphocholine contents were found in recipients with early graft-dysfunction and with virus-caused cirrhosis or high model for end-stage liver disease scores (≥30). Choline/phosphocholine deficiency in cultured human THLE-2 hepatocytes and animal models weakened hepatocellular cold tolerance and recovery of glutathione and ATP production, which was rescued by phosphocholine supplements. Among the biological processes impacted by choline/phosphocholine deficiency, 3 lipid-related metabolic processes were downregulated, whereas phosphocholine elevated the expression of genes in methylation processes. Consistently, in THLE-2, phosphocholine enhanced the overall RNA m6A methylation, among which the transcript stability of fatty acid desaturase 6 (FADS6) was improved. FADS6 functioned as a key phosphocholine effector in the production of polyunsaturated fatty acids, which may facilitate the hepatocellular recovery of energy and redox homeostasis. Thus, our study reveals the choline-phosphocholine metabolism and its downstream FADS6 functions in hepatic adaptation to cold and IR, which may inspire new strategies to monitor donor liver quality and improve recipient recovery from the liver transplantation process.
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Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.
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Diabetes Mellitus Experimental , Fatores de Transcrição Forkhead , Camundongos , Humanos , Animais , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Transporte Ativo do Núcleo Celular , Peixe-Zebra/metabolismo , Carioferinas/metabolismoRESUMO
Lenvatinib, a second-generation multi-receptor tyrosine kinase inhibitor approved by the FDA for first-line treatment of advanced liver cancer, facing limitations due to drug resistance. Here, we applied a multidimensional, high-throughput screening platform comprising patient-derived resistant liver tumor cells (PDCs), organoids (PDOs), and xenografts (PDXs) to identify drug susceptibilities for conquering lenvatinib resistance in clinically relevant settings. Expansion and passaging of PDCs and PDOs from resistant patient liver tumors retained functional fidelity to lenvatinib treatment, expediting drug repurposing screens. Pharmacological screening identified romidepsin, YM155, apitolisib, NVP-TAE684 and dasatinib as potential antitumor agents in lenvatinib-resistant PDC and PDO models. Notably, romidepsin treatment enhanced antitumor response in syngeneic mouse models by triggering immunogenic tumor cell death and blocking the EGFR signaling pathway. A combination of romidepsin and immunotherapy achieved robust and synergistic antitumor effects against lenvatinib resistance in humanized immunocompetent PDX models. Collectively, our findings suggest that patient-derived liver cancer models effectively recapitulate lenvatinib resistance observed in clinical settings and expedite drug discovery for advanced liver cancer, providing a feasible multidimensional platform for personalized medicine.
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Rationale: Liver resection and transplantation surgeries are accompanied by hepatic ischemia-reperfusion (HIR) injury that hampers the subsequent liver recovery. Given that the liver is the main organ for metabolism and detoxification, ischemia-reperfusion in essence bestows metabolic stress upon the liver and disrupts local metabolic and immune homeostasis. Most of the recent and current research works concerning HIR have been focusing on addressing HIR-induced hepatic injury and inflammation, instead of dealing with the metabolic reprogramming and restoration of redox homeostasis. As our previous work uncovers the importance of 5-aminolevulinate (5-ALA) synthesis during stress adaptation, here we evaluate the effects of supplementing 5-ALA to mitigate HIR injury. Methods: 5-ALA was supplemented into the mice or cultured cells during the ischemic or oxygen-glucose deprivation (OGD) phase. Following reperfusion or reoxygenation, cellular metabolism and energy homeostasis, mitochondrial production of reactive oxygen species (ROS) and transcriptomic changes were evaluated in HIR mouse models or cultured hepatocytes and macrophages. Liver injury, hepatocytic functional tests, and macrophagic M1/M2 polarization were assessed. Results: Dynamic changes in the expression of key enzymes in 5-ALA metabolism were first confirmed in donor and mouse liver samples following HIR. Supplemented 5-ALA modulated mouse hepatic lipid metabolism and reduced ATP production in macrophages following HIR, resulting in elevation of anti-inflammatory M2 polarization. Mechanistically, 5-ALA down-regulates macrophagic chemokine receptor CX3CR1 via the repression of RelA following OGD and reoxygenation (OGD/R). Cx3cr1 KO mice demonstrated milder liver injuries and more macrophage M2 polarization after HIR. M2 macrophage-secreted chitinase-like protein 3 (CHIL3; CHI3L1 in human) is an important HIR-induced effector downstream of CX3CR1 deficiency. Addition of CHIL3/CHI3L1 alone improved hepatocellular metabolism and reduced OGD/R-inflicted injuries in cultured mouse and human hepatocytes. Combined treatment with 5-ALA and CHIL3 during the ischemic phase facilitated lipid metabolism and ATP production in the mouse liver following HIR. Conclusion: Our results reveal that supplementing 5-ALA promotes macrophagic M2 polarization via downregulation of RelA and CX3CR1 in mice following HIR, while M2 macrophage-produced CHIL3/CHI3L1 also manifests beneficial effects to the recovery of hepatic metabolism. 5-ALA and CHIL3/CHI3L1 together mitigate HIR-induced mitochondrial dysfunction and hepatocellular injuries, which may be developed into safe and effective clinical treatments to attenuate HIR injuries.
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Ácido Aminolevulínico , Traumatismo por Reperfusão , Camundongos , Humanos , Animais , Ácido Aminolevulínico/farmacologia , Fígado/metabolismo , Traumatismo por Reperfusão/tratamento farmacológico , Traumatismo por Reperfusão/metabolismo , Isquemia/metabolismo , Trifosfato de Adenosina/metabolismo , Proteína 1 Semelhante à Quitinase-3/metabolismoRESUMO
Inadequate remnant volume and regenerative ability of the liver pose life-threatening risks to patients after partial liver transplantation (PLT) or partial hepatectomy (PHx), while few clinical treatments focus on safely accelerating regeneration. Recently, we discovered that supplementing 5-aminolevulinate (5-ALA) improves liver cold adaptation and functional recovery, leading us to uncover a correlation between 5-ALA metabolic activities and post-PLT recovery. In a mouse 2/3 PHx model, 5-ALA supplements enhanced liver regeneration, promoting infiltration and polarization of anti-inflammatory macrophages via P53 signaling. Intriguingly, chemokine receptor CX3CR1 functions to counterbalance these effects. Genetic ablation or pharmacological inhibition of CX3CR1 (AZD8797; phase II trial candidate) augmented the macrophagic production of insulin-like growth factor 1 (IGF-1) and subsequent hepatocyte growth factor (HGF) production by hepatic stellate cells. Thus, short-term treatments with both 5-ALA and AZD8797 demonstrated pro-regeneration outcomes superior to 5-ALA-only treatments in mice after PHx. Overall, our findings may inspire safe and effective strategies to better treat PLT and PHx patients.
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Fator de Crescimento Insulin-Like I , Regeneração Hepática , Animais , Camundongos , Ácido Aminolevulínico/farmacologia , Proliferação de Células , Modelos Animais de Doenças , Hepatócitos/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Fígado/metabolismo , Regeneração Hepática/fisiologiaRESUMO
Rationale: Hibernating thirteen-lined ground squirrels (GS; Ictidomys tridecemlineatus) are naturally adapted to prolonged periods of ultraprofound hypothermia (body temperature < 5 ºC) during torpor, and drastic oscillations of body temperature and ischemia/reperfusion-like stress during their short euthermic interbout arousals. Thus, their superior adaptability may hold tremendous promise for the advancement of donor organ cold preservation and subsequent organ transplantation. However, bridging hibernation research and translational medicine has been impeded by a dearth of in vitro research tools, till the recent establishment of the GS induced pluripotent stem cells (iPSCs). In this study, we reported the generation of functional hepatocyte-like cells (HLCs) from GS iPSCs. As temperature and oxygen supply affect cellular metabolism, we hypothesized that the GS HLCs can metabolically counter drastic temperature and oxygen supply changes. Differentially regulated metabolites can be evaluated and included into the preservation solution to mitigate temperature and ischemia/reperfusion-associated damage to donor livers. Methods: A protocol has been developed to produce GS iPSCs-derived HLCs. Comparative metabolomic analysis on GS HLCs and human donor liver samples revealed changes in metabolites caused by cold storage and rewarming. Human embryonic stem cell (ESC)-derived HLCs and ex vivo cold preservation and reperfusion of isolated rat livers were used to assess candidate metabolites that may have protective effects against preservation-related injuries. Results: GS iPSCs were efficiently differentiated into expandable, cryopreservation-compatible and functional HLCs. Metabolomic analysis unveiled distinct changes of mitochondrial metabolites between GS and human cells following cold storage and rewarming. GS and human HLC-based experiments indicated that the metabolism of 5-aminolevulinate (5-ALA) is key to restricting free radical production during rewarming. Survival of human HLCs was significantly increased following cold exposure and rewarming, as supplemented 5-ALA enhanced Complex III activity and improved mitochondrial respiration. Further, 5-ALA mitigated damage in rat livers following 48-h cold preservation and ex vivo reperfusion. Metabolomic and transcriptomic analyses revealed that supplemented 5-ALA promoted both anabolic and catabolic activities while alleviating cell death, inflammation, hypoxia and other stress responses in isolated perfused rat livers. Conclusion: In the liver, rewarming from ultraprofound hypothermia imposes complex metabolic challenges and stresses on the mitochondria. Metabolites such as 5-ALA can help alleviate mitochondrial stress. Supplementing 5-ALA to the liver preservation solution can substantially improve the functional recovery of rat livers following prolonged cold preservation, rewarming and reperfusion.
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Hipotermia , Transplante de Fígado , Ácido Aminolevulínico/farmacologia , Animais , Sobrevivência Celular , Criopreservação/métodos , Humanos , Hipotermia/metabolismo , Isquemia , Fígado/metabolismo , Doadores Vivos , Oxigênio/metabolismo , RatosRESUMO
Therapeutic hypothermia (TH) is potentially an important therapy for central nervous system (CNS) trauma. However, its clinical application remains controversial, hampered by two major factors: (1) Many of the CNS injury sites, such as the optic nerve (ON), are deeply buried, preventing access for local TH. The alternative is to apply TH systemically, which significantly limits the applicable temperature range. (2) Even with possible access for 'local refrigeration', cold-induced cellular damage offsets the benefit of TH. Here we present a clinically translatable model of traumatic optic neuropathy (TON) by applying clinical trans-nasal endoscopic surgery to goats and non-human primates. This model faithfully recapitulates clinical features of TON such as the injury site (pre-chiasmatic ON), the spatiotemporal pattern of neural degeneration, and the accessibility of local treatments with large operating space. We also developed a computer program to simplify the endoscopic procedure and expand this model to other large animal species. Moreover, applying a cold-protective treatment, inspired by our previous hibernation research, enables us to deliver deep hypothermia (4 °C) locally to mitigate inflammation and metabolic stress (indicated by the transcriptomic changes after injury) without cold-induced cellular damage, and confers prominent neuroprotection both structurally and functionally. Intriguingly, neither treatment alone was effective, demonstrating that in situ deep hypothermia combined with cold protection constitutes a breakthrough for TH as a therapy for TON and other CNS traumas.
Hypothermic therapy is a radical type of treatment that involves cooling a person's core body temperature several degrees below normal to protect against brain damage. Lowering body temperature slows blood flow, which reduces inflammation, and eases metabolic demands, similar to hibernation. It can also reduce lasting damage to the brain and aid recovery when used to treat people who have gone into cardiac arrest, where their heart suddenly stops beating. Recently, there has been renewed interest in using hypothermic therapy to treat people who have sustained traumatic brain injuries, which can cause brain swelling, and other nerve injuries. However, its use remains controversial because clinical trials have failed to show that inducing mild hypothermia provides any benefit for people with severe nerve injuries. This might be because cooling cells to near-freezing temperatures can damage their internal structural supports, called microtubules, thwarting any therapeutic benefit. Traumatic optical neuropathy is a type of injury in which the optic nerve the nerve that connects the eyes to the brain is damaged or severed, causing vision loss. There is currently no clinically proven treatment for this condition, nor is there a system that can test local treatments in large animals as a prior test to using the treatment in the clinic. Therefore, Zhang et al. wanted to establish such a animal model and test whether local hypothermic therapy could help protect the optic nerve. Zhang et al. used a surgical tool guided by an endoscope (a thin plastic tube with a light and camera attached to it) to injure the optic nerves of goats, and then deliver hypothermic therapy. To cool the surgically-injured nerves to a chilly 4C, Zhang et al. applied a deep-cooling agent, using a second reagent (a cocktail of protease inhibitors) to protect the cells' microtubules from cold-induced damage, an insight gained from a previous study of hibernating animals. This was critical, as the hypothermic therapy was only effective when the secondary protective agent was applied. The combination therapy developed by Zhang et al. relieved some aspects of nerve degeneration at the injury site and activated an anti-inflammatory response in cells, but did not restore vision. To simplify surgical techniques, Zhang et al. also developed a computer program which generates virtual surgical paths for up-the-nose endoscopic procedures based on brain scans of an animal's skull. This program was successfully applied in a range of large animals, including goats and macaque monkeys. Zhang et al.'s work establishes a method to study treatments for traumatic optical neuropathy using large animals, including hypothermic therapy. The methods developed could also be useful to study other optic nerve disorders, such as optic neuritis or ischemic optic neuropathy.
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Hipotermia Induzida , Hipotermia , Traumatismos do Nervo Óptico , Animais , Temperatura Baixa , Hipotermia Induzida/métodos , Nervo Óptico , Traumatismos do Nervo Óptico/terapiaRESUMO
The sympathetic nervous system-catecholamine-uncoupling protein 1 (UCP1) axis plays an essential role in non-shivering adaptive thermogenesis. However, whether there exists a direct effector that physically connects catecholamine signalling to UCP1 in response to acute cold is unknown. Here we report that outer mitochondrial membrane-located AIDA is phosphorylated at S161 by the catecholamine-activated protein kinase A (PKA). Phosphorylated AIDA translocates to the intermembrane space, where it binds to and activates the uncoupling activity of UCP1 by promoting cysteine oxidation of UCP1. Adipocyte-specific depletion of AIDA abrogates UCP1-dependent thermogenesis, resulting in hypothermia during acute cold exposure. Re-expression of S161A-AIDA, unlike wild-type AIDA, fails to restore the acute cold response in Aida-knockout mice. The PKA-AIDA-UCP1 axis is highly conserved in mammals, including hibernators. Denervation of the sympathetic postganglionic fibres abolishes cold-induced AIDA-dependent thermogenesis. These findings uncover a direct mechanistic link between sympathetic input and UCP1-mediated adaptive thermogenesis.
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Adipócitos Marrons/metabolismo , Tecido Adiposo Marrom/inervação , Proteínas de Transferência de Fosfolipídeos/metabolismo , Sistema Nervoso Simpático/fisiologia , Termogênese , Proteína Desacopladora 1/metabolismo , Adiponectina/genética , Adiponectina/metabolismo , Animais , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Metabolismo Energético , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Oxirredução , Proteínas de Transferência de Fosfolipídeos/deficiência , Proteínas de Transferência de Fosfolipídeos/genética , Fosforilação , Transdução de Sinais , Proteína Desacopladora 1/deficiência , Proteína Desacopladora 1/genéticaRESUMO
Comparative physiologists are often interested in adaptive physiological phenomena found in unconventional model organisms; however, research on these species is frequently constrained by the limited availability of investigative tools. Here, we propose that induced pluripotent stem cells (iPSCs) from unconventional model organisms may retain certain species-specific features that can consequently be investigated in depth in vitro; we use hibernating mammals as an example. Many species (including ground squirrels, bats and bears) can enter a prolonged state of physiological dormancy known as hibernation to survive unfavorable seasonal conditions. Our understanding of the mechanisms underpinning the rapid transition and adaptation to a hypothermic, metabolically suppressed winter torpor state remains limited partially because of the lack of an easily accessible model. To address the fascinating unanswered questions underlying hibernation biology, we have developed a powerful model system: iPSCs from a hibernating species, the thirteen-lined ground squirrel (Ictidomys tridecemlineatus). These stem cells can potentially be differentiated into any cell type, and can be used for the analysis of cell-autonomous mechanisms that facilitate adaptation to hibernation and for comparisons with non-hibernators. Furthermore, we can manipulate candidate molecular and cellular pathways underlying relevant physiological phenomena by pharmacological or RNAi-based methods, and CRISPR/Cas9 gene editing. Moreover, iPSC strategies can be applied to other species (e.g. seals, naked mole rats, humming birds) for in vitro studies on adaptation to extreme physiological conditions. In this Commentary, we discuss factors to consider when attempting to generate iPSCs from unconventional model organisms, based on our experience with the thirteen-lined ground squirrel.
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Células-Tronco Pluripotentes Induzidas/citologia , Fisiologia Comparada , Sciuridae/fisiologia , Animais , Técnicas de Cultura de Células , PesquisaRESUMO
Hibernating mammals survive hypothermia (<10°C) without injury, a remarkable feat of cellular preservation that bears significance for potential medical applications. However, mechanisms imparting cold resistance, such as cytoskeleton stability, remain elusive. Using the first iPSC line from a hibernating mammal (13-lined ground squirrel), we uncovered cellular pathways critical for cold tolerance. Comparison between human and ground squirrel iPSC-derived neurons revealed differential mitochondrial and protein quality control responses to cold. In human iPSC-neurons, cold triggered mitochondrial stress, resulting in reactive oxygen species overproduction and lysosomal membrane permeabilization, contributing to microtubule destruction. Manipulations of these pathways endowed microtubule cold stability upon human iPSC-neurons and rat (a non-hibernator) retina, preserving its light responsiveness after prolonged cold exposure. Furthermore, these treatments significantly improved microtubule integrity in cold-stored kidneys, demonstrating the potential for prolonging shelf-life of organ transplants. Thus, ground squirrel iPSCs offer a unique platform for bringing cold-adaptive strategies from hibernators to humans in clinical applications. VIDEO ABSTRACT.
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Adaptação Fisiológica , Células-Tronco Pluripotentes Induzidas/metabolismo , Neurônios/metabolismo , Animais , Diferenciação Celular , Temperatura Baixa , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Rim/efeitos dos fármacos , Rim/metabolismo , Lisossomos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/metabolismo , Neurônios/citologia , Estresse Oxidativo , Inibidores de Proteases/farmacologia , Ratos , Espécies Reativas de Oxigênio/metabolismo , Retina/metabolismo , Sciuridae , Transcriptoma , Tubulina (Proteína)/química , Tubulina (Proteína)/genética , Tubulina (Proteína)/metabolismoRESUMO
Hibernation is a seasonally adaptive strategy that allows hibernators to live through extremely cold conditions. Despite the profound reduction of blood flow to the retinas, hibernation causes no lasting retinal injury. Instead, hibernators show an increased tolerance to ischemic insults during the hibernation period. To understand the molecular changes of the retinas in response to hibernation, we applied an integrative transcriptome and metabolome analysis to explore changes in gene expression and metabolites of 13-lined ground squirrel retinas during hibernation. Metabolomic analysis showed a global decrease of ATP synthesis in hibernating retinas. Decreased glucose and galactose, increased beta-oxidation of carnitine and decreased storage of some amino acids in hibernating retinas indicated a shift of fuel use from carbohydrates to lipids and alternative usage of amino acids. Transcriptomic analysis revealed that the down-regulated genes were enriched in DNA-templated transcription and immune-related functions, while the up-regulated genes were enriched in mitochondrial inner membrane and DNA packaging-related functions. We further showed that a subset of genes underwent active alternative splicing events in response to hibernation. Finally, integrative analysis of the transcriptome and metabolome confirmed the shift of fuel use in the hibernating retina by the regulation of catabolism of amino acids and lipids. Through transcriptomic and metabolomic data, our analysis revealed the altered state of mitochondrial oxidative phosphorylation and the shift of energy source in the hibernating retina, advancing our understanding of the molecular mechanisms employed by hibernators. The data will also serve as a useful resource for the ocular and hibernation research communities.
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Metabolismo Energético , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Perfilação da Expressão Gênica/métodos , Hibernação , Metabolômica/métodos , Retina/metabolismo , Sciuridae/genética , Sciuridae/metabolismo , Transcriptoma , Adaptação Fisiológica , Trifosfato de Adenosina/metabolismo , Processamento Alternativo , Aminoácidos/metabolismo , Animais , Cromatografia Líquida , Carboidratos da Dieta/metabolismo , Gorduras na Dieta/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Sequenciamento de Nucleotídeos em Larga Escala , Masculino , Mitocôndrias/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Retina/imunologia , Sciuridae/imunologia , Análise de Sequência de RNA , Espectrometria de Massas em TandemRESUMO
Inflammation and oxidative stress are involved in age-related macular degeneration (AMD) and possibly associated with an activation of neuronal apoptosis inhibitor protein/class II transcription activator of the Major Histocompatibility Complex (MHC)/heterokaryon incompatibility/telomerase-associated protein 1, leucine-rich repeat or nucleotide-binding domain, leucine-rich repeat-containing family, and pyrin domain-containing 3 (NLRP3) inflammasome. In the present study, we used a translational approach to address this hypothesis. In patients with AMD, we observed increased mRNA levels of NLRP3, pro-interleukin-1 beta (IL-1ß) and pro-IL-18 in AMD lesions of the retinal pigment epithelium (RPE) and photoreceptor. In vitro, a similar increase was evoked by oxidative stress or lipopolysaccharide (LPS) stimulation in the adult retinal pigment epithelium (ARPE-19) cell line, and the increase was reduced in siRNA transfected cells to knockdown NLRP3. Ultrastructural studies of ARPE-19 cells showed a swelling of the cytoplasm, mitochondrial damage, and occurrence of autophagosome-like structures. NLRP3 positive dots were detected within autophagosome-like structures or in the extracellular space. Next, we used a mouse model of AMD, Ccl2/Cx3cr1 double knockout on rd8 background (DKO rd8) to ascertain the in vivo relevance. Ultrastructural studies of the RPE of these mice showed damaged mitochondria, autophagosome-like structures, and cytoplasmic vacuoles, which are reminiscent of the pathology seen in stressed ARPE-19 cells. The data suggest that the NLRP3 inflammasome may contribute in AMD pathogenesis.
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Proteínas de Transporte/genética , Degeneração Macular/genética , Degeneração Macular/patologia , Epitélio Pigmentado da Retina/patologia , Regulação para Cima , Adulto , Idoso de 80 Anos ou mais , Animais , Proteínas de Transporte/imunologia , Linhagem Celular , Células Cultivadas , Humanos , Inflamassomos/genética , Inflamassomos/imunologia , Inflamação/genética , Inflamação/imunologia , Inflamação/patologia , Macula Lutea/imunologia , Macula Lutea/metabolismo , Macula Lutea/patologia , Degeneração Macular/imunologia , Masculino , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR , Estresse Oxidativo , Interferência de RNA , RNA Interferente Pequeno/genética , Epitélio Pigmentado da Retina/imunologia , Epitélio Pigmentado da Retina/metabolismoRESUMO
Strategies aimed at invoking synaptic plasticity have therapeutic potential for several neurological conditions. The human retinal synaptic disease X-linked retinoschisis (XLRS) is characterized by impaired visual signal transmission through the retina and progressive visual acuity loss, and mice lacking retinoschisin (RS1) recapitulate human disease. Here, we demonstrate that restoration of RS1 via retina-specific delivery of adeno-associated virus type 8-RS1 (AAV8-RS1) vector rescues molecular pathology at the photoreceptor-depolarizing bipolar cell (photoreceptor-DBC) synapse and restores function in adult Rs1-KO animals. Initial development of the photoreceptor-DBC synapse was normal in the Rs1-KO retina; however, the metabotropic glutamate receptor 6/transient receptor potential melastatin subfamily M member 1-signaling (mGluR6/TRPM1-signaling) cascade was not properly maintained. Specifically, the TRPM1 channel and G proteins Gαo, Gß5, and RGS11 were progressively lost from postsynaptic DBC dendritic tips, whereas the mGluR6 receptor and RGS7 maintained proper synaptic position. This postsynaptic disruption differed from other murine night-blindness models with an electronegative electroretinogram response, which is also characteristic of murine and human XLRS disease. Upon AAV8-RS1 gene transfer to the retina of adult XLRS mice, TRPM1 and the signaling molecules returned to their proper dendritic tip location, and the DBC resting membrane potential was restored. These findings provide insight into the molecular plasticity of a critical synapse in the visual system and demonstrate potential therapeutic avenues for some diseases involving synaptic pathology.
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Moléculas de Adesão Celular/genética , Proteínas do Olho/genética , Terapia Genética/métodos , Retinosquise/patologia , Retinosquise/terapia , Animais , Sinalização do Cálcio , Moléculas de Adesão Celular/deficiência , Moléculas de Adesão Celular/metabolismo , Dependovirus/genética , Modelos Animais de Doenças , Eletrorretinografia , Proteínas do Olho/metabolismo , Vetores Genéticos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Plasticidade Neuronal , Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/patologia , Receptores de Glutamato Metabotrópico/metabolismo , Retinosquise/genética , Sinapses/metabolismo , Sinapses/patologia , Canais de Cátion TRPM/metabolismoRESUMO
During vertebrate eye development, the transcription factor MITF acts to promote the development of the retinal pigment epithelium (RPE). In embryos with Mitf mutations, the future RPE hyperproliferates and is respecified as retinal tissue but only in a small portion of the dorsal RPE. Using a series of genetic crosses, we show that this spatial restriction of RPE respecification is brought about by persistent expression of the anti-retinogenic ventral homeodomain gene Vax2 in the dorso-proximal and both Vax1 and Vax2 in the ventral RPE. We further show that dorso-proximal RPE respecification in Vax2/Mitf double mutants and dorso-proximal and ventral RPE respecification in Vax1/2/Mitf triple mutants result from increased FGF/MAP kinase signaling. In none of the mutants, however, does the distal RPE show signs of hyperproliferation or respecification, likely due to local JAGGED1/NOTCH signaling. Expression studies and optic vesicle culture experiments also suggest a role for NOTCH signaling within the mutant dorsal RPE domains, where ectopic JAGGED1 expression may partially counteract the effects of FGF/ERK1/2 signaling on RPE respecification. The results indicate the presence of complex interplays between distinct transcription factors and signaling molecules during eye development and show how RPE phenotypes associated with mutations in one gene are modulated by expression changes in other genes.
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Epistasia Genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fator de Transcrição Associado à Microftalmia/genética , Neuropeptídeos/genética , Epitélio Pigmentado da Retina/metabolismo , Animais , Proteínas de Ligação ao Cálcio/genética , Proteínas de Ligação ao Cálcio/metabolismo , Diferenciação Celular , Proliferação de Células , Cruzamentos Genéticos , Embrião de Mamíferos , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/genética , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteína Jagged-1 , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Fator de Transcrição Associado à Microftalmia/metabolismo , Mutação , Neuropeptídeos/metabolismo , Receptores Notch/genética , Receptores Notch/metabolismo , Epitélio Pigmentado da Retina/citologia , Epitélio Pigmentado da Retina/embriologia , Proteínas Serrate-Jagged , Transdução de SinaisRESUMO
The separation of the optic neuroepithelium into future retina and retinal pigment epithelium (RPE) is a critical event in early eye development in vertebrates. Here we show in mice that the transcription factor PAX6, well-known for its retina-promoting activity, also plays a crucial role in early pigment epithelium development. This role is seen, however, only in a background genetically sensitized by mutations in the pigment cell transcription factor MITF. In fact, a reduction in Pax6 gene dose exacerbates the RPE-to-retina transdifferentiation seen in embryos homozygous for an Mitf null allele, and it induces such a transdifferentiation in embryos that are either heterozygous for the Mitf null allele or homozygous for an RPE-specific hypomorphic Mitf allele generated by targeted mutation. Conversely, an increase in Pax6 gene dose interferes with transdifferentiation even in homozygous Mitf null embryos. Gene expression analyses show that, together with MITF or its paralog TFEC, PAX6 suppresses the expression of Fgf15 and Dkk3. Explant culture experiments indicate that a combination of FGF and DKK3 promote retina formation by inhibiting canonical WNT signaling and stimulating the expression of retinogenic genes, including Six6 and Vsx2. Our results demonstrate that in conjunction with Mitf/Tfec Pax6 acts as an anti-retinogenic factor, whereas in conjunction with retinogenic genes it acts as a pro-retinogenic factor. The results suggest that careful manipulation of the Pax6 regulatory circuit may facilitate the generation of retinal and pigment epithelium cells from embryonic or induced pluripotent stem cells.
Assuntos
Proteínas do Olho , Proteínas de Homeodomínio , Fator de Transcrição Associado à Microftalmia , Fatores de Transcrição Box Pareados , Proteínas Repressoras , Retina/crescimento & desenvolvimento , Epitélio Pigmentado da Retina/crescimento & desenvolvimento , Via de Sinalização Wnt , Proteínas Adaptadoras de Transdução de Sinal , Animais , Transdiferenciação Celular , Desenvolvimento Embrionário , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Dosagem de Genes , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/genética , Fator de Transcrição Associado à Microftalmia/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Retina/metabolismo , Epitélio Pigmentado da Retina/metabolismo , Via de Sinalização Wnt/genéticaRESUMO
Ion flow from intact tissue into epithelial wound sites results in lateral electric currents that may represent a major driver of wound healing cell migration. Use of applied electric fields (EF) to promote wound healing is the basis of Medicare-approved electric stimulation therapy. This study investigated the roles for EFs in wound re-epithelialization, using the Pax6(+/-) mouse model of the human ocular surface abnormality aniridic keratopathy (in which wound healing and corneal epithelial cell migration are disrupted). Both wild-type (WT) and Pax6(+/-) corneal epithelial cells showed increased migration speeds in response to applied EFs in vitro. However, only Pax6(+/+) cells demonstrated consistent directional galvanotaxis towards the cathode, with activation of pSrc signaling, polarized to the leading edges of cells. In vivo, the epithelial wound site normally represents a cathode, but 43% of Pax6(+/-) corneas exhibited reversed endogenous wound-induced currents (the wound was an anode). These corneas healed at the same rate as WT. Surprisingly, epithelial migration did not correlate with direction or magnitude of endogenous currents for WT or mutant corneas. Furthermore, during healing in vivo, no polarization of pSrc was observed. We found little evidence that Src-dependent mechanisms of cell migration, observed in response to applied EFs in vitro, normally exist in vivo. It is concluded that endogenous EFs do not drive long-term directionality of sustained healing migration in this mouse corneal epithelial model. Ion flow from wounds may nevertheless represent an important component of wound signaling initiation.
Assuntos
Eletricidade , Epitélio Corneano/lesões , Epitélio Corneano/patologia , Cicatrização , Animais , Western Blotting , Movimento Celular , Ativação Enzimática , Células Epiteliais/enzimologia , Células Epiteliais/patologia , Epitélio Corneano/enzimologia , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Humanos , Imuno-Histoquímica , Camundongos , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/metabolismo , Transporte Proteico , Proteínas Repressoras/metabolismo , Fatores de Tempo , Quinases da Família src/metabolismoRESUMO
Corneal development and structure were studied in the Iberian mole Talpa occidentalis, which has permanently closed eyelids, and the European mole Talpa europaea, in which the eyes are open. The vertebrate cornea typically maintains a three-layered structure - a stratified epithelium with protective and sensory function, an avascular, hypocellular, collagenous stroma, and an endothelium with both barrier and transport functions that regulates corneal hydration, hence maintaining transparency. Compared to mouse, both mole species had significant corneal specializations, but the Iberian mole had the most divergent phenotype, with no endothelium and a flattened monolayer epithelium. Nevertheless, normal epithelial cell junctions were observed and corneal transparency was maintained. Corneas of European moles have a dysmorphic phenotype that recapitulates the human disorder keratoconus for which no mouse model exists. Mole corneas are vascularized - a situation only previously observed in the manatee Trichechus- and have non-radial patterns of corneal innervation indicative of failure of corneal epithelial cell migration. The transcription factor Pax6 is required for corneal epithelial differentiation in mice, but was found to be dispensable in moles, which had mosaic patterns of PAX6 localization uniquely restricted, in European moles, to the apical epithelial cells. The apparently stalled or abnormal differentiation of corneas in adult moles is supported by their superficial similarity to the corneas of embryonic or neonatal mice, and their abnormal expression of cytokeratin-12 and cytokeratin-5. European moles seem to have maintained some barrier/protective function in their corneas. However, Iberian moles show a more significant corneal regression likely related to the permanent eyelid fusion. In this mole species, adaptation to the arid, harder, Southern European soils could have favoured the transfer of these functions to the permanently sealed eyelids.
Assuntos
Córnea/anatomia & histologia , Córnea/metabolismo , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Queratina-12/metabolismo , Queratina-5/metabolismo , Toupeiras/anatomia & histologia , Toupeiras/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Córnea/crescimento & desenvolvimento , Pálpebras/anatomia & histologia , Fator de Transcrição PAX6 , FenótipoRESUMO
Animals adapted to dark ecotopes may experience selective pressure for retinal reduction. No previous studies have explicitly addressed the molecular basis of retinal development in any fossorial mammal. We studied retinal development and function in the Iberian mole Talpa occidentalis, which was presumed to be blind because of its permanently closed eyes. Prenatal retina development was relatively normal, with specification of all cell types and evidence of dorsoventral regionalization. Severe developmental defects occurred after birth, subsequent to lens abnormalities. 'Blind' Iberian moles had rods, cones and rod nuclear ultrastructure typical of diurnal mammals. DiI staining revealed only contralateral projections through the optic chiasm. Y-maze experiments demonstrated that moles retain a photoavoidance response. Over-representation of melanopsin-positive retinal ganglion cells that mediate photoperiodicity was observed. Hence, molecular pathways of eye development in Iberian moles retain the adaptive function of rod/cone primary vision and photoperiodicity, with no evidence that moles are likely to completely lose their eyes on an evolutionary time scale.
Assuntos
Diferenciação Celular , Toupeiras/anatomia & histologia , Células Fotorreceptoras de Vertebrados/citologia , Retina , Células Ganglionares da Retina/citologia , Animais , Feminino , Microscopia Eletrônica de Transmissão , Toupeiras/metabolismo , Toupeiras/fisiologia , Células Fotorreceptoras de Vertebrados/fisiologia , Gravidez , Retina/citologia , Retina/embriologia , Retina/fisiologia , Retina/ultraestrutura , Células Fotorreceptoras Retinianas Cones/citologia , Células Fotorreceptoras Retinianas Cones/fisiologia , Células Ganglionares da Retina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/citologia , Células Fotorreceptoras Retinianas Bastonetes/fisiologia , Opsinas de Bastonetes/metabolismo , Vias Visuais/fisiologia , Vias Visuais/ultraestruturaRESUMO
PURPOSE: PAX6 heterozygosity (PAX6(+/-)) causes aniridia and aniridia-related keratopathy (ARK) in humans, but the pathway from gene dosage deficiency to clinical disease has not been fully characterized. Recently, the authors suggested a model of a chronic wound state exacerbated by oxidative stress, showed the barrier function of Pax6(+/-) corneas is compromised and suggested Pax6(+/-) corneas show the molecular signature of a perpetual wound-healing state. METHODS: Pax6(+/-) mice were used as a model for Pax6-related corneal diseases and in vivo wound-healing assays. Immunohistochemistry and electron microscopy analyses were performed on mutant and wounded corneas. RESULTS: This work reports defects in keratin, desmoplakin, and actin-based cytoskeletal structures in Pax6(+/-) cells. During wild-type corneal reepithelialization, cell fissures and desquamation, intracellular vesicles, intercellular gaps, and filopodialike structures were apparent, similar to the phenotypes seen in "unwounded" Pax6(+/-) corneal epithelia. Pax6(+/-) cells and wounded wild-type cells showed changed patterns of desmoplakin and actin localization. Protein oxidation and ERK1/2 and p38 MAPK phosphorylation were barely detected in the basal cells of intact wild-type corneal epithelia, but they were found in basal wild-type cells near the wound edge and throughout Pax6(+/-) corneal epithelia. CONCLUSIONS: These data show that cell junctions and cytoskeleton organization are dynamically remodeled in vivo by wounding and in Pax6(+/-) corneas. This apparent wound-healing phenotype contributes to the clinical aspects of ARK.
Assuntos
Aniridia/metabolismo , Adesão Celular/fisiologia , Doenças da Córnea/metabolismo , Proteínas do Citoesqueleto/metabolismo , Epitélio Corneano/lesões , Traumatismos Oculares/metabolismo , Proteínas do Olho/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição Box Pareados/fisiologia , Proteínas Repressoras/fisiologia , Actinas/metabolismo , Animais , Aniridia/patologia , Células Cultivadas , Doenças da Córnea/patologia , Desmoplaquinas/metabolismo , Epitélio Corneano/metabolismo , Epitélio Corneano/ultraestrutura , Traumatismos Oculares/patologia , Feminino , Queratinas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos CBA , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 6 Ativada por Mitógeno/metabolismo , Fator de Transcrição PAX6 , Fosforilação , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
PURPOSE: Corneal nerves play essential roles in maintaining the ocular surface through provision of neurotrophic support, but genetic control of corneal innervation is poorly understood. The possibility of a neurotrophic failure in ocular surface disease associated with heterozygosity at the Pax6 locus (aniridia-related keratopathy [ARK]) was investigated. METHODS: Patterns of corneal innervation were studied during development and aging in mice with different Pax6 dosages and in chimeras. Immunohistochemistry and ELISA-based assays were used to determine the molecular basis of defects seen in Pax6 mutants, and wound healing assays were performed. RESULTS: In adults, the Pax6(+/-) epithelium was less densely innervated than the wild-type epithelium, and radial projection of epithelial nerves was disrupted. Neurotrophic support of the corneal epithelium appeared normal. Directed nerve projection correlated with patterns of epithelial cell migration in adult wild-types, but innervation defects observed in Pax6(+/-) mice were not fully corrected in wound healing or chimeric models where directed epithelial migration was restored. CONCLUSIONS: Pax6 dosage nonautonomously controls robust directed radial projection of corneal neurons, and the guidance cues for growth cone guidance are not solely dependent on directed epithelial migration. There is little evidence that ARK represents neurotrophic keratitis.