RESUMO
Brain protein aggregates are a hallmark of neurodegenerative disease. Previous work indicates that specific protein components of these aggregates are toxic, including tau (encoded by MAPT) in Alzheimer's disease and related tauopathies. Increasing evidence also indicates that these toxic proteins traffic between cells in a prion-like fashion, thereby spreading pathology from one brain region to another. However, the mechanisms involved in trafficking are poorly understood. We therefore developed a transgenic Drosophila model to facilitate rapid evaluation of candidate tau trafficking modifiers. Our model uses the bipartite Q system to drive co-expression of tau and GFP in the fly eye. We found age-dependent spread of tau into the brain, represented by detection of tau, but not of GFP. We also found that tau trafficking was attenuated upon inhibition of the endocytic factor dynamin (encoded by shi) or knockdown of glycogen synthase kinase-3ß (GSK-3ß, encoded by sgg). Further work revealed that dynamin promoted tau uptake in recipient tissues, whereas GSK-3ß appeared to promote tau spread via direct phosphorylation of tau. Our robust and flexible system will promote the identification of tau-trafficking components involved in the pathogenesis of neurodegenerative diseases.
Assuntos
Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Drosophila , Drosophila melanogaster , Glicogênio Sintase Quinase 3 beta , Proteínas tau , Animais , Proteínas tau/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Fosforilação , Encéfalo/metabolismo , Encéfalo/patologia , Dinaminas/metabolismo , Transporte Proteico , Quinase 3 da Glicogênio Sintase/metabolismo , Humanos , Proteínas de Fluorescência Verde/metabolismoRESUMO
Diabetes mellitus (DM) and Alzheimer's disease (AD) rates are rising, mirroring the global trend of an aging population. Numerous epidemiological studies have shown that those with Type 2 diabetes (T2DM) have an increased risk of developing dementia. These degenerative and progressive diseases share some risk factors. To a large extent, the amyloid cascade is responsible for AD development. Neurofibrillary tangles induce neurodegeneration and brain atrophy; this chain reaction begins with hyperphosphorylation of tau proteins caused by progressive amyloid beta (Aß) accumulation. In addition to these processes, it seems that alterations in brain glucose metabolism and insulin signalling lead to cell death and reduced synaptic plasticity in AD, before the onset of symptoms, which may be years away. Due to the substantial evidence linking insulin resistance in the brain with AD, researchers have coined the name "Type 3 diabetes" to characterize the condition. We still know little about the processes involved, even though current animal models have helped illuminate the links between T2DM and AD. This brief overview discusses insulin and IGF-1 signalling disorders and the primary molecular pathways that may connect them. The presence of GSK-3ß in AD is intriguing. These proteins' association with T2DM and pancreatic ß-cell failure suggests they might be therapeutic targets for both disorders.
Assuntos
Doença de Alzheimer , Diabetes Mellitus Tipo 2 , Doença de Alzheimer/metabolismo , Doença de Alzheimer/patologia , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Humanos , Animais , Transdução de Sinais , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismoRESUMO
BACKGROUND: Androgenetic alopecia (AGA) is a common form of hair loss. Androgens, such as testosterone and dihydrotestosterone, are the main causes of AGA. Extracellular vesicles (EVs) derived from mesenchymal stem cells (MSCs) can reduce AGA. However, preparing therapeutic doses of MSCs for clinical use is challenging. Induced pluripotent stem cell-derived MSCs (iMSCs) are homogenous and easily expandable, enabling scalable production of EVs. Hyaluronic acid (HA) can exert various functions including free radical scavenging, immune regulation, and cell migration. Herein, we examined whether hyaluronic acid (HA) stimulation of iMSCs could produce EVs with enhanced therapeutic outcomes for AGA. METHODS: EVs were collected from iMSCs primed with HA (HA-iMSC-EVs) or without HA (iMSC-EVs). The characteristics of EVs were examined using dynamic light scattering, cryo-transmission electron microscopy, immunoblotting, flow cytometry, and proteomic analysis. In vitro, we compared the potential of EVs in stimulating the survival of hair follicle dermal papilla cells undergoing testosterone-mediated AGA. Additionally, the expression of androgen receptor (AR) and relevant growth factors as well as key proteins of Wnt/ß-catenin signaling pathway (ß-catenin and phosphorylated GSK3ß) was analyzed. Subsequently, AGA was induced in male C57/BL6 mice by testosterone administration, followed by repeated injections of iMSC-EVs, HA-iMSC-EVs, finasteride, or vehicle. Several parameters including hair growth, anagen phase ratio, reactivation of Wnt/ß-catenin pathway, and AR expression was examined using qPCR, immunoblotting, and immunofluorescence analysis. RESULTS: Both types of EVs showed typical characteristics for EVs, such as size distribution, markers, and surface protein expression. In hair follicle dermal papilla cells, the mRNA levels of AR, TGF-ß, and IL-6 increased by testosterone was blocked by HA-iMSC-EVs, which also contributed to the augmented expression of trophic genes related to hair regrowth. However, no notable changes were observed in the iMSC-EVs. Re-activation of Wnt/ß-catenin was observed in HA-iMSC-EVs but not in iMSC-EVs, as shown by ß-catenin stabilization and an increase in phosphorylated GSK3ß. Restoration of hair growth was more significant in HA-iMSC-EVs than in iMSC-EVs, and was comparable to that in mice treated with finasteride. Consistently, the decreased anagen ratio induced by testosterone was reversed by HA-iMSC-EVs, but not by iMSC-EVs. An increased expression of hair follicular ß-catenin protein, as well as the reduction of AR was observed in the skin tissue of AGA mice receiving HA-iMSC-EVs, but not in those treated with iMSC-EVs. CONCLUSIONS: Our results suggest that HA-iMSC-EVs have potential to improve AGA by regulating growth factors/cytokines and stimulating AR-related Wnt/ß-catenin signaling.
Assuntos
Alopecia , Vesículas Extracelulares , Folículo Piloso , Ácido Hialurônico , Células-Tronco Mesenquimais , Vesículas Extracelulares/metabolismo , Alopecia/terapia , Alopecia/metabolismo , Alopecia/tratamento farmacológico , Ácido Hialurônico/farmacologia , Ácido Hialurônico/metabolismo , Animais , Camundongos , Células-Tronco Mesenquimais/metabolismo , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Folículo Piloso/metabolismo , Folículo Piloso/efeitos dos fármacos , Humanos , Via de Sinalização Wnt/efeitos dos fármacos , Masculino , Receptores Androgênicos/metabolismo , Receptores Androgênicos/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Testosterona/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos Endogâmicos C57BLRESUMO
Glycogen synthase kinase 3ß (GSK-3ß) targets specific signaling pathways in response to distinct upstream signals. We used structural and functional studies to dissect how an upstream phosphorylation step primes the Wnt signaling component ß-catenin for phosphorylation by GSK-3ß and how scaffolding interactions contribute to this reaction. Our crystal structure of GSK-3ß bound to a phosphoprimed ß-catenin peptide confirmed the expected binding mode of the phosphoprimed residue adjacent to the catalytic site. An aspartate phosphomimic in the priming site of ß-catenin adopted an indistinguishable structure but reacted approximately 1000-fold slower than the native phosphoprimed substrate. This result suggests that substrate positioning alone is not sufficient for catalysis and that native phosphopriming interactions are necessary. We also obtained a structure of GSK-3ß with an extended peptide from the scaffold protein Axin that bound with greater affinity than that of previously crystallized Axin fragments. This structure neither revealed additional contacts that produce the higher affinity nor explained how substrate interactions in the GSK-3ß active site are modulated by remote Axin binding. Together, our findings suggest that phosphopriming and scaffolding produce small conformational changes or allosteric effects, not captured in the crystal structures, that activate GSK-3ß and facilitate ß-catenin phosphorylation. These results highlight limitations in our ability to predict catalytic activity from structure and have potential implications for the role of natural phosphomimic mutations in kinase regulation and phosphosite evolution.
Assuntos
Proteína Axina , Glicogênio Sintase Quinase 3 beta , beta Catenina , Humanos , Proteína Axina/metabolismo , Proteína Axina/química , Proteína Axina/genética , beta Catenina/metabolismo , beta Catenina/química , beta Catenina/genética , Domínio Catalítico , Cristalografia por Raios X , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/química , Glicogênio Sintase Quinase 3 beta/genética , Modelos Moleculares , Fosforilação , Ligação Proteica , Conformação Proteica , Via de Sinalização WntRESUMO
Maxillofacial bone defects can severely impact quality of life by impairing physiological functions such as chewing, breathing, swallowing, and pronunciation. Polyether ether ketone (PEEK) is commonly used for the repair of maxillofacial defects due to its mechanical adaptability, while its osteogenic properties still need refinement. Herein, we have utilized the piezoelectric effect exhibited by barium titanate (BTO) under low-intensity pulsed ultrasound (LIPUS) to develop an ultrasound responsive PEEK (PDA@BTO-SPEEK, PBSP) through the mediating effect of polydopamine (PDA), for repairing maxillofacial bone defects. After modification by PDA@BTO, PBSP possesses better hydrophilicity, which is conducive to cell growth and adhesion. Simultaneously, by virtue of the piezoelectric characteristics of BTO, PBSP obtains a piezoelectric coefficient that matches the bone cortex. Notably, when PBSP is stimulated by LIPUS, it can generate stable electricity and effectively accelerate the osteogenic differentiation of osteoblasts through the regulation of the Piezo1-induced calcium (Ca2+) influx and Akt/GSK3ß/ß-catenin pathway. In addition, PBSP presents satisfactory therapeutic effects in rat skull defect models, and its osteogenic efficiency can be further improved under LIPUS stimulation with high tissue penetration. Collectively, PBSP + LIPUS exhibits great potential as a promising alternative strategy for the repair of maxillofacial bone defects.
Assuntos
Benzofenonas , Glicogênio Sintase Quinase 3 beta , Cetonas , Osteogênese , Polietilenoglicóis , Polímeros , Proteínas Proto-Oncogênicas c-akt , Ratos Sprague-Dawley , beta Catenina , Animais , Glicogênio Sintase Quinase 3 beta/metabolismo , Polímeros/química , Osteogênese/efeitos dos fármacos , Ratos , Polietilenoglicóis/química , Proteínas Proto-Oncogênicas c-akt/metabolismo , Cetonas/química , Cetonas/farmacologia , beta Catenina/metabolismo , Diferenciação Celular/efeitos dos fármacos , Osteoblastos/efeitos dos fármacos , Ondas Ultrassônicas , Indóis/química , Indóis/farmacologia , Masculino , Transdução de Sinais/efeitos dos fármacos , Crânio/efeitos dos fármacos , Titânio/química , Titânio/farmacologia , Regeneração Óssea/efeitos dos fármacosRESUMO
5-Fluorouracil (5-FU) is widely used in the treatment of gastric cancer, and the emergence of drug resistance and toxic effects has limited its application. Therefore, there is an urgent need for safe and effective novel drugs or new therapies. ß-Ionone (BI) is found in vegetables and fruits and possesses an inhibitory proliferation of tumor cells in vitro and in vivo. In this study, we investigated whether BI could enhance the inhibitory effects of 5-FU on the proliferation of gastric adenocarcinoma cells and the growth of gastric cancer cell xenografts in a mouse model. The effects of BI and 5-FU alone or their combination on the cell viability, apoptosis, and mitochondrial membrane potential, the cell cycle, and its related proteins-Cyclin D1, and CDK4 as well as PCNA and GSK-3ß were evaluated in SGC-7901 cells and MKN45 cells by MTT, MB, flow cytometry and Western blot. In addition, the effects of BI and 5-FU alone or their combination on the growth of SGC-7901 cell xenografts in nude mice were investigated. The results showed that BI significantly enhanced the sensitivity of gastric adenocarcinoma cells to 5-FU in vitro and in vivo, i.e. proliferation inhibited, apoptosis induced and GSK-3ß protein activated. Therefore, our results suggest that BI increases the antitumor effect of 5-FU on gastric adenocarcinoma cells, at least partly from an activated GSK-3ß signaling pathway.
Assuntos
Adenocarcinoma , Apoptose , Proliferação de Células , Fluoruracila , Glicogênio Sintase Quinase 3 beta , Camundongos Nus , Norisoprenoides , Transdução de Sinais , Neoplasias Gástricas , Animais , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/patologia , Neoplasias Gástricas/metabolismo , Fluoruracila/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Humanos , Proliferação de Células/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Norisoprenoides/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Adenocarcinoma/metabolismo , Camundongos , Apoptose/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto , Sinergismo Farmacológico , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Camundongos Endogâmicos BALB C , Quinase 3 da Glicogênio Sintase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quinase 4 Dependente de Ciclina/metabolismoRESUMO
Fucoxanthin (Fx), a xanthophyll carotenoid abundant in brown algae, possesses several biological functions, such as antioxidant, anti-inflammatory, and cardiac-protective activities. However, the role of Fx in myocardial ischemia/reperfusion (MI/R) is still unclear. Thus, the aim of this study was to investigate the effect of Fx on MI/R-induced injury and explore the underlying mechanisms. Our results showed that in vitro, Fx treatment significantly suppressed inflammatory response, oxidative stress, and apoptosis in rat cardiomyocytes exposed to hypoxia/reoxygenation (H/R). In addition, Fx led to increased phosphorylation of AMPK, AKT, and GSK-3ß, and enhanced activation of Nrf2 in cardiomyocytes under H/R conditions. Notably, pretreatment with Compound C (AMPK inhibitor), partially reduced the beneficial effects of Fx in cardiomyocytes exposed to H/R. In vivo, Fx ameliorated myocardial damage, inhibited inflammatory response, oxidative stress, and apoptosis, and activated the AMPK/GSK-3ß/Nrf2 signaling in myocardial tissues in MI/R rat model. Taken together, these findings indicated that Fx attenuates MI/R-induced injury by inhibiting oxidative stress, inflammatory response, and apoptosis. The AMPK/GSK-3ß/Nrf2 pathway is involved in the cardioprotective effect of Fx in MI/R injury. Thus, Fx may be a promising drug for the treatment of MI/R.
Assuntos
Proteínas Quinases Ativadas por AMP , Apoptose , Glicogênio Sintase Quinase 3 beta , Traumatismo por Reperfusão Miocárdica , Miócitos Cardíacos , Fator 2 Relacionado a NF-E2 , Estresse Oxidativo , Transdução de Sinais , Xantofilas , Animais , Ratos , Proteínas Quinases Ativadas por AMP/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP/metabolismo , Apoptose/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/efeitos dos fármacos , Glicogênio Sintase Quinase 3 beta/metabolismo , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/patologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Fator 2 Relacionado a NF-E2/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Xantofilas/farmacologia , Xantofilas/químicaRESUMO
Temporal Lobe Epilepsy (TLE) is a chronic neurological disorder characterized by recurrent focal seizures originating in the temporal lobe. Despite the variety of antiseizure drugs currently available to treat TLE, about 30% of cases continue to have seizures. The etiology of TLE is complex and multifactorial. Increasing evidence indicates that Alzheimer's disease (AD) and drug-resistant TLE present common pathological features that may induce hyperexcitability, especially aberrant hyperphosphorylation of tau protein. Genetic polymorphic variants located in genes of the microtubule-associated protein tau (MAPT) and glycogen synthase kinase-3ß (GSK3B) have been associated with the risk of developing AD. The APOE ε4 allele is a major genetic risk factor for AD. Likewise, a gene-dose-dependent effect of ε4 seems to influence TLE. The present study aimed to investigate whether the APOE É4 allele and genetic variants located in the MAPT and GSK3B genes are associated with the risk of developing AD and drug-resistant TLE in a cohort of the Mexican population. A significant association with the APOE ε4 allele was observed in patients with AD and TLE. Additional genetic interactions were identified between this allele and variants of the MAPT and GSK3B genes.
Assuntos
Alelos , Doença de Alzheimer , Apolipoproteína E4 , Glicogênio Sintase Quinase 3 beta , Proteínas tau , Humanos , Proteínas tau/genética , Proteínas tau/metabolismo , Doença de Alzheimer/genética , Glicogênio Sintase Quinase 3 beta/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Masculino , Feminino , Pessoa de Meia-Idade , Apolipoproteína E4/genética , Adulto , Predisposição Genética para Doença , Idoso , Epilepsia Resistente a Medicamentos/genética , Epilepsia Resistente a Medicamentos/tratamento farmacológico , Epilepsia do Lobo Temporal/genética , Epilepsia do Lobo Temporal/tratamento farmacológico , Polimorfismo de Nucleotídeo ÚnicoRESUMO
INTRODUCTION: Alzheimer's disease (AD) is a neurodegenerative disease characterized by Amyloid plaques and neurofibrillary tangles. We explored the potential mechanism by which Danggui Shaoyao San (DSS) modulates central glucose metabolism via the insulin receptor substrate 1 (IRS1)/glycogen synthase kinase-3ß (GSK3ß)/Wnt3a-ß-catenin pathway, thereby exerting protective effects on cognitive functions. METHODS: In vitro, HT22 cells were induced with streptozotocin (STZ) to investigate the impact of GSK3ß on pathway transduction. The active components in the DSS stock solution were validated using mass spectrometry. Subsequently, an AD model in C57BL/6J mice was established through STZ injection into both ventricles. The success of the model was validated behaviorally and pathologically. The Morris Water Maze (MWM) test, immunohistochemistry, Western blotting, quantitative reverse transcription-PCR, and 18F-fluorodeoxyglucose-positron emission tomography (FDG-PET) were employed to evaluate the influence of DSS on memory and pathological changes in AD. RESULTS: The DSS stock solution, rich in active components, ameliorated the memory deficits in AD mice in the MWM. In vitro, GSK3ß exhibited regulatory control over Wnt and ß-catenin, with GSK3ß inhibition mitigating ß-amyloid and tau redundancies at protein and gene levels, facilitating signal transduction. In vivo, DSS impacted key targets in the IRS1/GSK3ß/Wnt3a-ß-catenin pathway, mitigated senile plaques resulting from amyloid ß (Aß) deposition and neurofiber tangles induced by tau hyperphosphorylation, and alleviated the decline in central glucose metabolism observed in FDG-PET. CONCLUSIONS: Our findings suggest that DSS potentially confers cognitive protection by alleviating central hypoglycemia through the IRS1/GSK3ß/Wnt3a-ß-catenin pathway. This may serve as a promising therapeutic avenue for AD.
Assuntos
Doença de Alzheimer , Disfunção Cognitiva , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas , Glicogênio Sintase Quinase 3 beta , Proteínas Substratos do Receptor de Insulina , Camundongos Endogâmicos C57BL , Proteína Wnt3A , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Proteínas Substratos do Receptor de Insulina/metabolismo , Camundongos , Medicamentos de Ervas Chinesas/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Proteína Wnt3A/metabolismo , Disfunção Cognitiva/tratamento farmacológico , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/etiologia , Masculino , beta Catenina/metabolismo , Via de Sinalização Wnt/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacosRESUMO
Our previous study proved that epicatechin (EC) and ß-glucan (BG) from whole-grain highland barley synergistically modulate glucose metabolism in insulin-resistant HepG2 cells. However, the main target and the mechanism underlying the modulation of glucose metabolism in vivo remain largely unknown. In this study, cell transfection assay and microscale thermophoresis analysis revealed that EC and BG could directly bind to the insulin receptor (IR) and mammalian receptor for rapamycin (mTOR), respectively. Molecular dynamic analysis indicated that the key amino acids of binding sites were Asp, Met, Val, Lys, Ser, and Tys. EC supplementation upregulated the IRS-1/PI3K/Akt pathway, while BG upregulated the mTOR/Akt pathway. Notably, supplementation with EC + BG significantly increased Akt and glucose transporter type 4 (GLUT4) protein expressions, while decreasing glycogen synthase kinase 3ß (GSK-3ß) expression in liver cells as compared to the individual effects of EC and BG, indicating their synergistic effect on improving hepatic glucose uptake and glycogen synthesis. Consistently, supplementation with EC + BG significantly decreased blood glucose levels and improved oral glucose tolerance compared to EC and BG. Therefore, combined supplementation with EC and BG may bind to corresponding receptors, targeting synergistic activation of Akt expression, leading to the improvement of hepatic glucose metabolism and thereby ameliorating hyperglycemia in vivo.
Assuntos
Catequina , Glucose , Hordeum , Hiperglicemia , Fígado , Camundongos Endogâmicos C57BL , beta-Glucanas , Hordeum/química , beta-Glucanas/farmacologia , beta-Glucanas/química , Animais , Camundongos , Catequina/farmacologia , Catequina/administração & dosagem , Fígado/metabolismo , Fígado/efeitos dos fármacos , Masculino , Humanos , Glucose/metabolismo , Hiperglicemia/tratamento farmacológico , Hiperglicemia/metabolismo , Sinergismo Farmacológico , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-akt/genética , Transportador de Glucose Tipo 4/metabolismo , Transportador de Glucose Tipo 4/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Glicemia/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Serina-Treonina Quinases TOR/genética , Extratos Vegetais/farmacologia , Extratos Vegetais/administração & dosagem , Células Hep G2RESUMO
Temporal Lobe Epilepsy (TLE) is a severe neurological condition characterized by recurrent seizures that often do not respond well to available anti-seizure medications. TLE has been associated with epileptogenesis, a process that starts during the latent period following a neurologic insult and is followed by chronic phase. Recent research has linked canonical Wnt signaling to the pathophysiology of epileptogenesis and TLE. Our previous study demonstrated differential regulation of canonical Wnt signaling during early and late stage post status epilepticus (SE) induction. Building on these findings, our current study utilized Wnt modulators: GSK-3ß inhibitor 6-bromoindirubin-3'-oxime (6-Bio) and disheveled inhibitor niclosamide and investigated their impact on canonical Wnt signaling during the early (30 days) and later stages (60 days) following SE induction. We assessed several parameters, including seizure frequency, astrogliosis, synaptic density, and neuronal counts in hippocampal tissue. We used immunohistochemistry and Nissl staining to evaluate gliosis, synaptic density, and neuronal counts in micro-dissected hippocampi. Western blotting was used to examine the expression of proteins involved in canonical Wnt/ß-catenin signaling, and real-time PCR was conducted to analyze their relative mRNA expression. Wnt modulators, 6-Bio and Niclosamide were found to reduce seizure frequency and various other parameters including behavioral parameters, hippocampal morphology, astrogliosis and synaptic density at different stages of TLE.
Assuntos
Epilepsia do Lobo Temporal , Gliose , Indóis , Fármacos Neuroprotetores , Niclosamida , Oximas , Via de Sinalização Wnt , Epilepsia do Lobo Temporal/tratamento farmacológico , Epilepsia do Lobo Temporal/metabolismo , Epilepsia do Lobo Temporal/patologia , Animais , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologia , Masculino , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Oximas/farmacologia , Oximas/uso terapêutico , Indóis/farmacologia , Indóis/uso terapêutico , Gliose/tratamento farmacológico , Gliose/patologia , Gliose/metabolismo , Niclosamida/farmacologia , Niclosamida/uso terapêutico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hipocampo/patologia , Ratos Sprague-Dawley , Glicogênio Sintase Quinase 3 beta/metabolismo , Sinapses/efeitos dos fármacos , Sinapses/metabolismo , Sinapses/patologia , RatosRESUMO
Friedreich ataxia (FA) is a rare neurodegenerative disease caused by decreased levels of the mitochondrial protein frataxin. Frataxin has been related in iron homeostasis, energy metabolism, and oxidative stress. Ferroptosis has recently been shown to be involved in FA cellular degeneration; however, its role in dorsal root ganglion (DRG) sensory neurons, the cells that are affected the most and the earliest, is mostly unknown. In this study, we used primary cultures of frataxin-deficient DRG neurons as well as DRG from the FXNI151F mouse model to study ferroptosis and its regulatory pathways. A lack of frataxin induced upregulation of transferrin receptor 1 and decreased ferritin and mitochondrial iron accumulation, a source of oxidative stress. However, there was impaired activation of NRF2, a key transcription factor involved in the antioxidant response pathway. Decreased total and nuclear NRF2 explains the downregulation of both SLC7A11 (a member of the system Xc, which transports cystine required for glutathione synthesis) and glutathione peroxidase 4, responsible for increased lipid peroxidation, the main markers of ferroptosis. Such dysregulation could be due to the increase in KEAP1 and the activation of GSK3ß, which promote cytosolic localization and degradation of NRF2. Moreover, there was a deficiency in the LKB1/AMPK pathway, which would also impair NRF2 activity. AMPK acts as a positive regulator of NRF2 and it is activated by the upstream kinase LKB1. The levels of LKB1 were reduced when frataxin decreased, in agreement with reduced pAMPK (Thr172), the active form of AMPK. SIRT1, a known activator of LKB1, was also reduced when frataxin decreased. MT-6378, an AMPK activator, restored NRF2 levels, increased GPX4 levels and reduced lipid peroxidation. In conclusion, this study demonstrated that frataxin deficiency in DRG neurons disrupts iron homeostasis and the intricate regulation of molecular pathways affecting NRF2 activation and the cellular response to oxidative stress, leading to ferroptosis.
Assuntos
Proteínas Quinases Ativadas por AMP , Modelos Animais de Doenças , Ferroptose , Frataxina , Ataxia de Friedreich , Gânglios Espinais , Glicogênio Sintase Quinase 3 beta , Proteínas de Ligação ao Ferro , Proteína 1 Associada a ECH Semelhante a Kelch , Fator 2 Relacionado a NF-E2 , Proteínas Serina-Treonina Quinases , Animais , Fator 2 Relacionado a NF-E2/metabolismo , Fator 2 Relacionado a NF-E2/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/genética , Ataxia de Friedreich/patologia , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/genética , Camundongos , Glicogênio Sintase Quinase 3 beta/metabolismo , Gânglios Espinais/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Proteínas de Ligação ao Ferro/metabolismo , Proteínas de Ligação ao Ferro/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Estresse Oxidativo , Transdução de Sinais , Ferro/metabolismo , Quinases Proteína-Quinases Ativadas por AMP/metabolismoRESUMO
Autophagy, a highly conserved self-digestion process crucial for cellular homeostasis, is triggered by various environmental signals, including nutrient scarcity. The regulation of lysosomal and autophagy-related processes is pivotal to maintaining cellular homeostasis and basal metabolism. The consequences of disrupting or diminishing lysosomal and autophagy systems have been investigated; however, information on the implications of hyperactivating lysosomal and autophagy genes on homeostasis is limited. Here, we present a mechanism of transcriptional repression involving upstream stimulatory factor 2 (USF2), which inhibits lysosomal and autophagy genes under nutrient-rich conditions. We find that USF2, together with HDAC1, binds to the CLEAR motif within lysosomal genes, thereby diminishing histone H3K27 acetylation, restricting chromatin accessibility, and downregulating lysosomal gene expression. Under starvation, USF2 competes with transcription factor EB (TFEB), a master transcriptional activator of lysosomal and autophagy genes, to bind to target gene promoters in a phosphorylation-dependent manner. The GSK3ß-mediated phosphorylation of the USF2 S155 site governs USF2 DNA-binding activity, which is involved in lysosomal gene repression. These findings have potential applications in the treatment of protein aggregation-associated diseases, including α1-antitrypsin deficiency. Notably, USF2 repression is a promising therapeutic strategy for lysosomal and autophagy-related diseases.
Assuntos
Autofagia , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Lisossomos , Fatores Estimuladores Upstream , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/genética , Lisossomos/metabolismo , Autofagia/genética , Humanos , Fatores Estimuladores Upstream/metabolismo , Fatores Estimuladores Upstream/genética , Fosforilação , Histona Desacetilase 1/metabolismo , Histona Desacetilase 1/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Regulação da Expressão Gênica , Regiões Promotoras Genéticas , Células HEK293 , Animais , Histonas/metabolismo , Células HeLa , Camundongos , AcetilaçãoRESUMO
In vivo and in vitro studies argue that concentration-dependent Wnt signaling regulates mammalian nephron progenitor cell (NPC) programs. Canonical Wnt signaling is regulated through the stabilization of ß-catenin, a transcriptional co-activator when complexed with Lef/Tcf DNA-binding partners. Using the GSK3ß inhibitor CHIR99021 (CHIR) to block GSK3ß-dependent destruction of ß-catenin, we examined dose-dependent responses to ß-catenin in mouse NPCs, using mRNA transduction to modify gene expression. Low CHIR-dependent proliferation of NPCs was blocked on ß-catenin removal, with evidence of NPCs arresting at the G2-M transition. While NPC identity was maintained following ß-catenin removal, mRNA-seq identified low CHIR and ß-catenin dependent genes. High CHIR activated nephrogenesis. Nephrogenic programming was dependent on Lef/Tcf factors and ß-catenin transcriptional activity. Molecular and cellular features of early nephrogenesis were driven in the absence of CHIR by a mutated stabilized form of ß-catenin. Chromatin association studies indicate low and high CHIR response genes are likely direct targets of canonical Wnt transcriptional complexes. Together, these studies provide evidence for concentration-dependent Wnt signaling in the regulation of NPCs and provide new insight into Wnt targets initiating mammalian nephrogenesis.
Assuntos
Néfrons , Células-Tronco , Via de Sinalização Wnt , beta Catenina , Animais , Néfrons/metabolismo , Néfrons/citologia , beta Catenina/metabolismo , Camundongos , Células-Tronco/metabolismo , Células-Tronco/citologia , Pirimidinas/farmacologia , Piridinas/farmacologia , Regulação da Expressão Gênica no Desenvolvimento , Proliferação de Células , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Organogênese/genética , Transcrição GênicaRESUMO
The prognosis of patients with high-risk neuroblastoma remains poor, partly due to inadequate immune recognition of the tumor. Neuroblastomas display extremely low surface MHC-I, preventing recognition by cytotoxic T lymphocytes (CTLs) and contributing to an immunosuppressive tumor microenvironment. Glycogen synthase kinase-3 beta (GSK-3ß) is involved in pathways that may affect the MHC-I antigen processing and presentation pathway. We proposed that therapeutic inhibition of GSK-3ß might improve the surface display of MHC-I molecules on neuroblastoma cells, and therefore tested if targeting of GSK-3ß using the inhibitor 9-ING-41 (Elraglusib) improves MHC-I-mediated CTL recognition. We analyzed mRNA expression data of neuroblastoma tumor datasets and found that non-MYCN-amplified neuroblastomas express higher GSK-3ß levels than MYCN-amplified tumors. In non-MYCN-amplified cells SH-SY5Y, SK-N-AS and SK-N-SH 9-ING-41 treatment enhanced MHC-I surface display and the expression levels of a subset of genes involved in MHC-I antigen processing and presentation. Further, 9-ING-41 treatment triggered increased STAT1 pathway activation, upstream of antigen presentation pathways in two of the three non-MYCN-amplified cell lines. Finally, in co-culture experiments with CD8 + T cells, 9-ING-41 improved immune recognition of the neuroblastoma cells, as evidenced by augmented T-cell activation marker levels and T-cell proliferation, which was further enhanced by PD-1 immune checkpoint inhibition. Our preclinical study provides experimental support to further explore the GSK-3ß inhibitor 9-ING-41 as an immunomodulatory agent to increase tumor immune recognition in neuroblastoma.
Assuntos
Linfócitos T CD8-Positivos , Glicogênio Sintase Quinase 3 beta , Neuroblastoma , Humanos , Neuroblastoma/imunologia , Neuroblastoma/patologia , Neuroblastoma/genética , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/antagonistas & inibidores , Linfócitos T CD8-Positivos/imunologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T Citotóxicos/imunologia , Proteína Proto-Oncogênica N-Myc/genética , Proteína Proto-Oncogênica N-Myc/metabolismoRESUMO
BACKGROUND: Gastric cancer (GC), a prevalent malignant tumor which is a leading cause of death from malignancy around the world. Peritoneal metastasis accounts for the major cause of mortality in patients with GC. Despite hyperthermia intraperitoneal chemotherapy (HIPEC) improves the therapeutic effect of GC, it's equivocal about the mechanism under HIPEC. METHODS: MiR-183-5p expression was sifted from miRNA chip and detected in both GC patients and cell lines by qRT-PCR. Gene interference and rescue experiments were performed to identified biological function in vitro and vivo. Next, we affirmed PPP2CA as targeted of miR-183-5p by dual luciferase reporter assay. Finally, the potential relationship between HIPEC and miR-183-5p was explored. RESULTS: MiR-183-5p is up-regulated in GC and associated with advanced stage and poor prognosis. MiR-183-5p accelerate GC migration in vitro which is influenced by miR-183-5p/PPP2CA/AKT/GSK3ß/ß-catenin Axis. HIPEC exerts migration inhibition via attenuating miR-183-5p expression. CONCLUSION: MiR-183-5p can be used as a potential HIPEC biomarker in patients with CC.
Assuntos
Movimento Celular , Glicogênio Sintase Quinase 3 beta , Hipertermia Induzida , MicroRNAs , Proteínas Proto-Oncogênicas c-akt , Neoplasias Gástricas , beta Catenina , Humanos , Neoplasias Gástricas/patologia , Neoplasias Gástricas/genética , Neoplasias Gástricas/metabolismo , MicroRNAs/genética , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Hipertermia Induzida/métodos , beta Catenina/metabolismo , beta Catenina/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Camundongos , Animais , Masculino , Feminino , Proteína Fosfatase 2/metabolismo , Proteína Fosfatase 2/genética , Linhagem Celular Tumoral , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Regulação Neoplásica da Expressão Gênica , Prognóstico , Pessoa de Meia-Idade , Camundongos Endogâmicos BALB C , Antineoplásicos Fitogênicos/farmacologiaRESUMO
Acquired resistance is inevitable in the treatment of non-small cell lung cancer (NSCLC) with osimertinib, and one of the primary mechanisms responsible for this resistance is the epithelial-mesenchymal transition (EMT). We identify upregulation of the proviral integration site for Moloney murine leukemia virus 1 (PIM1) and functional inactivation of glycogen synthase kinase 3ß (GSK3ß) as drivers of EMT-associated osimertinib resistance. Upregulation of PIM1 promotes the growth, invasion, and resistance of osimertinib-resistant cells and is significantly correlated with EMT molecules expression. Functionally, PIM1 suppresses the ubiquitin-proteasome degradation of snail family transcriptional repressor 1 (SNAIL) and snail family transcriptional repressor 2 (SLUG) by deactivating GSK3ß through phosphorylation. The stability and accumulation of SNAIL and SLUG facilitate EMT and encourage osimertinib resistance. Furthermore, treatment with PIM1 inhibitors prevents EMT progression and re-sensitizes osimertinib-resistant NSCLC cells to osimertinib. PIM1/GSK3ß signaling is activated in clinical samples of osimertinib-resistant NSCLC, and dual epidermal growth factor receptor (EGFR)/PIM1 blockade synergistically reverse osimertinib-resistant NSCLC in vivo. These data identify PIM1 as a driver of EMT-associated osimertinib-resistant NSCLC cells and predict that PIM1 inhibitors and osimertinib combination therapy will provide clinical benefit in patients with EGFR-mutant NSCLC.
Assuntos
Acrilamidas , Compostos de Anilina , Carcinoma Pulmonar de Células não Pequenas , Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Receptores ErbB , Glicogênio Sintase Quinase 3 beta , Neoplasias Pulmonares , Proteínas Proto-Oncogênicas c-pim-1 , Transdução de Sinais , Humanos , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Acrilamidas/farmacologia , Acrilamidas/uso terapêutico , Proteínas Proto-Oncogênicas c-pim-1/metabolismo , Proteínas Proto-Oncogênicas c-pim-1/genética , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Compostos de Anilina/farmacologia , Compostos de Anilina/uso terapêutico , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , Receptores ErbB/metabolismo , Receptores ErbB/genética , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Animais , Camundongos , Linhagem Celular Tumoral , Mutação/genética , Camundongos Nus , Fatores de Transcrição da Família Snail/metabolismo , Fatores de Transcrição da Família Snail/genética , Indóis , PirimidinasRESUMO
Triple-negative breast cancer (TNBC) represents a significant health concern for women worldwide, and the overproduction of MMP9 and CD151 is associated with various cancers, influencing tumour growth and progression. This study aimed to investigate how CD151 and MMP9 affect TNBC cell migration, apoptosis, proliferation, and invasion. Immunohistochemical experiments revealed that CD151 and MMP9 were positively expressed in triple-negative breast cancer, and lymph node metastasis, the histological grade, and CD151 and MMP9 expression were found to be independent prognostic factors for the survival of patients with triple-negative breast cancer. Cytological experiments indicated that the knockdown of CD151 or MMP9 slowed triple-negative breast cancer cell growth, migration, and invasion and increased the apoptosis rate. Compared with CD151 knockdown, double MMP9 and CD151 knockdown further promoted cell death and inhibited TNBC cell proliferation, migration, and invasion. Moreover, ß-catenin and p-GSK-3ß were significantly downregulated. In summary, simultaneously silencing CD151 and MMP9 further suppressed the proliferation, migration and invasion of TNBC cells and promoted their apoptosis. One possible strategy for inducing this effect is to block the GSK-3ß/ß-catenin pathway.
Assuntos
Apoptose , Movimento Celular , Proliferação de Células , Técnicas de Silenciamento de Genes , Glicogênio Sintase Quinase 3 beta , Metaloproteinase 9 da Matriz , Tetraspanina 24 , Neoplasias de Mama Triplo Negativas , beta Catenina , Humanos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Neoplasias de Mama Triplo Negativas/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/genética , beta Catenina/metabolismo , beta Catenina/genética , Feminino , Metaloproteinase 9 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/genética , Tetraspanina 24/metabolismo , Tetraspanina 24/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Apoptose/genética , Pessoa de Meia-Idade , Transdução de Sinais , Regulação Neoplásica da Expressão Gênica , Invasividade Neoplásica , PrognósticoRESUMO
BACKGROUND: The abnormal aggregation of α-synuclein (α-syn) in the substantia nigra pars compacta (SNpc) region of the brain is characteristic of Parkinson's disease (PD), leading to the selective demise of neurons. Modifications in the post-translational processing of α-syn, phosphorylation at Ser129 in particular, are implicated in α-syn aggregation and are considered key hallmarks of PD. Furthermore, dysregulated Wnt/ß-catenin signaling, influenced by glycogen synthase kinase-3 beta (GSK-3ß), is implicated in PD pathogenesis. Inhibition of GSK-3ß holds promise in promoting neuroprotection by enhancing the Wnt/ß-catenin pathway. METHODS: In our previous study utilizing 1-methyl-4-phenylpyridinium (MPP+)-administered differentiated SH-SY5Y cells and a PD mouse model, we explored Vanillin's neuroprotective properties and related mechanisms against neuronal loss induced by MPP+/1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) administration. In the current study, we elucidated the mitigating effects of Vanillin on motor impairments, P-Ser129-α-syn expression, Wnt/ß-catenin signaling, and autophagic neuron death induced by MPTP in a mouse model of PD by performing motor function tests, western blot analysis and immunostaining. RESULTS: Our results show that Vanillin effectively modulated the motor dysfunctions, GSK-3ß expression, and activity, activated the Wnt/ß-catenin signaling, and reduced autophagic neuronal demise in the MPTP-lesioned mice, highlighting its neuroprotective effects. CONCLUSIONS: These findings underscore the complex interplay between α-syn pathology, GSK-3ß, Wnt/ß-catenin signaling, and autophagic-cell death in PD pathogenesis. Targeting these pathways, particularly with Vanillin, can be a promising therapeutic strategy for restoring dopaminergic (DA-ergic) neuronal homeostasis and slowing the progression of PD. Further research is crucial to resolving existing disputes and translating these discoveries into effective therapeutic interventions for PD patients.
Assuntos
Benzaldeídos , Modelos Animais de Doenças , Fármacos Neuroprotetores , Via de Sinalização Wnt , alfa-Sinucleína , Animais , Masculino , Camundongos , alfa-Sinucleína/metabolismo , alfa-Sinucleína/efeitos dos fármacos , Benzaldeídos/farmacologia , Benzaldeídos/administração & dosagem , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/metabolismo , Glicogênio Sintase Quinase 3 beta/metabolismo , Glicogênio Sintase Quinase 3 beta/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/administração & dosagem , Transtornos Parkinsonianos/metabolismo , Transtornos Parkinsonianos/tratamento farmacológico , Sinucleinopatias/metabolismo , Sinucleinopatias/tratamento farmacológico , Via de Sinalização Wnt/efeitos dos fármacos , Via de Sinalização Wnt/fisiologiaRESUMO
BACKGROUND: Intestinal damage is a common and serious complication in patients with graft-versus-host disease (GVHD). Human placental mesenchymal stromal cells (hPMSCs) ameliorate GVHD tissue damage by exerting anti-oxidative effects; however, the underlying mechanisms remain not fully clear. METHODS: A GVHD mouse model and tumor necrosis factor-α (TNF-α)-stimulated human colon epithelial cell lines NCM460 and HT-29 cells were used to investigate the mechanisms of hPMSCs alleviating GVHD-induced intestinal oxidative damage. RESULTS: hPMSCs reduced TNF-α concentrations and the number of CD3+TNF-α+ T-cells, which were negatively correlated with the expression of claudin-1, occludin, and ZO-1, through CD73 in the colon tissue of GVHD mice. Meanwhile, hPMSCs reduced the mean fluorescence intensity (MFI) of reactive oxygen species (ROS) and the concentration of malondialdehyde (MDA), promoted superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) activities, as well as claudin-1, occludin, and ZO-1 expression, in colonic epithelial cells of GVHD mice and TNF-α-stimulated cells via CD73. Moreover, hPMSCs upregulated adenosine (ADO) concentrations in GVHD mice and TNF-α-stimulated cells and mitigated the loss of tight junction proteins via the CD73/ADO/ADO receptors. Further analysis showed that hPMSCs diminished Fyn expression and enhanced Nrf2, GCLC, and HO-1 expression in both TNF-α-stimulated cells and colonic epithelial cells of GVHD mice by activating PI3K/Akt/GSK-3ß pathway. CONCLUSIONS: The results suggested that hPMSC-mediated redox metabolism balance and promoted tight junction protein expression were achieved via CD73/ADO/PI3K/Akt/GSK-3ß/Fyn/Nrf2 axis, by which alleviating intestinal oxidative injury in GVHD mice.