RESUMO
Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with the loss of dopaminergic neurons and neuroinflammation. Recent studies have identified a role of T cells in the pathogenesis of PD. Additionally, these studies suggested that α-synuclein (α-Syn) is related to abnormal T-cell responses and may act as an epitope and trigger autoimmune T-cell responses. However, it is unclear whether the α-Syn-mediated autoimmune response occurs and whether it is related to neuronal cell death and glial cell activation. In this study, we investigated the autoimmune T-cell response induced by α-Syn peptides and evaluated the neurotoxic effect of the α-Syn peptide-mediated autoimmune response. The immunization of mice with α-Syn peptides resulted in enhanced autoimmune responses, such as the peptide recall response, polarization toward Th1/Th17 cells, and regulatory T cell imbalance. Furthermore, the α-Syn autoimmune response led to the death of primary neurons cocultured with splenocytes. Treatment with conditioned media from α-Syn peptide-immunized splenocytes induced microglia and toxic A1-type astrocyte activation. Taken together, our results provide evidence of the potential role of the α-Syn-initiated autoimmune response and its contribution to neuronal cell death and glial cell activation.
Assuntos
Autoimunidade , Morte Celular , Neurônios , alfa-Sinucleína , Animais , alfa-Sinucleína/imunologia , alfa-Sinucleína/metabolismo , Camundongos , Morte Celular/efeitos dos fármacos , Neurônios/imunologia , Neurônios/metabolismo , Neurônios/patologia , Neuroglia/imunologia , Neuroglia/metabolismo , Neuroglia/efeitos dos fármacos , Doença de Parkinson/imunologia , Doença de Parkinson/patologia , Doença de Parkinson/metabolismo , Camundongos Endogâmicos C57BL , Humanos , Ativação Linfocitária/imunologia , Ativação Linfocitária/efeitos dos fármacos , Peptídeos/imunologia , Células Cultivadas , Feminino , Linfócitos T Reguladores/imunologiaRESUMO
Severe fever with thrombocytopenia syndrome (SFTS) has a high mortality rate compared to other infectious diseases. SFTS is particularly associated with a high risk of mortality in immunocompromised individuals, while most patients who die of SFTS exhibit symptoms of severe encephalitis before death. However, the region of brain damage and mechanisms by which the SFTS virus (SFTSV) causes encephalitis remains unknown. Here, we revealed that SFTSV infects the brainstem and spinal cord, which are regions of the brain associated with respiratory function, and motor nerves in IFNAR1-/- mice. Further, we show that A1-reactive astrocytes are activated, causing nerve cell death, in infected mice. Primary astrocytes of SFTSV-infected IFNAR1-/- mice also induced neuronal cell death through the activation of A1-reactive astrocytes. Herein, we showed that SFTSV induces fatal neuroinflammation in the brain regions important for respiratory function and motor nerve, which may underlie mortality in SFTS patients. This study provides new insights for the treatment of SFTS, for which there is currently no therapeutic approach.
Assuntos
Astrócitos , Infecções por Bunyaviridae , Camundongos Knockout , Phlebovirus , Receptor de Interferon alfa e beta , Animais , Astrócitos/virologia , Astrócitos/patologia , Camundongos , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/deficiência , Phlebovirus/genética , Phlebovirus/fisiologia , Phlebovirus/patogenicidade , Infecções por Bunyaviridae/virologia , Infecções por Bunyaviridae/patologia , Infecções por Bunyaviridae/imunologia , Encéfalo/virologia , Encéfalo/patologia , Encéfalo/imunologia , Medula Espinal/virologia , Medula Espinal/patologia , Modelos Animais de Doenças , Neurônios/virologia , Neurônios/patologia , Camundongos Endogâmicos C57BL , Tronco Encefálico/virologia , Tronco Encefálico/patologia , Morte CelularRESUMO
Human embryonic stem cells (hESCs) have unique characteristics, such as self-renewal and pluripotency, which are distinct from those of other cell types. These characteristics of hESCs are tightly regulated by complex signaling mechanisms. In this study, we demonstrate that yes-associated protein (YAP) functions in an hESC-specific manner to maintain self-renewal and survival in hESCs. hESCs were highly sensitive to YAP downregulation to promote cell survival. Interestingly, hESCs displayed dynamic changes in YAP expression in response to YAP downregulation. YAP was critical for the maintenance of self-renewal. Additionally, the function of YAP in maintenance of self-renewal and cell survival was hESC-specific. Doxycycline upregulated YAP in hESCs and attenuated the decreased cell survival induced by YAP downregulation. However, decreased expression of self-renewal markers triggered by YAP downregulation and neural/cardiac differentiation were affected by doxycycline treatment. Collectively, the results reveal the mechanism underlying the role of YAP and the novel function of doxycycline in hESCs.
Assuntos
Células-Tronco Embrionárias Humanas , Diferenciação Celular/fisiologia , Doxiciclina/metabolismo , Doxiciclina/farmacologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Transdução de Sinais , Proteínas de Sinalização YAPRESUMO
Renal ischemia reperfusion (IR) injury is a major cause of acute kidney injury (AKI) that is often complicated by multiple organ failure of the liver and intestine. The mineralocorticoid receptor (MR) is activated in patients with renal failure associated with glomerular and tubular damage. We thus investigated whether canrenoic acid (CA), a mineralocorticoid receptor (MR) antagonist, protects against AKI-induced hepatic and intestinal injury, suggesting the underlying mechanisms. Mice were divided into five groups: sham mice, mice subjected to renal IR, and mice pretreated with canrenoic acid (CA; 1 or 10 mg/kg) 30 min prior to renal IR. At 24 h after renal IR, the levels of plasma creatinine, alanine aminotransferase and aldosterone were measured, and structural changes and inflammatory responses of the kidney, liver, and intestine were analyzed. We found that CA treatment reduced plasma creatinine levels, tubular cell death and oxidative stress induced by renal IR. CA treatment also decreased renal neutrophil infiltration and inflammatory cytokine expression and inhibited the release of high-mobility group box 1 induced by renal IR. Consistently, CA treatment reduced renal IR-induced plasma alanine transaminase, hepatocellular injury and neutrophil infiltration, and inflammatory cytokine expression. CA treatment also decreased small intestinal cell death, neutrophil infiltration and inflammatory cytokine expression induced by renal IR. Taken together, we conclude that MR antagonism by CA treatment protects against multiple organ failure in the liver and intestine after renal IR.
Assuntos
Injúria Renal Aguda , Traumatismo por Reperfusão , Camundongos , Animais , Antagonistas de Receptores de Mineralocorticoides , Ácido Canrenoico/metabolismo , Insuficiência de Múltiplos Órgãos/complicações , Creatinina/metabolismo , Receptores de Mineralocorticoides/metabolismo , Rim/metabolismo , Injúria Renal Aguda/metabolismo , Isquemia/metabolismo , Traumatismo por Reperfusão/metabolismo , Citocinas/metabolismo , Reperfusão/efeitos adversosRESUMO
Tagetes erecta and Ocimum basilicum are medicinal plants that exhibit anti-inflammatory effects against various diseases. However, their individual and combined effects on osteoarthritis (OA) are unknown. Herein, we aimed to demonstrate the effects of T. erecta, O. basilicum, and their mixture, WGA-M001, on OA pathogenesis. The administration of total extracts of T. erecta and O. basilicum reduced cartilage degradation and inflammation without causing cytotoxicity. Although WGA-M001 contained lower concentrations of the individual extracts, it strongly inhibited the expression of pathogenic factors. In vivo OA studies also supported that WGA-M001 had protective effects against cartilage destruction at lower doses than those of T. erecta and O. basilicum. Moreover, its effects were stronger than those observed using Boswellia and Perna canaliculus. WGA-M001 effectively inhibited the interleukin (IL)-1ß-induced nuclear factor kappa-light-chain-enhancer of the activated B cell (NF-κB) pathway and ERK phosphorylation. Furthermore, RNA-sequence analysis also showed that WGA-M001 decreased the expression of genes related to the IL-1ß-induced NF-κB and ERK signaling pathways. Therefore, WGA-M001 is more effective than the single total extracts of T. erecta and O. basilicum in attenuating OA progression by regulating ERK and NF-κB signaling. Our results open new possibilities for WGA-M001 as a potential therapeutic agent for OA treatment.
Assuntos
Ocimum basilicum , Osteoartrite , Tagetes , NF-kappa B/metabolismo , Tagetes/metabolismo , Condrócitos/metabolismo , Cartilagem/metabolismo , Osteoartrite/patologiaRESUMO
Every year, hundreds of thousands of people die because of metastatic brain cancer. Most metastatic cancer research uses 2D cell culture or animal models, but they have a few limitations, such as difficulty reproducing human tissue structures. This study developed a simple 3D in vitro model to better replicate brain metastasis using human cancer cells and human embryonic stem cell-derived cerebral organoids (metastatic brain cancer cerebral organoid [MBCCO]). The MBCCO model successfully reproduced metastatic cancer processes, including cell adhesion, proliferation, and migration, in addition to cell-cell interactions. Using the MBCCO model, we demonstrated that lung-specific X protein (LUNX) plays an important role in cell proliferation and migration or invasion. We also observed astrocyte accumulation around and their interaction with cancer cells through connexin 43 in the MBCCO model. We analyzed whether the MBCCO model can be used to screen drugs by measuring the effects of gefitinib, a well-known anticancer agent. We also examined the toxicity of gefitinib using normal cerebral organoids (COs). Therefore, the MBCCO model is a powerful tool for modeling human metastatic brain cancer in vitro and can also be used to screen drugs.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Células-Tronco Embrionárias Humanas/patologia , Organoides/patologia , Células A549 , Antineoplásicos/farmacologia , Encéfalo/efeitos dos fármacos , Neoplasias Encefálicas/tratamento farmacológico , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Células HEK293 , Células-Tronco Embrionárias Humanas/efeitos dos fármacos , Humanos , Neurônios/efeitos dos fármacos , Neurônios/patologia , Organoides/efeitos dos fármacosRESUMO
Accumulating evidence suggests that α-synuclein (α-syn) occurs physiologically as a helically folded tetramer that resists aggregation. However, the mechanisms underlying the regulation of formation of α-syn tetramers are still mostly unknown. Cellular membrane lipids are thought to play an important role in the regulation of α-syn tetramer formation. Since glucocerebrosidase 1 (GBA1) deficiency contributes to the aggregation of α-syn and leads to changes in neuronal glycosphingolipids (GSLs) including gangliosides, we hypothesized that GBA1 deficiency may affect the formation of α-syn tetramers. Here, we show that accumulation of GSLs due to GBA1 deficiency decreases α-syn tetramers and related multimers and increases α-syn monomers in CRISPR-GBA1 knockout (KO) SH-SY5Y cells. Moreover, α-syn tetramers and related multimers are decreased in N370S GBA1 Parkinson's disease (PD) induced pluripotent stem cell (iPSC)-derived human dopaminergic (hDA) neurons and murine neurons carrying the heterozygous L444P GBA1 mutation. Treatment with miglustat to reduce GSL accumulation and overexpression of GBA1 to augment GBA1 activity reverse the destabilization of α-syn tetramers and protect against α-syn preformed fibril-induced toxicity in hDA neurons. Taken together, these studies provide mechanistic insights into how GBA1 regulates the transition from monomeric α-syn to α-syn tetramers and multimers and suggest unique therapeutic opportunities for PD and dementia with Lewy bodies.
Assuntos
Neurônios Dopaminérgicos/metabolismo , Glucosilceramidase/deficiência , Glicoesfingolipídeos/metabolismo , Doença de Parkinson/metabolismo , alfa-Sinucleína/metabolismo , 1-Desoxinojirimicina/análogos & derivados , Linhagem Celular Tumoral , Glucosilceramidase/genética , Humanos , Multimerização ProteicaRESUMO
Non-alcoholic fatty liver disease (NAFLD) is a chronic metabolic liver disease associated with obesity and insulin resistance. Activation of the purinergic receptor P2Y2R has been reported to promote adipogenesis, inflammation and dyslipidemia in adipose tissues in obese mice. However, the role of P2Y2R and its mechanisms in NAFLD remain unknown. We hypothesized that P2Y2R deficiency may play a protective role in NAFLD by modulating lipid metabolism in the liver. In this study, we fed wild type and P2Y2R knockout mice with a high-fat diet (HFD) for 12 weeks and analyzed metabolic phenotypes. First, P2Y2R deficiency effectively improved insulin resistance with a reduction in body weight and plasma insulin. Second, P2Y2R deficiency attenuated hepatic lipid accumulation and injury with reduced alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels. Third, P2Y2R deficiency decreased the expression of fatty acid synthesis mediators (cluster of differentiation (CD36), fatty acid synthase (FAS), and stearoyl-CoA desaturase 1 (SCD1)); and increased the expression of adipose triglyceride lipase (ATGL), a lipolytic enzyme. Mechanistically, P2Y2R deficiency increased the AMP-activated protein kinase (AMPK) activity to improve mitochondrial fatty acid ß-oxidation (FAO) by regulating acetyl-CoA carboxylase (ACC) and carnitine palmitoyltransferase 1A (CPT1A)-mediated FAO pathway. In addition, P2Y2R deficiency increased peroxisome proliferator-activated gamma co-activator-1α (PGC-1α)-mediated mitochondrial biogenesis. Conclusively, P2Y2R deficiency ameliorated HFD-induced hepatic steatosis by enhancing FAO through AMPK signaling and PGC-1α pathway, suggesting P2Y2R as a promising therapeutic target for NAFLD.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ácidos Graxos/metabolismo , Lipogênese/genética , Hepatopatia Gordurosa não Alcoólica/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Receptores Purinérgicos P2Y2/metabolismo , Acetil-CoA Carboxilase/metabolismo , Alanina Transaminase/metabolismo , Animais , Aspartato Aminotransferases/metabolismo , Peso Corporal , Antígenos CD36/metabolismo , Carnitina O-Palmitoiltransferase/metabolismo , Dieta Hiperlipídica , Ácido Graxo Sintases/metabolismo , Insulina/sangue , Resistência à Insulina/fisiologia , Lipase/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Obesos , Mitocôndrias/metabolismo , Hepatopatia Gordurosa não Alcoólica/enzimologia , Obesidade/metabolismo , Receptores Purinérgicos P2Y2/deficiência , Receptores Purinérgicos P2Y2/genética , Estearoil-CoA Dessaturase/metabolismoRESUMO
Lipid dysregulation in diabetes mellitus escalates endothelial dysfunction, the initial event in the development and progression of diabetic atherosclerosis. In addition, lipid-laden macrophage accumulation in the arterial wall plays a significant role in the pathology of diabetes-associated atherosclerosis. Therefore, inhibition of endothelial dysfunction and enhancement of macrophage cholesterol efflux is the important antiatherogenic mechanism. Rosmarinic acid (RA) possesses beneficial properties, including its anti-inflammatory, antioxidant, antidiabetic and cardioprotective effects. We previously reported that RA effectively inhibits diabetic endothelial dysfunction by inhibiting inflammasome activation in endothelial cells. However, its effect on cholesterol efflux remains unknown. Therefore, in this study, we aimed to assess the effect of RA on cholesterol efflux and its underlying mechanisms in macrophages. RA effectively reduced oxLDL-induced cholesterol contents under high glucose (HG) conditions in macrophages. RA enhanced ATP-binding cassette transporter A1 (ABCA1) and G1 (ABCG1) expression, promoting macrophage cholesterol efflux. Mechanistically, RA differentially regulated ABCA1 expression through JAK2/STAT3, JNK and PKC-p38 and ABCG1 expression through JAK2/STAT3, JNK and PKC-ERK1/2/p38 in macrophages. Moreover, RA primarily stabilized ABCA1 rather than ABCG1 protein levels by impairing protein degradation. These findings suggest RA as a candidate therapeutic to prevent atherosclerotic cardiovascular disease complications related to diabetes by regulating cholesterol efflux in macrophages.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , Cinamatos/farmacologia , Depsídeos/farmacologia , Glucose/efeitos adversos , Lipoproteínas LDL/efeitos adversos , Macrófagos/citologia , Transportador 1 de Cassete de Ligação de ATP/química , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Modelos Biológicos , Proteólise/efeitos dos fármacos , Transdução de Sinais , Células THP-1 , Ácido RosmarínicoRESUMO
Diabetic nephropathy (DN) is a common pathological feature in patients with diabetes and the leading cause of end-stage renal disease. Although several pharmacological agents have been developed, the management of DN remains challenging. Geniposide, a natural compound has been reported for anti-inflammatory and anti-diabetic effects; however, its role in DN remains poorly understood. This study investigated the protective effects of geniposide on DN and its underlying mechanisms. We used a C57BL/6 mouse model of DN in combination with a high-fat diet and streptozotocin after unilateral nephrectomy and treated with geniposide by oral gavage for 5 weeks. Geniposide effectively improves DN-induced renal structural and functional abnormalities by reducing albuminuria, podocyte loss, glomerular and tubular injury, renal inflammation and interstitial fibrosis. These changes induced by geniposide were associated with an increase of AMPK activity to enhance ULK1-mediated autophagy response and a decrease of AKT activity to block oxidative stress, inflammation and fibrosis in diabetic kidney. In addition, geniposide increased the activities of PKA and GSK3ß, possibly modulating AMPK and AKT pathways, efficiently improving renal dysfunction and ameliorating the progression of DN. Conclusively, geniposide enhances ULK1-mediated autophagy and reduces oxidative stress, inflammation and fibrosis, suggesting geniposide as a promising treatment for DN.
Assuntos
Anti-Inflamatórios/farmacologia , Proteína Homóloga à Proteína-1 Relacionada à Autofagia/metabolismo , Autofagia/efeitos dos fármacos , Diabetes Mellitus Experimental/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Iridoides/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Complicações do Diabetes/patologia , Diabetes Mellitus Experimental/patologia , Dieta Hiperlipídica , Modelos Animais de Doenças , Fibrose/tratamento farmacológico , Fibrose/prevenção & controle , Glicogênio Sintase Quinase 3 beta/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Estresse Oxidativo/efeitos dos fármacosRESUMO
We investigated the role of nuclear factor of activated T cells 5 (NFAT5) under hyperosmotic conditions in human lens epithelial cells (HLECs). Hyperosmotic stress decreased the viability of human lens epithelial B-3 cells and significantly increased NFAT5 expression. Hyperosmotic stress-induced cell death occurred to a greater extent in NFAT5-knockout (KO) cells than in NFAT5 wild-type (NFAT5 WT) cells. Bcl-2 and Bcl-xl expression was down-regulated in NFAT5 WT cells and NFAT5 KO cells under hyperosmotic stress. Pre-treatment with a necroptosis inhibitor (necrostatin-1) significantly blocked hyperosmotic stress-induced death of NFAT5 KO cells, but not of NFAT5 WT cells. The phosphorylation levels of receptor-interacting protein kinase 1 (RIP1) and RIP3, which indicate the occurrence of necroptosis, were up-regulated in NFAT5 KO cells, suggesting that death of these cells is predominantly related to the necroptosis pathway. This finding is the first to report that necroptosis occurs when lens epithelial cells are exposed to hyperosmolar conditions, and that NFAT5 is involved in this process.
Assuntos
Células Epiteliais/metabolismo , Células Epiteliais/patologia , Cristalino/patologia , Pressão Osmótica , Estresse Fisiológico , Fatores de Transcrição/metabolismo , Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Células Epiteliais/efeitos dos fármacos , Humanos , Soluções Hipertônicas/toxicidade , Inflamação/patologia , Complexo de Proteínas Formadoras de Poros Nucleares/metabolismo , Pressão Osmótica/efeitos dos fármacos , Proteínas de Ligação a RNA/metabolismo , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Estresse Fisiológico/efeitos dos fármacosRESUMO
Seomae mugwort, a Korean native variety of Artemisia argyi, exhibits physiological effects against various diseases. However, its effects on osteoarthritis (OA) are unclear. In this study, a Seomae mugwort extract prevented cartilage destruction in an OA mouse model. In vitro and ex vivo analyses revealed that the extract suppressed MMP3, MMP13, ADAMTS4 and ADAMTS5 expression induced by IL-1ß, IL-6 and TNF-α and inhibited the loss of extracellular sulphated proteoglycans. In vivo analysis revealed that oral administration of the extract suppressed DMM-induced cartilage destruction. We identified jaceosidin in Seomae mugwort and showed that this compound decreased MMP3, MMP13, ADAMTS4 and ADAMTS5 expression levels, similar to the action of the Seomae mugwort extract in cultured chondrocytes. Interestingly, jaceosidin and eupatilin combined had similar effects to Seomae mugwort in the DMM-induced OA model. Induction of IκB degradation by IL-1ß was blocked by the extract and jaceosidin, whereas JNK phosphorylation was only suppressed by the extract. These results suggest that the Seomae mugwort extract and jaceosidin can attenuate cartilage destruction by suppressing MMPs, ADAMTS4/5 and the nuclear factor-κB signalling pathway by blocking IκB degradation. Thus, the findings support the potential application of Seomae mugwort, and particularly jaceosidin, as natural therapeutics for OA.
Assuntos
Artemisia/química , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/metabolismo , Flavonoides/farmacologia , Proteínas I-kappa B/metabolismo , Osteoartrite/metabolismo , Extratos Vegetais/farmacologia , Animais , Artrite Experimental , Biomarcadores , Cartilagem Articular/patologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Modelos Animais de Doenças , Flavonoides/química , Expressão Gênica , Imuno-Histoquímica , Interleucina-1beta/farmacologia , Metaloproteinases da Matriz/metabolismo , Camundongos , Modelos Biológicos , NF-kappa B/metabolismo , Osteoartrite/tratamento farmacológico , Osteoartrite/etiologia , Osteoartrite/patologia , Extratos Vegetais/química , Proteoglicanas/metabolismo , Proteólise , Transdução de Sinais/efeitos dos fármacosRESUMO
Accumulating evidence suggests that the non-receptor tyrosine kinase c-Abl plays an important role in the progression of Parkinson's disease (PD) and c-Abl inhibition could be neuroprotective in PD and related α-synucleinopathies. Nilotinib, a c-Abl inhibitor, has shown improved motor and cognitive symptoms in PD patients. However, issues concerning blood-brain barrier (BBB) penetration, lack of selectivity and safety still remain. Radotinib HCl is a selective Bcr-Abl kinase inhibitor that not only effectively access the brain, but also exhibits greater pharmacokinetic properties and safety profiles compared to Nilotinib and other c-Abl inhibitors. Here, we show the neuroprotective efficacy of Radotinib HCl, a brain penetrant c-Abl inhibitor, in a pre-clinical model of PD. Importantly, in vitro studies demonstrate that the treatment of Radotinib HCl protects the α-synuclein preformed fibrils (PFF)-induced neuronal toxicity, reduces the α-synuclein PFF-induced Lewy bodies (LB)/Lewy neurites (LN)-like pathology and inhibits the α-synuclein PFF-induced c-Abl activation in primary cortical neurons. Furthermore, administration of Radotinib HCl inhibits c-Abl activation and prevents dopaminergic neuron loss, neuroinflammation and behavioral deficits following α-synuclein PFF-induced toxicity in vivo. Taken together, our findings indicate that Radotinib HCl has beneficial neuroprotective effects in PD and provides an evidence that selective and brain permeable c-Abl inhibitors can be potential therapeutic agents for the treatment of PD and related α-synucleinopathies.
Assuntos
Encéfalo/efeitos dos fármacos , Degeneração Neural/tratamento farmacológico , Doença de Parkinson/tratamento farmacológico , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , alfa-Sinucleína/genética , Animais , Barreira Hematoencefálica , Encéfalo/metabolismo , Modelos Animais de Doenças , Dopamina/metabolismo , Neurônios Dopaminérgicos/efeitos dos fármacos , Neurônios Dopaminérgicos/patologia , Humanos , Corpos de Lewy/efeitos dos fármacos , Camundongos , Degeneração Neural/genética , Fármacos Neuroprotetores/administração & dosagem , Doença de Parkinson/genética , Doença de Parkinson/patologia , Proteínas Proto-Oncogênicas c-abl/genética , Pirimidinas/administração & dosagem , Sesquiterpenos/administração & dosagemRESUMO
α-Synuclein misfolding and aggregation plays a major role in the pathogenesis of Parkinson's disease. Although loss of function mutations in the ubiquitin ligase, parkin, cause autosomal recessive Parkinson's disease, there is evidence that parkin is inactivated in sporadic Parkinson's disease. Whether parkin inactivation is a driver of neurodegeneration in sporadic Parkinson's disease or a mere spectator is unknown. Here we show that parkin in inactivated through c-Abelson kinase phosphorylation of parkin in three α-synuclein-induced models of neurodegeneration. This results in the accumulation of parkin interacting substrate protein (zinc finger protein 746) and aminoacyl tRNA synthetase complex interacting multifunctional protein 2 with increased parkin interacting substrate protein levels playing a critical role in α-synuclein-induced neurodegeneration, since knockout of parkin interacting substrate protein attenuates the degenerative process. Thus, accumulation of parkin interacting substrate protein links parkin inactivation and α-synuclein in a common pathogenic neurodegenerative pathway relevant to both sporadic and familial forms Parkinson's disease. Thus, suppression of parkin interacting substrate protein could be a potential therapeutic strategy to halt the progression of Parkinson's disease and related α-synucleinopathies.
Assuntos
Doença de Parkinson/metabolismo , Proteínas Repressoras/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , alfa-Sinucleína/metabolismo , Animais , Feminino , Humanos , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Doença de Parkinson/patologiaRESUMO
The progressive neurodegeneration in Parkinson's disease (PD) is accompanied by neuroinflammation and endothelial vascular impairment. Although the vitamin D receptor (VDR) is expressed in both dopamine neurons and brain endothelial cells, its role in the regulation of endothelial biology has not been explored in the context of PD. In a 6-hydroxydopamine (6-OHDA)-induced PD mouse model, we observed reduced transcription of the VDR and its downstream target genes, CYP24 and MDR1a. The 6-OHDA-induced transcriptional repression of these genes were recovered after the VDR ligand-1α,25-dihydroxyvitamin D3 (1,25(OH)2D3) treatment. Similarly, reduced vascular protein expression of P-glycoprotein (P-gp), encoded by MDR1a, after 6-OHDA administration was reversed by 1,25(OH)2D3. Moreover, marked reduction of endothelial P-gp expression with concomitant α-synuclein aggregation was found in a combinatorial AAV-αSyn/αSyn preformed fibril (PFF) injection mouse model and postmortem PD brains. Supporting the direct effect of α-synuclein aggregation on endothelial biology, PFF treatment of human umbilical vein endothelial cells (HUVECs) was sufficient to induce α-synuclein aggregation and repress transcription of the VDR. PFF-induced P-gp downregulation and impaired functional activity in HUVECs completely recovered after 1,25(OH)2D3 treatment. Taken together, our results suggest that a dysfunctional VDR-P-gp pathway could be a potential target for the maintenance of vascular homeostasis in PD pathological conditions.
Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Encéfalo/metabolismo , Endotélio Vascular/metabolismo , Doença de Parkinson/metabolismo , Receptores de Calcitriol/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Idoso de 80 Anos ou mais , Animais , Calcitriol/metabolismo , Circulação Cerebrovascular , Família 24 do Citocromo P450/metabolismo , Modelos Animais de Doenças , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Ligantes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Doenças Neurodegenerativas/metabolismo , Lobo Temporal/patologia , Vitamina D3 24-Hidroxilase/metabolismo , alfa-Sinucleína/metabolismoRESUMO
Cardiac differentiation of human pluripotent stem cells may be induced under chemically defined conditions, wherein the regulation of Wnt/ß-catenin pathway is often desirable. Here, we examined the effect of trolox, a vitamin E analog, on the cardiac differentiation of human embryonic stem cells (hESCs). 6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox) significantly enhanced cardiac differentiation in a time- and dose-dependent manner after the mesodermal differentiation of hESCs. Trolox promoted hESC cardiac differentiation through its inhibitory activity against the Wnt/ß-catenin pathway. This study demonstrates an efficient cardiac differentiation method and reveals a novel Wnt/ß-catenin regulator.
RESUMO
BACKGROUND/AIMS: Stem cell transplantation has emerged as a promising therapeutic strategy, but the exact mechanisms by which stem cells exposed to hypoxic conditions increase the survival rate and rescue ischemic injury at the graft site are not well known. In this study, we aimed to determine if c-Met-activated mesenchymal stem cells (MSCs) pre-exposed to hypoxia promote therapeutic efficacy when transplanted to ischemic models, and whether c-Met interacts with cellular prion protein (PrPC) present in the ischemic tissue. METHODS: Western blot analysis was performed to determine the expression levels of PrPC, C-caspase-3, and C-PARP-1, as well as the phosphorylation of Akt, p38, JNK, and BAX. A co-immunoprecipitation assay was performed to show that PrPC binds with c-Met in vitro. An adhesion assay was performed to explore the alterations in MSCs attached to myoblasts (in vitro), and an invasion assay was performed to determine the effect on MSC invasion capacity upon interaction with myoblast-induced c-Met and PrPC. CD31-positive capillaries and αSMA-positive arterioles in in vivo hindlimb ischemic tissue were quantified by immunofluorescence staining. The level of apoptosis in the tissue of each group was assessed by quantifying the number of C-caspase-3-positive cells. Finally, laser Doppler technology was utilized to detect the enhanced angiogenic effects in vivo. RESULTS: We showed that hypoxic conditions increased PrPC levels in vivo (hindlimb ischemic tissue) and in vitro (myoblasts) and increased c-Met levels in MSCs. To identify the relationship between c-Met from MSCs and PrPC from myoblasts, we used a co-culturing system with myoblasts and MSCs pre-exposed to hypoxia. Hypoxia increased the phosphorylation of mitogen-activated protein kinases. Transplantation of hypoxia-pre-exposed MSCs to the ischemic site increased anti-apoptosis and enhanced the survival and proliferation of transplanted MSCs in a murine hindlimb model, resulting in improved functional recovery of the ischemic tissue. All the aforementioned effects were inhibited by the pretreatment of MSCs with the c-Met-neutralizing antibody Conclusion: c-Met-activated MSCs pre-exposed to hypoxia interact with PrPC at the site of ischemic injury to increase the efficiency of MSC transplantation. Hence, our study demonstrated that c-Met is a potential target for MSC-based therapies.
Assuntos
Membro Posterior/irrigação sanguínea , Isquemia/metabolismo , Isquemia/terapia , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Proteínas PrPC/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Hipóxia Celular , Células Cultivadas , Membro Posterior/metabolismo , Masculino , Células-Tronco Mesenquimais/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Mioblastos/citologia , Mioblastos/metabolismo , Mapas de Interação de ProteínasRESUMO
Mesenchymal stem cells (MSCs) could be a promising solution in the treatment of various diseases including chronic kidney disease (CKD). However, endoplasmic reticulum (ER) stress induced by ischemia in the area of application limits the integration and survival of MSCs in patients. In our study, we generated ER stress-induced conditions in MSCs using P-cresol. As P-cresol is a toxic compound accumulated in the body of CKD patients and induces apoptosis and inflammation through reactive oxygen species (ROS), we observed ER stress-induced MSC apoptosis activated by oxidative stress, which in turn resulted from ROS generation. To overcome stress-induced apoptosis, we investigated the protective effects of tauroursodeoxycholic acid (TUDCA), a bile acid, on ER stress in MSCs. In ER stress, TUDCA treatment of MSCs reduced ER stress-associated protein activation, including GRP78, PERK, eIF2α, ATF4, IRE1α, and CHOP. Next, to explore the protective mechanism adopted by TUDCA, TUDCA-mediated cellular prion protein (PrPC) activation was assessed. We confirmed that PrPC expression significantly increased ROS, which was eliminated by superoxide dismutase and catalase in MSCs. These findings suggest that TUDCA protects from inflammation and apoptosis in ER stress via PrPC expression. Our study demonstrates that TUDCA protects MSCs against inflammation and apoptosis in ER stress by PrPC expression in response to P-cresol exposure.
Assuntos
Antioxidantes/farmacologia , Células-Tronco Mesenquimais/metabolismo , Proteínas PrPC/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ácido Tauroquenodesoxicólico/farmacologia , Tecido Adiposo/citologia , Apoptose , Células Cultivadas , Cresóis/toxicidade , Chaperona BiP do Retículo Endoplasmático , Estresse do Retículo Endoplasmático , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Estresse Oxidativo , Proteínas PrPC/genéticaRESUMO
In order to realize the practical use of human pluripotent stem cell (hPSC)-derived cardiomyocytes for the purpose of clinical use or cardiovascular research, the generation of large numbers of highly purified cardiomyocytes should be achieved. Here, we show an efficient method for cardiac differentiation of human induced pluripotent stem cells (hiPSCs) in chemically defined conditions and purification of hiPSC-derived cardiomyocytes using a reporter system. Regulation of the Wnt/ß-catenin signaling pathway is implicated in the induction of the cardiac differentiation of hPSCs. We increased cardiac differentiation efficiency of hiPSCs in chemically defined conditions through combined treatment with XAV939, a tankyrase inhibitor and IWP2, a porcupine inhibitor and optimized concentrations. Although cardiac differentiation efficiency was high (>80%), it was difficult to suppress differentiation into non-cardiac cells, Therefore, we applied a lentiviral reporter system, wherein green fluorescence protein (GFP) and Zeocin-resistant gene are driven by promoter activation of a gene (TNNT2) encoding cardiac troponin T (cTnT), a cardiac-specific protein, to exclude non-cardiomyocytes from differentiated cell populations. We transduced this reporter construct into differentiated cells using a lentiviral vector and then obtained highly purified hiPSC-derived cardiomyocytes by treatment with the lowest effective dose of Zeocin. We significantly increased transgenic efficiency through manipulation of the cells in which the differentiated cells were simultaneously infected with virus and re-plated after single-cell dissociation. Purified cells specifically expressed GFP, cTnT, displayed typical properties of cardiomyocytes. This study provides an efficient strategy for obtaining large quantities of highly purified hPSC-derived cardiomyocytes for application in regenerative medicine and biomedical research.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular , Separação Celular , Genes Reporter , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Miócitos Cardíacos/metabolismo , Via de Sinalização WntRESUMO
Acacetin (5,7-dihydroxy-4'-methoxyflavone), a flavonoid compound isolated from Flos Chrysanthemi Indici, chrysanthemum, safflower, and Calamintha and Linaria species has been shown to have anti-cancer activity, indicating its potential clinical value in cancer treatment. In this study, we sought to study the potentials of acacetin in preventing human dopaminergic neuronal death via inhibition of 6-hydroxydopamine (6-OHDA)-induced neuronal cell death in the SH-SY5Y cells. Our results suggest that acacetin was effective in preventing 6-OHDA-induced neuronal cell death through regulation of mitochondrial-mediated cascade apoptotic cell death. Pretreatment with acacetin significantly inhibited neurotoxicity and neuronal cell death through reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP) dysfunction. Acacetin also markedly acted on key molecules in apoptotic cell death pathways and reduced phosphorylation of c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinases (PI3K)/Akt, and glycogen synthase kinase-3beta (GSK-3ß). These results suggested that acacetin could inhibit 6-OHDA-induced neuronal cell death originating from ROS-mediated cascade apoptosis pathway. Thus, the results of our study suggest that acacetin is a potent therapeutic agent for PD progression.