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Existing prognostic models to predict the neurological recovery in patients with cardiac arrest receiving targeted temperature management (TTM) either exhibit moderate accuracy or are too complicated for clinical application. This necessitates the development of a simple and generalizable prediction model to inform clinical decision-making for patients receiving TTM. The present study explores the predictive validity of the Cardiac Arrest Survival Post-resuscitation In-hospital (CASPRI) score in cardiac arrest patients receiving TTM, regardless of cardiac event location, and uses artificial neural network (ANN) algorithms to boost the prediction performance. This retrospective observational study evaluated the prognostic relevance of the CASPRI score and applied ANN to develop outcome prediction models in a cohort of 570 patients with cardiac arrest and treated with TTM between 2014 and 2019 in a nationwide multicenter registry in Taiwan. In univariate logistic regression analysis, the CASPRI score was significantly associated with neurological outcome, with the area under the receiver operating characteristics curve (AUC) of 0.811. The generated ANN model, based on 10 items of the CASPRI score, achieved a training AUC of 0.976 and validation AUC of 0.921, with the accuracy, precision, sensitivity, and specificity of 89.2%, 91.6%, 87.6%, and 91.2%, respectively, for the validation set. CASPRI score has prognostic relevance in patients who received TTM after cardiac arrest. The generated ANN-boosted, CASPRI-based model exhibited good performance for predicting TTM neurological outcome, thus, we propose its clinical application to improve outcome prediction, facilitate decision-making, and formulate individualized therapeutic plans for patients receiving TTM.
Assuntos
Reanimação Cardiopulmonar , Hipotermia Induzida , Parada Cardíaca Extra-Hospitalar , Hospitais , Humanos , Hipotermia Induzida/efeitos adversos , Redes Neurais de Computação , Parada Cardíaca Extra-Hospitalar/etiologia , Parada Cardíaca Extra-Hospitalar/terapia , Ressuscitação , Estudos RetrospectivosRESUMO
Temozolomide (TMZ) monotherapy is known to be insufficient for resistant/relapsed glioblastoma (GBM), thus seeking a sensitization agent for TMZ is necessary. It was found that regorafenib may improve the overall survival of relapsed GBM patients. We aimed to discover whether regorafenib can enhance the anti-GBM effects of TMZ, and elucidate underlying mechanism. Our analysis of The Cancer Genome Atlas database revealed that the increased expression of CXCR4 is linked to poor survival of GBM patients. Additionally, TMZ treatment may trigger CXCR4/CXCL12 axis of GBM. We used two GBM cell lines, two primary GBM cells, and animal model to identify underlying mechanism and treatment efficacy of regorafenib combined with TMZ by cytotoxicity, apoptosis, reporter gene and invasion/migration assays, chemokine array, Western blotting, MRI, microarray, and immunohistochemistry. We observed that the chemokine CXCL-12 and its receptor CXCR4 regulate the resistance to TMZ, whereas the inhibition of CXCL-12/CXCR4 signaling sensitizes GBM cells to TMZ. The TMZ-induced CXCL-12/CXCR4 signaling, phosphor-extracellular signal-regulated kinases 1 and 2 (ERK1/2) and nuclear factor kappa light chain enhancer of activated B cells (NF-κB), and NF-κB-related proteins can effectively diminish when combining with regorafenib. Regorafenib significantly enhanced the TMZ-induced extrinsic/intrinsic apoptotic pathways, and facilitated the suppression of invasion and migration potential in GBM. Orthotopic tumor experiments demonstrated tumor size reduction and prolonged survival in combination group even with half-dose of TMZ. Our findings provide promising evidence that regorafenib may sensitize GBM to TMZ treatment through inhibition of the CXCL12/CXCR4/ERK/NF-κB signaling.
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Neoplasias Encefálicas , Glioblastoma , Compostos de Fenilureia , Piridinas , Animais , Apoptose , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Quimiocina CXCL12/farmacologia , Quimiocina CXCL12/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/genética , Glioblastoma/metabolismo , Humanos , NF-kappa B/metabolismo , Compostos de Fenilureia/farmacologia , Compostos de Fenilureia/uso terapêutico , Piridinas/farmacologia , Piridinas/uso terapêutico , Receptores CXCR4/uso terapêutico , Temozolomida/farmacologia , Temozolomida/uso terapêuticoRESUMO
Human platelet lysates (HPLs) made from clinical-grade platelet concentrates are currently evaluated in the preclinical models of Parkinson's disease, Alzheimer's disease, traumatic brain injury, and others, as a new polyvalent neuroprotective biotherapy of the central nervous system. However, the presence and content of extracellular vesicles (EVs) in HPLs and their potential contribution to the neuroprotective and neurorestorative activities of HPLs are still unknown. We, therefore, characterized the EVs present in four different HPL preparations and after purification by size-exclusion chromatography. We then tested the effect of the isolated EVs on neuronal cell repair. We identified that all four HPLs contained a high and similar amount of EVs (1011 to 1012/mL) with a mean size ranging from ca. 50 to 300 nm and a negative zeta potential as determined by nanoparticle tracking analysis and dynamic light scattering. Western blot analysis revealed that the EVs present in HPLs expressed the clusters of differentiation 41 (CD41) and 61 (CD61) characteristic of platelets. These EVs were efficiently isolated from HPL proteins by Sepharose CL-2B size-exclusion column chromatography as confirmed by total protein determination and protein profile by sodium dodecyl sulfate polyacrylamide gel electrophoresis, with 73-85% recovery and maintenance of their size, negative zeta potential, and CD41 and CD61 expression. Interestingly, the EVs purified from the four HPLs exhibited a differential capacity to promote cell growth and migration in a wound-healing assay using SH-SY5Y neuronal cells, and one EV preparation stimulated network formation in primary neuronal cultures. These data indicated that the EVs present in HPLs have different neuroregenerative capacities and that some EV preparations may have interesting applications as a stand-alone therapy for usage in neuroregenerative medicine.
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Plaquetas , Vesículas Extracelulares , Proliferação de Células , Cromatografia , HumanosRESUMO
Traumatic brain injury (TBI) is a prevalent head injury worldwide which increases the risk of neurodegenerative diseases. Increased reactive oxygen species (ROS) and inflammatory chemokines after TBI induces secondary effects which damage neurons. Targeting NADPH oxidase or increasing redox systems are ways to reduce ROS and damage. Earlier studies show that C-C motif chemokine ligand 5 (CCL5) has neurotrophic functions such as promoting neurite outgrowth as well as reducing apoptosis. Although CCL5 levels in blood are associated with severity in TBI patients, the function of CCL5 after brain injury is unclear. In the current study, we induced mild brain injury in C57BL/6 (wildtype, WT) mice and CCL5 knockout (CCL5-KO) mice using a weight-drop model. Cognitive and memory functions in mice were analyzed by Novel-object-recognition and Barnes Maze tests. The memory performance of both WT and KO mice were impaired after mild injury. Cognition and memory function in WT mice quickly recovered after 7 days but recovery took more than 14 days in CCL5-KO mice. FJC, NeuN and Hypoxyprobe staining revealed large numbers of neurons damaged by oxidative stress in CCL5-KO mice after mTBI. NADPH oxidase activity show increased ROS generation together with reduced glutathione peroxidase-1 (GPX1) and glutathione (GSH) activity in CCL5-KO mice; this was opposite to that seen in WT mice. CCL5 increased GPX1 expression and reduced intracellular ROS levels which subsequently increased cell survival both in primary neuron cultures and in an overexpression model using SHSY5Y cell. Memory impairment in CCL5-KO mice induced by TBI could be rescued by i.p. injection of the GSH precursor - N-acetylcysteine (NAC) or intranasal delivery of recombinant CCL5 into mice after injury. We conclude that CCL5 is an important molecule for GPX1 antioxidant activation during post-injury day 1-3, and protects hippocampal neurons from ROS as well as improves memory function after trauma.
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Concussão Encefálica , Animais , Quimiocina CCL5 , Glutationa Peroxidase/metabolismo , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glutationa Peroxidase GPX1RESUMO
Traumatic brain injury (TBI) leads to major brain anatomopathological damages underlined by neuroinflammation, oxidative stress and progressive neurodegeneration, ultimately leading to motor and cognitive deterioration. The multiple pathological events resulting from TBI can be addressed not by a single therapeutic approach, but rather by a synergistic biotherapy capable of activating a complementary set of signalling pathways and providing synergistic neuroprotective, anti-inflammatory, antioxidative, and neurorestorative activities. Human platelet lysate might fulfil these requirements as it is composed of a plethora of biomolecules readily accessible as a TBI biotherapy. In the present study, we tested the therapeutic potential of human platelet lysate using in vitro and in vivo models of TBI. We first prepared and characterized platelet lysate from clinical-grade human platelet concentrates. Platelets were pelletized, lysed by three freeze-thaw cycles, and centrifuged. The supernatant was purified by 56°C 30 min heat treatment and spun to obtain the heat-treated platelet pellet lysate that was characterized by ELISA and proteomic analyses. Two mouse models were used to investigate platelet lysate neuroprotective potential. The injury was induced by an in-house manual controlled scratching of the animals' cortex or by controlled cortical impact injury. The platelet lysate treatment was performed by topical application of 60 µl in the lesioned area, followed by daily 60 µl intranasal administration from Day 1 to 6 post-injury. Platelet lysate proteomics identified over 1000 proteins including growth factors, neurotrophins, and antioxidants. ELISA detected several neurotrophic and angiogenic factors at â¼1-50 ng/ml levels. We demonstrate, using two mouse models of TBI, that topical application and intranasal platelet lysate consistently improved mouse motor function in the beam and rotarod tests, mitigated cortical neuroinflammation, and oxidative stress in the injury area, as revealed by downregulation of pro-inflammatory genes and the reduction in reactive oxygen species levels. Moreover, platelet lysate treatment reduced the loss of cortical synaptic proteins. Unbiased proteomic analyses revealed that heat-treated platelet pellet lysate reversed several pathways promoted by both controlled cortical impact and cortical brain scratch and related to transport, postsynaptic density, mitochondria or lipid metabolism. The present data strongly support, for the first time, that human platelet lysate is a reliable and effective therapeutic source of neurorestorative factors. Therefore, brain administration of platelet lysate is a therapeutical strategy that deserves serious and urgent consideration for universal brain trauma treatment.
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Terapia Biológica/métodos , Plaquetas/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/terapia , Administração Intranasal , Animais , Lesões Encefálicas Traumáticas/patologia , Linhagem Celular Tumoral , Córtex Cerebral/metabolismo , Córtex Cerebral/patologia , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Glucoregulatory efficiency and ATP production are key regulators for neuronal plasticity and memory formation. Besides its chemotactic and neuroinflammatory functions, the CC chemokine--CCL5 displays neurotrophic activity. We found impaired learning-memory and cognition in CCL5-knockout mice at 4 months of age correlated with reduced hippocampal long-term potentiation and impaired synapse structure. Re-expressing CCL5 in knockout mouse hippocampus restored synaptic protein expression, neuronal connectivity and cognitive function. Using metabolomics coupled with FDG-PET imaging and seahorse analysis, we found that CCL5 participates in hippocampal fructose and mannose degradation, glycolysis, gluconeogenesis as well as glutamate and purine metabolism. CCL5 additionally supports mitochondrial structural integrity, purine synthesis, ATP generation, and subsequent aerobic glucose metabolism. Overexpressing CCL5 in WT mice also enhanced memory-cognition performance as well as hippocampal neuronal activity and connectivity through promotion of de novo purine and glutamate metabolism. Thus, CCL5 actions on glucose aerobic metabolism are critical for mitochondrial function which contribute to hippocampal spine and synapse formation, improving learning and memory.
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Memória , Sinapses , Animais , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Camundongos , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Sinapses/metabolismoRESUMO
INTRODUCTION: Diabetes increases the risk of Parkinson's disease (PD). The phosphorylation of type 1 insulin receptor substrate (IRS-1) determines the function of insulin signaling pathway. Extracellular vesicles (EVs) are emerging as biomarkers of human diseases. The present study investigated whether PD patients exert altered phosphorylation IRS-1 (p-IRS-1) inside the blood neuron-derived extracellular vesicles (NDEVs). RESEARCH DESIGN AND METHODS: In total, there were 94 patients with PD and 63 healthy controls recruited and their clinical manifestations were evaluated. Blood NDEVs were isolated using the immunoprecipitation method, and Western blot analysis was conducted to assess total IRS-1, p-IRS-1, and downstream substrates level in blood NDEVs. Statistical analysis was performed using SPSS 19.0, and p < 0.05 was considered significant. RESULTS: The isolated blood EVs were validated according to the presence of CD63 and HSP70, nanoparticle tracking analysis and transmission electron microscopy. NDEVs were positive with neuronal markers. PD patients exerted significantly higher level of p-IRS-1S312 in blood NDEVs than controls. In addition, the p-IRS-1S312 levels in blood NDEVs was positively associated with the severity of tremor in PD patients after adjusting of age, sex, hemoglobin A1c, and body mass index (BMI). CONCLUSION: PD patients exerted altered p-IRS-1S312 in the blood NDEVs, and also correlated with the severity of tremor. These findings suggested the association between dysfunctional insulin signaling pathway with PD. The role of altered p-IRS-1S312 in blood NDEVs as a segregating biomarker of PD required further cohort study to assess the association with the progression of PD.
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BACKGROUND: Curcumin is a polyphenol natural product of the plant Curcuma longa. Recent studies suggest that curcumin inhibit mTOR activity in vitro, which prompts us to investigate curcumin function as a new class of mTOR inhibitor suitable for tuberous sclerosis complex (TSC) treatment. PURPOSE: We aim to investigate the efficacy of curcumin in the treatment of TSC related manifestations in animal model. STUDY DESIGN: Solid lipid curcumin particle (SLCP), a novel curcumin formulation, was used to treat TSC related manifestations in Tsc2 knockout mice. METHODS: The novel object recognition test was used to analyze the recognition memory function. The long-term potentiation was studied using electrophysiological analysis. Western blotting was used to assess the protein expression and activation status. RESULTS: Recognition memory deficit began as early as 4 weeks of age in both male and female Tsc2+/- mice. Oral administration with SLCP activates AMPK activity and inhibits mTOR activity in the brain tissue of Tsc2+/- mice, and can rescue the electrophysiological abnormality and object recognition memory loss in the mice. CONCLUSIONS: Our results suggest that SLCP could be an effective treatment for TSC patients.
Assuntos
Curcumina/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Esclerose Tuberosa/tratamento farmacológico , Administração Oral , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Curcumina/administração & dosagem , Modelos Animais de Doenças , Feminino , Humanos , Potenciação de Longa Duração/efeitos dos fármacos , Masculino , Transtornos da Memória/tratamento farmacológico , Camundongos Knockout , Inibidores de Proteínas Quinases/farmacologia , Esclerose Tuberosa/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/metabolismoRESUMO
The accumulation of reactive oxygen species (ROS) commonly occurs during normal aging and during some acute/chronic progressive disorders. In order to avoid oxidative damage, scavenging of these radicals is important. Previously, we identified zinc finger protein 179 (Znf179) as a neuroprotector that increases antioxidant enzymes against superoxide radicals. However, the molecular mechanisms involved in the activation and regulation of Znf179 remain unresolved. Here, by performing sequence alignment, bioinformatics analysis, immunoprecipitation using two specific acetyl-lysine antibodies, and treatment with the histone deacetylase (HDAC) inhibitor SAHA, we determined the lysine-specific acetylation of Znf179. Furthermore, we investigated Znf179 interaction with HDACs and revealed that peroxide insult induced a dissociation of Znf179-HDAC1/HDAC6, causing an increase in Znf179 acetylation. Importantly, HDAC inhibition by SAHA further prompted Znf179 hyperacetylation, which promoted Znf179 to form a transcriptional complex with Sp1 and increased antioxidant gene expression against oxidative attack. In summary, the results obtained in this study showed that Znf179 was regulated by HDACs and that Znf179 acetylation was a critical mechanism in the induction of antioxidant defense systems. Additionally, HDAC inhibitors may have therapeutic potential for induction of Znf179 acetylation, strengthening the Znf179 protective functions against neurodegenerative processes.
Assuntos
Acetilação/efeitos dos fármacos , Antioxidantes/farmacologia , Proteínas de Ligação a DNA/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Linhagem Celular , Proteínas de Ligação a DNA/química , Humanos , Camundongos , Alinhamento de SequênciaRESUMO
Leiomyomas (myomas) are the most common benign smooth muscle cell tumor of the myometrium. Resveratrol, a stilbene, has been used as an anti-inflammatory and antitumor agent. In the current study, we investigated the inhibitory effect of resveratrol on the proliferation of primary human myoma cell cultures. Resveratrol arrested cell proliferation via integrin αvß3. It also inhibited integrin αvß3 expression and protein accumulation. Concurrently, constitutive AKT phosphorylation in myoma cells was inhibited by resveratrol. Expressions of proapoptotic genes, such as cyclooxygenase (COX)-2, p21 and CDKN2, were induced by resveratrol in myoma cells. On the other hand, expressions of proliferative (anti-apoptotic) genes were either inhibited, as in BCL2, or unchanged, as in cyclin D1 and proliferating cell nuclear antigen (PCNA). The accumulation of insulin-like growth factor (IGF)-1 receptor (IGF-1R) was inhibited by resveratrol in primary myoma cells. IGF-1-induced cell proliferation was inhibited by co-incubation with resveratrol. Therefore, growth modulation of myoma cells occurs via mechanisms dependent on cross-talk between integrin αvß3 and IGF-1R. Our findings suggest that resveratrol can be considered an alternative therapeutic agent for myomas.
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Proliferação de Células/efeitos dos fármacos , Integrina alfaVbeta3/metabolismo , Leiomioma/patologia , Receptor Cross-Talk , Receptor IGF Tipo 1/metabolismo , Estilbenos/farmacologia , Neoplasias Uterinas/patologia , Feminino , Citometria de Fluxo , Humanos , Marcação In Situ das Extremidades Cortadas , Leiomioma/metabolismo , Fosforilação , Resveratrol , Neoplasias Uterinas/metabolismoRESUMO
Programmed death-ligand 1 (PD-L1) is a critical regulator to defend tumor cells against immune surveillance. Thyroid hormone has been shown to induce PD-L1 expression in cancer cells. Its nano-particulated analogue, nano-diamino-tetrac (NDAT; Nanotetrac) is an anticancer/anti-angiogenic agent. In the current study, the inhibitory mechanism by which NDAT inhibited PD-L1 mRNA abundance and PD-L1 protein content in oral cancer cells was investigated. NDAT inhibited inducible PD-L1 expression and protein accumulation by the inhibition of activated ERK1/2 and PI3K. Knockdown PD-L1 also inhibited the proliferation of oral cancer cells which suggests that the inhibitory effect of NDAT on PD-L1 expression maybe is one of the critical mechanisms for NDAT-induced anti-proliferative effect in oral cancer cells.
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Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/genética , Carcinoma de Células Escamosas/patologia , Proliferação de Células/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Neoplasias Bucais/patologia , Nanopartículas , Tiroxina/análogos & derivados , Antineoplásicos/farmacologia , Antígeno B7-H1/metabolismo , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Linhagem Celular Tumoral , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neoplasias Bucais/genética , Neoplasias Bucais/metabolismo , Inibidores de Fosfoinositídeo-3 Quinase , Inibidores de Proteínas Quinases/farmacologia , RNA Mensageiro/metabolismo , Tiroxina/farmacologiaRESUMO
Glioblastoma multiforme (GBM) is a highly malignant type of brain tumor found in humans. GBM cells reproduce quickly, and the median survival time for patients after therapy is approximately 1 year with a high relapse rate. Current therapies and diagnostic tools for GBM are limited; therefore, we searched for a more favorable therapeutic target or marker protein for both therapy and diagnosis. We used mass spectrometry (MS) analysis to identify GBM-associated marker proteins from human plasma and GBM cell cultures. Additional plasma and 52 brain tissues obtained from patients with gliomas were used to validate the association rate of serum amyloid A1 (SAA1) in different grades of gliomas and its distribution in tumors. Microarray database analysis further validated the coefficient of SAA1 levels in gliomas. The cellular mechanisms of SAA1 in GBM proliferation and infiltration were investigated in vitro. We analyzed the correlation between SAA1 and patients' medication requirement to demonstrate the clinical effects of SAA1 in GBM. SAA1 was identified from MS analysis, and its level was revealed to be correlated with the disease grade, clinical severity, and survival rate of patients with gliomas. In vitro cultures, including GBM cells and normal astrocytes, revealed that SAA1 promotes cell migration and invasion through integrin αVß3 to activate the Erk signaling pathway. Magnetic resonance imaging and tumor region-specific microarray analysis identified a correlation between SAA1 and GBM cell infiltration in patients. In summary, our results demonstrate that SAA1 in combination with integrin αV and ß3 can serve as an indicator of high glioblastoma risk. We also identified the cellular mechanisms of SAA1 contributing to GBM progression, which can serve as the basis for future GBM therapy.
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Movimento Celular , Progressão da Doença , Glioblastoma/metabolismo , Glioblastoma/patologia , Integrina alfaVbeta3/metabolismo , Proteína Amiloide A Sérica/metabolismo , Astrócitos/metabolismo , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Feminino , Glioblastoma/sangue , Glioblastoma/diagnóstico , Humanos , Masculino , Pessoa de Meia-Idade , Invasividade Neoplásica , Análise de SobrevidaRESUMO
The precise assembly of a functional nervous system relies on the guided migration of axonal growth cones, which is made possible by signals transmitted to the cytoskeleton by cell surface-expressed guidance receptors. We investigated the function of ephexin1, a Rho guanine nucleotide exchange factor, as an essential growth-cone guidance intermediary in the context of spinal lateral motor column (LMC) motor axon trajectory selection in the limb mesenchyme. Using in situ mRNA detection, we first show that ephexin1 is expressed in LMC neurons of chick and mouse embryos at the time of spinal motor axon extension into the limb. Ephexin1 loss of function and gain of function using in ovo electroporation in chick LMC neurons, of either sex, perturbed LMC axon trajectory selection, demonstrating an essential role of ephexin1 in motor axon guidance. In addition, ephexin1 loss in mice of either sex led to LMC axon trajectory selection errors. We also show that ephexin1 knockdown attenuates the growth preference of LMC neurites against ephrins in vitro and Eph receptor-mediated retargeting of LMC axons in vivo, suggesting that ephexin1 is required in Eph-mediated LMC motor axon guidance. Finally, both ephexin1 knockdown and ectopic expression of nonphosphorylatable ephexin1 mutant attenuated the retargeting of LMC axons caused by Src overexpression, implicating ephexin1 as an Src target in Eph signal relay in this context. In summary, our findings demonstrate that ephexin1 is essential for motor axon guidance and suggest an important role in relaying ephrin:Eph signals that mediate motor axon trajectory selection.SIGNIFICANCE STATEMENT The proper development of functioning neural circuits requires precise nerve connections among neurons or between neurons and their muscle targets. The Eph tyrosine kinase receptors expressed in neurons are important in many contexts during neural-circuit formation, such as axon outgrowth, axon guidance, and synaptic formation, and have been suggested to be involved in neurodegenerative disorders, including amyotrophic lateral sclerosis and Alzheimer's disease. To dissect the mechanism of Eph signal relay, we studied ephexin1 gain of function and loss of function and found ephexin1 essential for the development of limb nerves toward their muscle targets, concluding that it functions as an intermediary to relay Eph signaling in this context. Our work could thus shed new light on the molecular mechanisms controlling neuromuscular connectivity during embryonic development.
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Orientação de Axônios/fisiologia , Axônios/ultraestrutura , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neurônios Motores/citologia , Animais , Axônios/metabolismo , Embrião de Galinha , Efrinas/metabolismo , Extremidades/inervação , Camundongos , Neurônios Motores/metabolismo , Músculo Esquelético/inervaçãoRESUMO
Insulin regulates systematic metabolism in the hypothalamus and the peripheral insulin response. An inflammatory reaction in peripheral adipose tissues contributes to type 2 diabetes mellitus (T2DM) development and appetite regulation in the hypothalamus. Chemokine CCL5 and C-C chemokine receptor type 5 (CCR5) levels have been suggested to mediate arteriosclerosis and glucose intolerance in type 2 diabetes mellitus (T2DM). In addition, CCL5 plays a neuroendocrine role in the hypothalamus by regulating food intake and body temperature, thus, prompting us to investigate its function in hypothalamic insulin signaling and the regulation of peripheral glucose metabolism. The micro-osmotic pump brain infusion system is a quick and precise way to manipulate CCL5 function and study its effect in the brain. It also provides a convenient alternative approach to generating a transgenic knockout animal. In this system, CCL5 signaling was blocked by intracerebroventricular (ICV) infusion of its antagonist, MetCCL5, using a micro-osmotic pump. The peripheral glucose metabolism and insulin responsiveness was detected by the Oral Glucose Tolerance Test (OGTT) and Insulin Tolerance Test (ITT). Insulin signaling activity was then analyzed by protein blot from tissue samples derived from the animals. After 7-14 days of MetCCL5 infusion, the glucose metabolism and insulin responsiveness was impaired in mice, as seen in the results of the OGTT and ITT. The IRS-1 serine302 phosphorylation was increased and the Akt activity was reduced in mice hypothalamic neurons following CCL5 inhibition. Altogether, our data suggest that blocking CCL5 in the mouse brain increases the phosphorylation of IRS-1 S302 and interrupts hypothalamic insulin signaling, leading to a decrease in insulin function in peripheral tissues as well as the impairment of glucose metabolism.
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Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Quimiocina CCL5/administração & dosagem , Intolerância à Glucose/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Insulina/metabolismo , Animais , Glicemia/metabolismo , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/metabolismo , Bombas de Infusão , Masculino , Camundongos , Proteínas Recombinantes/administração & dosagem , Transdução de SinaisRESUMO
Type 2 diabetes mellitus (T2DM) is a global health crisis which is characterized by insulin signaling impairment and chronic inflammation in peripheral tissues. The hypothalamus in the central nervous system (CNS) is the control center for energy and insulin signal response regulation. Chronic inflammation in peripheral tissues and imbalances of certain chemokines (such as CCL5, TNFα, and IL-6) contribute to diabetes and obesity. However, the functional mechanism(s) connecting chemokines and hypothalamic insulin signal regulation still remain unclear. In vitro primary neuron culture models are convenient and simple models which can be used to investigate insulin signal regulation in hypothalamic neurons. In this study, we introduced exogeneous GLUT4 protein conjugated with GFP (GFP-GLUT4) into primary hypothalamic neurons to track GLUT4 membrane translocation upon insulin stimulation. Time-lapse images of GFP-GLUT4 protein trafficking were recorded by deconvolution microscopy, which allowed users to generate high-speed, high-resolution images without damaging the neurons significantly while conducting the experiment. The contribution of CCR5 in insulin regulated GLUT4 translocation was observed in CCR5 deficient hypothalamic neurons, which were isolated and cultured from CCR5 knockout mice. Our results demonstrated that the GLUT4 membrane translocation efficiency was reduced in CCR5 deficient hypothalamic neurons after insulin stimulation.
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Transportador de Glucose Tipo 4/metabolismo , Hipotálamo/diagnóstico por imagem , Microscopia/métodos , Neurônios/metabolismo , Animais , Transportador de Glucose Tipo 4/genética , Hipotálamo/citologia , Hipotálamo/metabolismo , Masculino , Camundongos , Transporte ProteicoRESUMO
Recently, a novel substrate known as an electrospun polylactic acid (PLLA) microtube array membrane (MTAM) was successfully developed as a cell coculture platform. Structurally, this substrate is made up of one-to-one connected, ultrathin, submicron scale fibers that are arranged in an arrayed formation. Its unique structure confers several key advantages which are beneficial in a cell coculture system. In this study, the interaction between rat fetal neural stem cells (NSC) and astrocytes was examined by comparing the outcome of a typical Transwell-based coculture system and that of an electrospun PLLA MTAM-based coculture system. Compared to tissue culture polystyrene (TCP) and Transwell coculture inserts, a superior cell viability of NSC was observed when cultured in lumens of electrospun PLLA MTAM (with supportive immunostaining images). Reverse transcription polymerase chain reaction revealed a strong interaction between astrocytes and NSC through a higher expression of doublecortin and a lower expression of nestin. These data demonstrate that MTAM is clearly a better coculture platform than the traditional Transwell system.
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Astrócitos/química , Regeneração Nervosa/fisiologia , Engenharia Tecidual/métodos , Animais , Técnicas de Cocultura , Proteína Duplacortina , Humanos , RatosRESUMO
Many neurodegenerative diseases are accompanied by metabolic disorders. CCL5/RANTES, and its receptor CCR5 are known to contribute to neuronal function as well as to metabolic disorders such as type 2 diabetes mellitus, obesity, atherosclerosis and metabolic changes after HIV infection. Herein, we found that the lack of CCR5 or CCL5 in mice impaired regulation of energy metabolism in hypothalamus. Immunostaining and co-immunoprecipitation revealed the specific expression of CCR5, associated with insulin receptors, in the hypothalamic arcuate nucleus (ARC). Both ex vivo stimulation and in vitro tissue culture studies demonstrated that the activation of insulin, and PI3K-Akt pathways were impaired in CCR5 and CCL5 deficient hypothalamus. The inhibitory phosphorylation of insulin response substrate-1 at Ser302 (IRS-1S302) but not IRS-2, by insulin was markedly increased in CCR5 and CCL5 deficient animals. Elevating CCR5/CCL5 activity induced GLUT4 membrane translocation and reduced phospho-IRS-1S302 through AMPKα-S6 Kinase. Blocking CCR5 using the antagonist, MetCCL5, abolished the de-phosphorylation of IRS-1S302 and insulin signal activation. In addition, intracerebroventricular delivery of MetCCL5 interrupted hypothalamic insulin signaling and elicited peripheral insulin responsiveness and glucose intolerance. Taken together, our data suggest that CCR5 regulates insulin signaling in hypothalamus which contributes to systemic insulin sensitivity and glucose metabolism.
Assuntos
Quimiocina CCL5/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Metabolismo Energético/genética , Insulina/metabolismo , Animais , Núcleo Arqueado do Hipotálamo/metabolismo , Núcleo Arqueado do Hipotálamo/patologia , Quimiocina CCL5/antagonistas & inibidores , Quimiocina CCL5/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/patologia , Modelos Animais de Doenças , Transportador de Glucose Tipo 4/genética , Humanos , Hipotálamo/metabolismo , Hipotálamo/patologia , Insulina/genética , Camundongos , Neurônios/metabolismo , Neurônios/patologia , Fosfatidilinositol 3-Quinases/genética , Fosforilação , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Transdução de Sinais/genéticaRESUMO
BACKGROUND: Traumatic brain injury (TBI) is a global health concern that typically causes emotional disturbances and cognitive dysfunction. Secondary pathologies following TBI may be associated with chronic neurodegenerative disorders and an enhanced likelihood of developing dementia-like disease in later life. There are currently no approved drugs for mitigating the acute or chronic effects of TBI. METHODS: The effects of the drug pomalidomide (Pom), an FDA-approved immunomodulatory agent, were evaluated in a rat model of moderate to severe TBI induced by controlled cortical impact. Post-TBI intravenous administration of Pom (0.5 mg/kg at 5 or 7 h and 0.1 mg/kg at 5 h) was evaluated on functional and histological measures that included motor function, fine more coordination, somatosensory function, lesion volume, cortical neurodegeneration, neuronal apoptosis, and the induction of pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6). RESULTS: Pom 0.5 mg/kg administration at 5 h, but not at 7 h post-TBI, significantly mitigated the TBI-induced injury volume and functional impairments, neurodegeneration, neuronal apoptosis, and cytokine mRNA and protein induction. To evaluate underlying mechanisms, the actions of Pom on neuronal survival, microglial activation, and the induction of TNF-α were assessed in mixed cortical cultures following a glutamate challenge. Pom dose-dependently ameliorated glutamate-mediated cytotoxic effects on cell viability and reduced microglial cell activation, significantly attenuating the induction of TNF-α. CONCLUSIONS: Post-injury treatment with a single Pom dose within 5 h significantly reduced functional impairments in a well-characterized animal model of TBI. Pom decreased the injury lesion volume, augmented neuronal survival, and provided anti-inflammatory properties. These findings strongly support the further evaluation and optimization of Pom for potential use in clinical TBI.
Assuntos
Encefalite/tratamento farmacológico , Fatores Imunológicos/uso terapêutico , Transtornos Motores/tratamento farmacológico , Degeneração Neural/tratamento farmacológico , Transtornos Psicomotores/tratamento farmacológico , Distúrbios Somatossensoriais/tratamento farmacológico , Talidomida/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Lesões Encefálicas Traumáticas/complicações , Lesões Encefálicas Traumáticas/tratamento farmacológico , Lesões Encefálicas Traumáticas/patologia , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/patologia , Citocinas/genética , Citocinas/metabolismo , Modelos Animais de Doenças , Encefalite/etiologia , Lateralidade Funcional/efeitos dos fármacos , Proteína Glial Fibrilar Ácida/metabolismo , Masculino , Transtornos Motores/etiologia , Degeneração Neural/etiologia , Fosfopiruvato Hidratase/metabolismo , Transtornos Psicomotores/etiologia , Ratos , Ratos Sprague-Dawley , Distúrbios Somatossensoriais/etiologia , Talidomida/uso terapêuticoRESUMO
The accumulation of reactive oxygen species (ROS) have implicated the pathogenesis of several human diseases including neurodegenerative disorders, stroke, and traumatic brain injury, hence protecting neurons against ROS is very important. In this study, we focused on sigma-1 receptor (Sig-1R), a chaperone at endoplasmic reticulum, and investigated its protective functions. Using hydrogen peroxide (H2O2)-induced ROS accumulation model, we verified that apoptosis-signaling pathways were elicited by H2O2 treatment. However, the Sig-1R agonists, dehydroepiandrosterone (DHEA) and DHEA sulfate (DHEAS), reduced the activation of apoptotic pathways significantly. By performing protein-protein interaction assays and shRNA knockdown of Sig-1R, we identified the brain Zinc finger protein 179 (Znf179) as a downstream target of Sig-1R regulation. The neuroprotective effect of Znf179 overexpression was similar to that of DHEAS treatment, and likely mediated by affecting the levels of antioxidant enzymes. We also quantified the levels of peroxiredoxin 3 (Prx3) and superoxide dismutase 2 (SOD2) in the hippocampi of wild-type and Znf179 knockout mice, and found both enzymes to be reduced in the knockout versus the wild-type mice. In summary, these results reveal that Znf179 plays a novel role in neuroprotection, and Sig-1R agonists may be therapeutic candidates to prevent ROS-induced damage in neurodegenerative and neurotraumatic diseases.