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1.
J Biomed Sci ; 31(1): 91, 2024 Sep 17.
Artigo em Inglês | MEDLINE | ID: mdl-39285280

RESUMO

BACKGROUND: Traumatic brain injury (TBI) causes axon tearing and synapse degradation, resulting in multiple neurological dysfunctions and exacerbation of early neurodegeneration; the repair of axonal and synaptic structures is critical for restoring neuronal function. C-C Motif Chemokine Ligand 5 (CCL5) shows many neuroprotective activities. METHOD: A close-head weight-drop system was used to induce mild brain trauma in C57BL/6 (wild-type, WT) and CCL5 knockout (CCL5-KO) mice. The mNSS score, rotarod, beam walking, and sticker removal tests were used to assay neurological function after mTBI in different groups of mice. The restoration of motor and sensory functions was impaired in CCL5-KO mice after one month of injury, with swelling of axons and synapses from Golgi staining and reduced synaptic proteins-synaptophysin and PSD95. Administration of recombinant CCL5 (Pre-treatment: 300 pg/g once before injury; or post-treatment: 30 pg/g every 2 days, since 3 days after injury for 1 month) through intranasal delivery into mouse brain improved the motor and sensory neurological dysfunctions in CCL5-KO TBI mice. RESULTS: Proteomic analysis using LC-MS/MS identified that the "Nervous system development and function"-related proteins, including axonogenesis, synaptogenesis, and myelination signaling pathways, were reduced in injured cortex of CCL5-KO mice; both pre-treatment and post-treatment with CCL5 augmented those pathways. Immunostaining and western blot analysis confirmed axonogenesis and synaptogenesis related Semaphorin, Ephrin, p70S6/mTOR signaling, and myelination-related Neuregulin/ErbB and FGF/FAK signaling pathways were up-regulated in the cortical tissue by CCL5 after brain injury. We also noticed cortex redevelopment after long-term administration of CCL5 after brain injury with increased Reelin positive Cajal-Rerzius Cells and CXCR4 expression. CCL5 enhanced the growth of cone filopodia in a primary neuron culture system; blocking CCL5's receptor CCR5 by Maraviroc reduced the intensity of filopodia in growth cone and also CCL5 mediated mTOR and Rho signalling activation. Inhibiting mTOR and Rho signaling abolished CCL5 induced growth cone formation. CONCLUSIONS: CCL5 plays a critical role in starting the intrinsic neuronal regeneration system following TBI, which includes growth cone formation, axonogenesis and synaptogensis, remyelination, and the subsequent proper wiring of cortical circuits. Our study underscores the potential of CCL5 as a robust therapeutic stratagem in treating axonal injury and degeneration during the chronic phase after mild brain injury.


Assuntos
Axônios , Quimiocina CCL5 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Animais , Camundongos , Quimiocina CCL5/metabolismo , Axônios/metabolismo , Axônios/fisiologia , Lesões Encefálicas Traumáticas/metabolismo , Lesões Encefálicas Traumáticas/fisiopatologia , Masculino , Neurônios/metabolismo , Lesões Encefálicas/metabolismo , Neurogênese
2.
J Biomed Sci ; 31(1): 87, 2024 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-39237980

RESUMO

BACKGROUND: The burgeoning field of regenerative medicine has significantly advanced with recent findings on biotherapies using human platelet lysates (HPLs), derived from clinical-grade platelet concentrates (PCs), for treating brain disorders. These developments have opened new translational research avenues to explore the neuroprotective effects of platelet-extracellular vesicles (PEVs). Their potential in managing neurodegenerative conditions like traumatic brain injury (TBI) and Parkinson's disease (PD) warrants further exploration. We aimed here to characterize the composition of a PEV preparation isolated from platelet concentrate (PC) supernatant, and determine its neuroprotective potential and neurorestorative effects in cellular and animal models of TBI and PD. METHODS: We isolated PEVs from the supernatant of clinical-grade PC collected from healthy blood donors utilizing high-speed centrifugation. PEVs were characterized by biophysical, biochemical, microscopic, and LC-MS/MS proteomics methods to unveil biological functions. Their functionality was assessed in vitro using SH-SY5Y neuronal cells, LUHMES dopaminergic neurons, and BV-2 microglial cells, and in vivo by intranasal administration in a controlled cortical impact (CCI)-TBI model using 8-weeks-old male C57/BL6 mice, and in a PD model induced by MPTP in 5-month-old male C57/BL6 mice. RESULTS: PEVs varied in size from 50 to 350 nm, predominantly around 200 nm, with concentrations ranging between 1010 and 1011/mL. They expressed specific platelet membrane markers, exhibited a lipid bilayer by cryo-electron microscopy and, importantly, showed low expression of pro-coagulant phosphatidylserine. LC-MS/MS indicated a rich composition of trophic factors, including neurotrophins, anti-inflammatory agents, neurotransmitters, and antioxidants, unveiling their multifaceted biological functions. PEVs aided in the restoration of neuronal functions in SH-SY5Y cells and demonstrated remarkable neuroprotective capabilities against erastin-induced ferroptosis in dopaminergic neurons. In microglial cells, they promoted anti-inflammatory responses, particularly under inflammatory conditions. In vivo, intranasally delivered PEVs showed strong anti-inflammatory effects in a TBI mouse model and conserved tyrosine hydroxylase expression of dopaminergic neurons of the substantia nigra in a PD model, leading to improved motor function. CONCLUSIONS: The potential of PEV-based therapies in neuroprotection opens new therapeutic avenues for neurodegenerative disorders. The study advocates for clinical trials to establish the efficacy of PEV-based biotherapies in neuroregenerative medicine.


Assuntos
Plaquetas , Lesões Encefálicas Traumáticas , Vesículas Extracelulares , Camundongos Endogâmicos C57BL , Fármacos Neuroprotetores , Doença de Parkinson , Vesículas Extracelulares/metabolismo , Animais , Humanos , Lesões Encefálicas Traumáticas/metabolismo , Camundongos , Plaquetas/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/administração & dosagem , Masculino , Doença de Parkinson/terapia , Administração Intranasal , Modelos Animais de Doenças
3.
Mol Psychiatry ; 26(11): 6451-6468, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-33931731

RESUMO

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.


Assuntos
Memória , Sinapses , Animais , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Memória/fisiologia , Camundongos , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Sinapses/metabolismo
4.
Brain ; 144(10): 3142-3158, 2021 11 29.
Artigo em Inglês | MEDLINE | ID: mdl-34086871

RESUMO

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 ∼1-50 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.


Assuntos
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 C57BL
5.
J Neurosci ; 38(8): 2043-2056, 2018 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-29363583

RESUMO

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.


Assuntos
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/inervação
6.
Cells Tissues Organs ; 204(3-4): 179-190, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28848167

RESUMO

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.


Assuntos
Astrócitos/química , Regeneração Nervosa/fisiologia , Engenharia Tecidual/métodos , Animais , Técnicas de Cocultura , Proteína Duplacortina , Humanos , Ratos
7.
J Neuroinflammation ; 13(1): 168, 2016 06 28.
Artigo em Inglês | MEDLINE | ID: mdl-27353053

RESUMO

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 terapêutico
8.
Eur J Immunol ; 41(5): 1221-30, 2011 May.
Artigo em Inglês | MEDLINE | ID: mdl-21469130

RESUMO

Chloride intracellular channel (CLIC) 4 has diverse functions in membrane trafficking, apoptosis, angiogenesis and cell differentiation. CLIC4 is abundantly expressed in macrophages, but its role in innate immune functions is unclear. Here, we show that primary murine macrophages express increased amounts of CLIC4 after exposure to bacterial lipopolysaccharide (LPS). Endogenous CLIC4 level was significantly elevated in the brain, heart, lung, kidney, liver and spleen after LPS injection of mice. Stable macrophage lines overexpressing CLIC4 produced more TNF, IL-6, IL-12 and CCL5 than mock transfectants when exposed to LPS. To explore the role of CLIC4 in vivo, we generated CLIC4-null mice. These mice were protected from LPS-induced death, and had reduced serum levels of inflammatory cytokines. Upon infection with Listeria monocytogenes, CLIC4-deficient mice were impaired in their ability to clear infection, and their macrophages responded to Listeria by producing less inflammatory cytokines and chemokines than the WT controls. When challenged with LPS in vitro, deletion of clic4 gene had little effect on MAPK and NF-κB activation, but led to a reduced accumulation of phosphorylated interferon response factor 3 (IRF3) within macrophages. Conversely, overexpression of CLIC4 enhanced LPS-mediated IRF3. Thus, these findings suggest that CLIC4 is an LPS-induced product that can serve as a positive regulator of LPS signaling.


Assuntos
Canais de Cloreto/metabolismo , Imunidade Inata , Lipopolissacarídeos/imunologia , Macrófagos/imunologia , Proteínas Mitocondriais/metabolismo , Animais , Western Blotting , Linhagem Celular , Quimiocinas/sangue , Quimiocinas/imunologia , Canais de Cloreto/biossíntese , Canais de Cloreto/genética , Citocinas/sangue , Citocinas/imunologia , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Escherichia coli/imunologia , Regulação da Expressão Gênica/efeitos dos fármacos , Genótipo , Fator Regulador 3 de Interferon/metabolismo , Lipopolissacarídeos/metabolismo , Listeria monocytogenes/imunologia , Listeria monocytogenes/patogenicidade , Listeriose/imunologia , Listeriose/microbiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Mitocondriais/biossíntese , Proteínas Mitocondriais/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Reação em Cadeia da Polimerase , Choque Séptico/imunologia , Transdução de Sinais
9.
Sci Rep ; 12(1): 7254, 2022 05 04.
Artigo em Inglês | MEDLINE | ID: mdl-35508580

RESUMO

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 Retrospectivos
10.
Neurotherapeutics ; 19(2): 616-634, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-35267171

RESUMO

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.


Assuntos
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 terapêutico
11.
Hum Mol Genet ; 18(16): 2929-42, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-19443488

RESUMO

Huntington's disease (HD) is an autosomal dominant neurodegenerative disease caused by a CAG trinucleotide expansion in the Huntingtin (Htt) gene. The resultant mutant Htt protein (mHtt) forms aggregates in the brain and several peripheral tissues (e.g. the liver) and causes devastating neuronal degeneration. Metabolic defects resulting from Htt aggregates in peripheral tissues also contribute to HD pathogenesis. Simultaneous improvement of defects in both the CNS and peripheral tissues is thus the most effective therapeutic strategy and is highly desirable. We earlier showed that an agonist of the A(2A) adenosine receptor (A(2A) receptor), CGS21680 (CGS), attenuates neuronal symptoms of HD. We found herein that the A(2A) receptor also exists in the liver, and that CGS ameliorated the urea cycle deficiency by reducing mHtt aggregates in the liver. By suppressing aggregate formation, CGS slowed the hijacking of a crucial transcription factor (HSF1) and two protein chaperons (Hsp27 and Hsp70) into hepatic Htt aggregates. Moreover, the abnormally high levels of high-molecular-mass ubiquitin conjugates in the liver of an HD mouse model (R6/2) were also ameliorated by CGS. The protective effect of CGS against mHtt-induced aggregate formation was reproduced in two cells lines and was prevented by an antagonist of the A(2A) receptor and a protein kinase A (PKA) inhibitor. Most importantly, the mHtt-induced suppression of proteasome activity was also normalized by CGS through PKA. Our findings reveal a novel therapeutic pathway of A(2A) receptors in HD and further strengthen the concept that the A(2A) receptor can be a drug target in treating HD.


Assuntos
Doença de Huntington/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Receptor A2A de Adenosina/metabolismo , Ubiquitina/metabolismo , Ureia/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Antagonistas do Receptor A2 de Adenosina , Animais , Linhagem Celular , Modelos Animais de Doenças , Feminino , Humanos , Doença de Huntington/tratamento farmacológico , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Fenetilaminas/farmacologia , Receptor A2A de Adenosina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Proteínas da Membrana Plasmática de Transporte de Serotonina/metabolismo
13.
ACS Biomater Sci Eng ; 7(12): 5823-5835, 2021 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-34846835

RESUMO

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.


Assuntos
Plaquetas , Vesículas Extracelulares , Proliferação de Células , Cromatografia , Humanos
14.
Redox Biol ; 46: 102067, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34315111

RESUMO

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.


Assuntos
Concussão Encefálica , Animais , Quimiocina CCL5 , Glutationa Peroxidase/metabolismo , Hipocampo/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Glutationa Peroxidase GPX1
15.
Front Cell Dev Biol ; 8: 564641, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33344443

RESUMO

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.

16.
J Neurosci ; 28(13): 3277-90, 2008 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-18367595

RESUMO

Huntington's disease (HD) is a hereditary neurological disease caused by expended CAG repeats in the HD gene, which codes for a protein called Huntingtin (Htt). The resultant mutant Huntingtin (mHtt) forms aggregates in neurons and causes neuronal dysfunction. In astrocytes, the largest population of brain cells, mHtt also exists. We report herein that astrocyte-conditioned medium (ACM) collected from astrocytes of R6/2 mice (a mouse model of HD) caused primary cortical neurons to grow less-mature neurites, migrate more slowly, and exhibit lower calcium influx after depolarization than those maintained in wild-type (WT) ACM. Using a cytokine antibody array and ELISA assays, we demonstrated that the amount of a chemokine [chemokine (C-C motif) ligand 5 (CCL5)/regulated on activation normal T cell expressed and secreted (RANTES)] released by R6/2 astrocytes was much less than that by WT astrocytes. When cortical neurons were treated with the indicated ACM, supplementation with recombinant CCL5/RANTES ameliorated the neuronal deficiency caused by HD-ACM, whereas removing CCL5/RANTES from WT-ACM using an anti-CCL5/RANTES antibody mimicked the effects evoked by HD-ACM. Quantitative PCR and promoter analyses demonstrated that mHtt hindered the activation of the CCL5/RANTES promoter by reducing the availability of nuclear factor kappaB-p65 and, hence, reduced the transcript level of CCL5/RANTES. Moreover, ELISA assays and immunocytochemical staining revealed that mHtt retained the residual CCL5/RANTES inside R6/2 astrocytes. In line with the above findings, elevated cytosolic CCL5/RANTES levels were also observed in the brains of two mouse models of HD [R6/2 and Hdh((CAG)150)] and human HD patients. These findings suggest that mHtt hinders one major trophic function of astrocytes which might contribute to the neuronal dysfunction of HD.


Assuntos
Astrócitos/metabolismo , Quimiocina CCL5/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Neurônios/metabolismo , Proteínas Nucleares/fisiologia , Expansão das Repetições de Trinucleotídeos/fisiologia , Idoso , Idoso de 80 Anos ou mais , Animais , Animais Recém-Nascidos , Astrócitos/química , Encéfalo/patologia , Cálcio/metabolismo , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Quimiocina CCL2/metabolismo , Imunoprecipitação da Cromatina/métodos , Meios de Cultivo Condicionados/farmacologia , Embrião de Mamíferos , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Humanos , Proteína Huntingtina , Doença de Huntington/metabolismo , Doença de Huntington/patologia , Masculino , Camundongos , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Neurônios/efeitos dos fármacos , Proteínas Nucleares/genética , Ratos , Ratos Sprague-Dawley , Transfecção/métodos , Expansão das Repetições de Trinucleotídeos/genética
17.
Phytomedicine ; 54: 132-139, 2019 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-30668362

RESUMO

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/metabolismo
18.
J Vis Exp ; (131)2018 01 04.
Artigo em Inglês | MEDLINE | ID: mdl-29364220

RESUMO

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.


Assuntos
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 Sinais
19.
Food Chem Toxicol ; 120: 346-355, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30026090

RESUMO

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.


Assuntos
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/metabolismo
20.
Redox Biol ; 19: 74-80, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30121389

RESUMO

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 Sequência
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