RESUMO
Development of physiologically relevant cellular models with strong translatability to human pathophysiology is critical for identification and validation of novel therapeutic targets. Herein we describe a detailed protocol for generation of an advanced 3-dimensional kidney cellular model using induced pluripotent stem cells, where differentiation and maturation of kidney progenitors and podocytes can be monitored in live cells due to CRISPR/Cas9-mediated fluorescent tagging of kidney lineage markers (SIX2 and NPHS1). Utilizing these cell lines, we have refined the previously published procedures to generate a new, higher throughput protocol suitable for drug discovery. Using paraffin-embedded sectioning and whole-mount immunostaining, we demonstrated that organoids grown in suspension culture express key markers of kidney biology (WT1, ECAD, LTL, nephrin) and vasculature (CD31) within renal cortical structures with microvilli, tight junctions and podocyte foot processes visualized by electron microscopy. Additionally, the organoids resemble the adult kidney transcriptomics profile, thereby strengthening the translatability of our in vitro model. Thus, development of human nephron-like structures in vitro fills a major gap in our ability to assess the effect of potential treatment on key kidney structures, opening up a wide range of possibilities to improve clinical translation.
Assuntos
Sistemas CRISPR-Cas , Descoberta de Drogas/métodos , Edição de Genes/métodos , Células-Tronco Pluripotentes Induzidas/fisiologia , Rim/fisiologia , Organoides/fisiologia , Podócitos/fisiologia , Biomarcadores/metabolismo , Diferenciação Celular , Linhagem Celular , Linhagem da Célula , Regulação da Expressão Gênica , Genótipo , Ensaios de Triagem em Larga Escala , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/ultraestrutura , Rim/efeitos dos fármacos , Rim/metabolismo , Rim/ultraestrutura , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/ultraestrutura , Fenótipo , Podócitos/efeitos dos fármacos , Podócitos/metabolismo , Podócitos/ultraestrutura , Fatores de Tempo , TranscriptomaRESUMO
Diabetic nephropathy (DN) remains an unmet medical challenge as its prevalence is projected to continue to increase and specific medicines for treatment remain undeveloped. Activation of the immune system, in particular T-cells, is emerging as a possible mechanism underlying DN disease progression in humans and animal models. We hypothesized that inhibition of T-cell activation will ameliorate DN. Interaction of B7-1 (CD80) on the surface of antigen presenting cells with its binding partners, CTLA4 (CD152) and CD28 on T-cells, is essential for T-cell activation. In this study we used the soluble CTLA4-Fc fusion protein Abatacept to block cell surface B7-1, preventing the cellular interaction and inhibiting T-cell activation. When Abatacept was dosed in an animal model of diabetes-induced albuminuria, it reduced albuminuria in both prevention and intervention modes. The number of T-cells infiltrating the kidneys of DN animals correlated with the degree of albuminuria, and treatment with Abatacept reduced the number of renal T-cells. As B7-1 induction has been recently proposed to underlie podocyte damage in DN, Abatacept could be efficacious in DN by protecting podocytes. However, this does not appear to be the case as B7-1 was not expressed in 1) kidneys of DN animals; 2) stimulated human podocytes in culture; or 3) glomeruli of DN patients. We conclude that Abatacept ameliorates DN by blocking systemic T-cell activation and not by interacting with podocytes.
Assuntos
Abatacepte/farmacologia , Albuminúria/tratamento farmacológico , Nefropatias Diabéticas/tratamento farmacológico , Imunossupressores/farmacologia , Rim/efeitos dos fármacos , Ativação Linfocitária/efeitos dos fármacos , Linfócitos T/efeitos dos fármacos , Albuminúria/imunologia , Albuminúria/metabolismo , Albuminúria/patologia , Animais , Antígeno B7-1/imunologia , Antígeno B7-1/metabolismo , Linhagem Celular , Colágeno Tipo IV/metabolismo , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/imunologia , Diabetes Mellitus Tipo 1/induzido quimicamente , Diabetes Mellitus Tipo 1/imunologia , Nefropatias Diabéticas/imunologia , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Dieta Hiperlipídica , Humanos , Rim/imunologia , Rim/metabolismo , Rim/patologia , Camundongos Endogâmicos C57BL , Podócitos/efeitos dos fármacos , Podócitos/imunologia , Podócitos/metabolismo , Estreptozocina , Linfócitos T/imunologia , Fatores de TempoRESUMO
BACKGROUND: The role of myeloperoxidase in chronic kidney disease (CKD) and its association with coronary artery disease (CAD) is controversial. In this study, we compared myeloperoxidase and protein-bound 3-chlorotyrosine (ClY) levels in subjects with varying degrees of CKD and tested their associations with CAD. METHODS: From Clinical Phenotyping Resource and Biobank Core, 111 patients were selected from CKD stages 1 to 5. Plasma myeloperoxidase level was measured using enzyme-linked-immunosorbent assay. Plasma protein-bound 3-ClY, a specific product of hypochlorous acid generated by myeloperoxidase was measured by liquid chromatography mass spectrometry. RESULTS: We selected 29, 20, 24, 22, and 16 patients from stages 1 to 5 CKD, respectively. In a sex-adjusted general linear model, mean ± SD of myeloperoxidase levels decreased from 18.1 ± 12.3 pmol in stage 1 to 10.9 ± 4.7 pmol in stage 5 (p = 0.011). In patients with and without CAD, the levels were 19.1 ± 10.1 and 14.8 ± 8.7 pmol (p = 0.036). There was an increase in 3-ClY mean from 0.81 ± 0.36 mmol/mol-tyrosine in stage 1 to 1.42 ± 0.41 mmol/mol-tyrosine in stage 5 (p < 0.001). The mean 3-ClY levels in patients with and without CAD were 1.25 ± 0.44 and 1.04 ± 0.42 mmol/mol-tyrosine (p = 0.023), respectively. C-statistic of ClY when added to myeloperoxidase level to predict CKD stage 5 was 0.86, compared to 0.79 for the myeloperoxidase level alone (p = 0.0097). CONCLUSION: The myeloperoxidase levels decrease from stages 1 to 5, whereas activity increases. In contrast, both myeloperoxidase and ClY levels rise in the presence of CAD at various stages of CKD. Measuring both plasma myeloperoxidase and 3-CLY levels provide added value to determine the burden of myeloperoxidase-mediated oxidative stress.
Assuntos
Doença da Artéria Coronariana/sangue , Peroxidase/sangue , Insuficiência Renal Crônica/sangue , Tirosina/análogos & derivados , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores/sangue , Doença da Artéria Coronariana/etiologia , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estresse Oxidativo , Peroxidase/metabolismo , Insuficiência Renal Crônica/complicações , Fatores de Risco , Índice de Gravidade de Doença , Tirosina/sangue , Tirosina/metabolismo , Adulto JovemRESUMO
BACKGROUND: To develop medicines that are safe and efficacious to all patients, clinical trials must enroll appropriate target populations, but imbalances related to race, ethnicity and sex have been reported. A comprehensive analysis and improvement in understanding representativeness of patient enrollment in industry-sponsored trials are key public health needs. METHODS: We assessed race/ethnicity and sex representation in AstraZeneca (AZ)-sponsored clinical trials in the United States (US) from 2010 to 2022, compared with the 2019 US Census. RESULTS: In total, 246 trials representing 95,372 patients with complete race/ethnicity and sex records were analyzed. The proportions of different race/ethnicity subgroups in AZ-sponsored clinical trials and the US Census were similar (White: 69.5% vs 60.1%, Black or African American: 13.3% vs 12.5%, Asian: 1.8% vs 5.8%, Hispanic: 14.4% vs 18.5%). We also observed parity in the proportions of males and females between AZ clinical trials and US Census (males: 52.4% vs 49.2%, females: 47.6% vs 50.8%). Comparisons of four distinct therapy areas within AZ (Respiratory and Immunology [R&I]; Cardiovascular, Renal, and Metabolism [CVRM]; Solid Tumors; and Hematological Malignancies), including by trial phases, revealed greater variability, with proportions observed above and below US Census levels. CONCLUSION: This analysis provides the first detailed insights into the representativeness of AZ trials. Overall, the proportions of different race/ethnicity and sex subgroups in AZ-sponsored clinical trials were broadly aligned with the US Census. We outline some of AZ's planned health equity initiatives that are intended to continue to improve equitable patient enrollment.
Assuntos
Ensaios Clínicos como Assunto , Feminino , Humanos , Masculino , Ensaios Clínicos como Assunto/estatística & dados numéricos , Indústria Farmacêutica , Etnicidade/estatística & dados numéricos , Seleção de Pacientes , Grupos Raciais/estatística & dados numéricos , Fatores Sexuais , Estados Unidos , Brancos , Negro ou Afro-Americano , Asiático , Hispânico ou LatinoRESUMO
5-Hydroxytryptamine 6 (5-HT6) receptors are involved in learning and memory processes and are discussed as promising targets for the treatment of cognitive impairment in central nervous system disorders. A number of 5-HT6 antagonists are currently in the clinical development for schizophrenia and Alzheimer's disease (AD). There is some discrepancy regarding cognitive efficacy in subjects, and only limited data are available on the role of the 5-HT6 receptor in animal models of psychosis. The aim of this study was to investigate the efficacy of the selective 5-HT6 antagonists, Ro-4368554 (1-10 mg/kg, intraperitoneally) and SB-258585 (3-30 mg/kg, intraperitoneally), in animal models for schizophrenia and AD. Both compounds showed cognition-enhancing effects in object recognition, whereas only SB-258585 was able to prevent the scopolamine-induced deficit in the Morris water-maze test. Neither Ro-4368554 nor SB-258585 prevented scopolamine-induced impairment in contextual fear conditioning. Similarly, both compounds were ineffective on MK-801-induced deficits in contextual fear conditioning and spatial working memory. Ro-4368554, but not SB-258585 reversed the apomorphine-induced deficit in prepulse inhibition. Amphetamine-induced hyperlocomotion was not affected by either compound. Taken together, the overall efficacy of Ro-4368554 and SB-258585 in animal models for AD and schizophrenia is rather limited. These data show moderate efficacy in some models for AD but do not support the therapeutic potential of 5-HT6 antagonists for schizophrenia.
Assuntos
Indóis/farmacologia , Piperazinas/farmacologia , Receptores de Serotonina/efeitos dos fármacos , Antagonistas da Serotonina/farmacologia , Sulfonamidas/farmacologia , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/fisiopatologia , Anfetamina/toxicidade , Animais , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Medo/efeitos dos fármacos , Hipercinese/induzido quimicamente , Indóis/administração & dosagem , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Piperazinas/administração & dosagem , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Esquizofrenia/tratamento farmacológico , Esquizofrenia/fisiopatologia , Antagonistas da Serotonina/administração & dosagem , Sulfonamidas/administração & dosagemRESUMO
Phosphoinositol 3-kinases (PI3Ks) γ and δ are key enzymes in hematopoietic cells and have been seen as high-value targets for the treatment of diseases with inflammatory and immunomodulatory components since their discovery and the identification of their roles. In this Perspective we review progress in the application of inhibitors of PI3Kγ and δ to inflammatory and immunological conditions over the past 6 years. We consider progress in the understanding of the roles of PI3Kγ and PI3Kδ in immunology and inflammation, the experience from clinical trials where inhibitors have been tested, and what has been learned about the safety of their use. The extensive medicinal chemistry efforts to discover both isoform selective and dual PI3Kγδ inhibitors are analyzed and detailed. Developments in understanding the structural chemistry of the PI3K enzymes and the factors that govern isoform selectivity are discussed. The effects observed with the known inhibitor compounds in animal models are described.
Assuntos
Doenças Autoimunes/tratamento farmacológico , Classe I de Fosfatidilinositol 3-Quinases/antagonistas & inibidores , Classe Ib de Fosfatidilinositol 3-Quinase/metabolismo , Inflamação/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase/uso terapêutico , Animais , Linhagem Celular Tumoral , Classe I de Fosfatidilinositol 3-Quinases/metabolismo , Ensaios Clínicos como Assunto , Humanos , Estrutura Molecular , Inibidores de Fosfoinositídeo-3 Quinase/química , Inibidores de Fosfoinositídeo-3 Quinase/metabolismo , Ligação ProteicaRESUMO
BACKGROUND: We report that R- and S-phenibut (ß-phenyl-γ-aminobutyric acid) - derivatives of GABA - bind with an affinity of c.a. 90µM to the gabapentin binding site in a competitive assay, a value comparable to that for previously claimed targets for this enantioermic molecule. This finding implied potential activity in neuropathic pain, this being one of the clinically validated indications for gabapentin. METHODS: The effect of phenibut on tactile allodynia was tested in a chronic constriction nerve injury (CCI) neuropathic pain model and against hypersensitivity following inflammation induced by inoculation using complete Freund's adjuvant (CFA) model. RESULTS: Indeed, a significant inhibitory effect on tactile allodynia was detected in rats in both employed chronic pain models with stronger and clearly dose dependent effect with R isomer. CONCLUSIONS: The results confirm activity in chronic pain models predicted from affinity for the gabapentin site and suggests, at least partially, that α2δ-subunits of presynaptic voltage-gated calcium channels are involved in mediating this effect.
Assuntos
Aminas/antagonistas & inibidores , Dor Crônica/tratamento farmacológico , Ácidos Cicloexanocarboxílicos/antagonistas & inibidores , Ácido gama-Aminobutírico/análogos & derivados , Animais , Modelos Animais de Doenças , Adjuvante de Freund , Gabapentina , Hipersensibilidade/tratamento farmacológico , Masculino , Medição da Dor/efeitos dos fármacos , Ensaio Radioligante , Ratos , Estereoisomerismo , Ácido gama-Aminobutírico/farmacologia , Ácido gama-Aminobutírico/uso terapêuticoRESUMO
Several studies have demonstrated that C57 and DBA mice exhibit behavioural differences in diverse learning tasks as well as variations in the expression of long-term potentiation (LTP) in the hippocampus. In the present investigation, we tested the possibility that these differences between the two strains might be attributable to differential regulation of hippocampal alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors by calcium-dependent mechanisms. Using in vitro receptor autoradiography, we found that calcium treatment of C57 mice sections resulted in a marked increase of 3H-AMPA binding in areas CA3 and CA1 of the hippocampus and in the dentate gyrus. However, we discovered that the ability of calcium to upregulate 3H-AMPA binding in the DBA strain was much lower than in corresponding regions from the C57 strain. Western blot and immunohistochemical experiments indicated that truncation of AMPA receptor subunits by calcium-dependent mechanisms was possibly not responsible for the binding differences, as no significant variations in glutamate receptor subunit 1 (GluR1) and GluR2/3 immunoreactivity were observed between the two strains after calcium treatment. Interestingly, we found that strain-related variations in the regulation of 3H-AMPA binding by calcium were totally eliminated when brain sections were preincubated with preferential inhibitors of lipoxygenase (LO) pathways of arachidonic acid (AA) metabolism. Taken together, these results suggest that calcium-induced regulation of AMPA receptors varies between the two strains and that this variation might be linked to the production of specific AA metabolites.
Assuntos
Ácido Araquidônico/metabolismo , Sinalização do Cálcio/fisiologia , Cálcio/metabolismo , Hipocampo/metabolismo , Lipoxigenase/metabolismo , Receptores de AMPA/metabolismo , Animais , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Cálcio/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Hipocampo/efeitos dos fármacos , Hipocampo/enzimologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Ensaio Radioligante , Receptores de AMPA/efeitos dos fármacos , Especificidade da Espécie , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiônico/farmacologiaRESUMO
Oxidative stress is currently considered a mediator of cell death in several neurodegenerative diseases. Notably, it may play an important role in the degeneration of dopamine neurons of the substantia nigra in Parkinson's disease. We examined the effect of a strong oxidant, the herbicide paraquat, on cell distress using native and neuronal pheochromocytoma PC12 cells. Paraquat administration for 8 hours induced a significant cellular death in both native and in neuronal PC12 cells. Since the anti-oxidant properties of estrogens may promote neuroprotection in vitro and in vivo, we then investigated the ability of estradiol stereoisomers, 17alpha-estradiol and 17- beta-estradiol, to rescue PC12 cells submitted to paraquat-induced oxidative stress. Our results show a protective effect of both estradiol stereoisomers in neuronal PC12 cells treated with paraquat, whereas this effect could not be observed in native PC12 cells. We also demonstrate that estrogen receptor beta protein expression is modulated by paraquat administration in native PC12 cells, while paraquat does not change estrogen receptor beta ?expression in neuronal PC12 cells. Paraquat also decreases estrogen receptor alpha in neuronal PC12 cells, thus suggesting new routes for paraquat to collapse cellular metabolism. Besides, the oxidation of dihydrodhodamine-123 into fluorescent rhodamine in the presence of paraquat but not in presence of paraquat and 17 alpha-estradiol or 17 beta-estradiol, sustain a possible direct scavenging role of both estradiol stereoisomers.
Assuntos
Estradiol/farmacologia , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Paraquat/toxicidade , Animais , Isomerismo , Neurônios/patologia , Estresse Oxidativo/fisiologia , Células PC12 , RatosRESUMO
L-DOPA-induced dyskinesia (LID) is among the motor complications that arise in Parkinson patients after a prolonged treatment with levodopa (L-DOPA). Since previous transcriptome and proteomic studies performed in the rat model of LID suggested important changes in striatal energy-related components, we hypothesize that oral creatine supplementation could prevent or attenuate the occurrence of LID. In this study, 6-hydroxydopamine-lesioned rats received a 2% creatine-supplemented diet for 1 month prior to L-DOPA therapy. During the 21 days of L-DOPA treatment, significant reductions in abnormal involuntary movements (AIMs) have been observed in the creatine-supplemented group, without any worsening of parkinsonism. In situ hybridization histochemistry and immunohistochemistry analysis of the striatum also showed a reduction in the levels of prodynorphin mRNA and FosB/DeltaFosB-immunopositive cells in creatine-supplemented diet group, an effect that was dependant on the development of AIMs. Further investigation of the bioenergetics' status of the denervated striatum revealed significant changes in the levels of creatine both after L-DOPA alone and with the supplemented diet. In conclusion, we demonstrated that combining L-DOPA therapy with a diet enriched in creatine could attenuate LID, which may represent a new way to control the motor complications associated with L-DOPA therapy.
Assuntos
Creatina/metabolismo , Discinesia Induzida por Medicamentos/metabolismo , Metabolismo Energético/fisiologia , Neostriado/metabolismo , Fármacos Neuroprotetores/metabolismo , Administração Oral , Análise de Variância , Animais , Creatina/administração & dosagem , Suplementos Nutricionais , Modelos Animais de Doenças , Discinesia Induzida por Medicamentos/complicações , Discinesia Induzida por Medicamentos/prevenção & controle , Encefalinas/genética , Encefalinas/metabolismo , Feminino , Técnicas In Vitro , Levodopa/efeitos adversos , Levodopa/uso terapêutico , Neostriado/efeitos dos fármacos , Fármacos Neuroprotetores/administração & dosagem , Oxidopamina , Transtornos Parkinsonianos/induzido quimicamente , Transtornos Parkinsonianos/complicações , Transtornos Parkinsonianos/tratamento farmacológico , Fosfocreatina/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/análise , Ratos , Ratos Sprague-Dawley , Estatísticas não ParamétricasRESUMO
Subchronic treatment with memantine using osmotic pumps in male rats was used to verify whether plasma levels significantly blocking L-N-methyl-D-aspartate (NMDA) receptors (and shown previously to be neuroprotective) may impair learning. Treatment with 6.27, 12.5 and 18.8 mg/rat/day provided plasma levels of 1.03+/-0.08, 5.07+/-0.68 and 11.68+/-0.90 micromol/l. Only the lowest plasma level is therapeutically relevant and has previously been shown to be neuroprotective. Significant deficits in a passive avoidance task were only observed at the highest dose. Working memory, tested as spontaneous alternation in the cross maze, was impaired by the middle and highest doses, and these doses also induced hyperlocomotion. Microdialysis experiments with in-vivo recovery (27.4%) showed that infusion of memantine at 6.27 mg/rat/day (ca. 23 mg/kg/day) produced a concentration of 990+/-105 nmol/l in extracellular fluid. In-vivo NMDA receptor occupancy experiments demonstrated significant, dose-dependent receptor occupancy of 32.7 and 65.7% by memantine at the doses producing 1 and 5 micromol/l plasma levels, respectively. Moreover, acute administration (2.5 mg/kg intraperitoneally) of memantine to mature female rats produced approximately two-fold higher plasma levels than in young male rats. In conclusion, a dose of memantine which produces a plasma level (1 micromol/l) within the therapeutic range, reported previously to be neuroprotective, leads to intracellular brain levels similar to the affinity of memantine for NMDA receptors (receptor binding, patch clamp). This has been also extended by the experiments showing that at this plasma concentration, memantine occupies ca. 30% NMDA receptors in the brain and produces no cognitive impairment.
Assuntos
Aprendizagem da Esquiva/efeitos dos fármacos , Medo/efeitos dos fármacos , Aprendizagem em Labirinto/efeitos dos fármacos , Memantina/farmacologia , Memantina/farmacocinética , Memória de Curto Prazo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/farmacocinética , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Animais , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Feminino , Masculino , Microdiálise , Atividade Motora/efeitos dos fármacos , Plasticidade Neuronal/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Tempo de Reação/efeitos dos fármacos , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Retenção Psicológica/efeitos dos fármacos , Fatores SexuaisRESUMO
Differential in-gel electrophoresis (DIGE) experiments allow three protein samples to be run per gel. The three samples are labeled with the spectrally resolvable fluorescent dyes, Cy2, Cy3, and Cy5, respectively. Here, we show that protein-specific dye effects exist, and we present a linear mixed model for analysis of DIGE data which takes dye effects into account. A Java implementation of the model, called DIGEanalyzer, is freely available at http://bioinfo.thep.lu.se/digeanalyzer.html. Three DIGE experiments from our laboratory, with 173, 64, and 24 gels, respectively, were used to quantify and verify the dye effects. DeCyder 5.0 and 6.5 were used for spot detection and matching. The fractions of proteins with a statistically significant (0.001 level) dye effect were 19, 34, and 23%, respectively. The fractions of proteins with a dye effect above 1.4-fold change were 1, 4, and 6%, respectively. The median magnitude of the dye effect was 1.07-fold change for Cy5 versus Cy3 and 1.16-fold change for Cy3 versus Cy2. The maximal dye effect was a seven-fold change. The dye effects of spots corresponding to the same protein tend to be similar within each of the three experiments, and to a smaller degree across experiments.
Assuntos
Biologia Computacional/métodos , Corantes Fluorescentes/química , Processamento de Imagem Assistida por Computador/métodos , Proteínas/análise , Proteômica/métodos , Algoritmos , Animais , Química Encefálica , Neoplasias da Mama/química , Carbocianinas/química , Eletroforese em Gel Bidimensional/instrumentação , Eletroforese em Gel Bidimensional/métodos , Feminino , Humanos , Internet , Modelos Lineares , Neoplasias Ovarianas/química , Proteínas/química , Ratos , Software , Espectrometria de Massas em TandemRESUMO
L-DOPA-induced dyskinesia (LID) is among the motor complications that arise in Parkinson's disease (PD) patients after a prolonged treatment with L-DOPA. To this day, transcriptome analysis has been performed in a rat model of LID [Neurobiol. Dis., 17 (2004), 219] but information regarding the proteome is still lacking. In the present study, we investigated the changes occurring at the protein level in striatal samples obtained from the unilaterally 6-hydroxydopamine-lesion rat model of PD treated with saline, L-DOPA or bromocriptine using two-dimensional difference gel electrophoresis and mass spectrometry (MS). Rats treated with L-DOPA were allocated to two groups based on the presence or absence of LID. Among the 2000 spots compared for statistical difference, 67 spots were significantly changed in abundance and identified using matrix-assisted laser desorption/ionization time-of-flight MS, atmospheric pressure matrix-assisted laser desorption/ionization and HPLC coupled tandem MS (LC/MS/MS). Out of these 67 proteins, LID significantly changed the expression level of five proteins: alphabeta-crystalin, gamma-enolase, guanidoacetate methyltransferase, vinculin, and proteasome alpha-2 subunit. Complementary techniques such as western immunoblotting and immunohistochemistry were performed to investigate the validity of the data obtained using the proteomic approach. In conclusion, this study provides new insights into the protein changes occurring in LID.
Assuntos
Corpo Estriado/metabolismo , Discinesia Induzida por Medicamentos/metabolismo , Levodopa/efeitos adversos , Proteínas/metabolismo , Proteômica/métodos , Análise de Variância , Animais , Comportamento Animal , Modelos Animais de Doenças , Discinesia Induzida por Medicamentos/etiologia , Discinesia Induzida por Medicamentos/patologia , Eletroforese em Gel Bidimensional/métodos , Masculino , Espectrometria de Massas , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
The present investigation provides the first indication that constitutive, calcium-independent phospholipase A2 activity (iPLA2) modulates phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) subtype of glutamate receptors. Preincubation of frozen-thawed brain sections with two iPLA2 inhibitors, bromoenol lactone (BEL) or palmitoyl trifluoromethyl ketone (PACO), produced a dose-dependent enhancement in phosphorylation at both Ser831 and Ser845 sites on the GluR1 subunit of AMPA receptors. This effect was not associated with changes in phosphorylation at the Ser sites of either the GluR2/3 subunits of AMPA receptors or the NR1 subunits of N-methyl-D-aspartate (NMDA) receptors, nor was it reproduced by inhibition of the calcium-dependent form of PLA2 activity. These results suggest that the effects of these inhibitors are selective to GluR1 subunits and that they are dependent on iPLA2 activity. The ability of iPLA2 inhibitors to increase GluR1 phosphorylation was mimicked by the 5-lipoxygenase (5-LO) inhibitor MK-886, but not by blockers of 12-lipoxygenase (12-LO) or cyclooxygenase. Additional experiments indicated that calcium-mediated truncation of GluR1 subunits was reduced by iPLA2 inhibitors, an effect that was not correlated with overall changes in the distribution of AMPA receptors between intracellular and membrane compartments prepared from whole brain sections. However, quantitative autoradiographic analysis indicated enhanced 3H-AMPA binding to the CA1 stratum radiatum of the hippocampus in BEL-treated sections. Saturation kinetics experiments demonstrated that this binding augmentation was due to an increase in the maximal number of AMPA binding sites. Altogether, our results point to the conclusion that basal iPLA2 activity, through the generation of 5-LO metabolites, regulates AMPA receptor phosphorylation of GluR1 subunits, an effect that might selectively influence the number of membrane receptors in area CA1 of the hippocampus.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Hipocampo/enzimologia , Neurônios/enzimologia , Fosfolipases A/metabolismo , Receptores de AMPA/metabolismo , Animais , Ácido Araquidônico/metabolismo , Sítios de Ligação/efeitos dos fármacos , Sítios de Ligação/fisiologia , Ligação Competitiva/efeitos dos fármacos , Ligação Competitiva/fisiologia , Sinalização do Cálcio/efeitos dos fármacos , Sinalização do Cálcio/fisiologia , Relação Dose-Resposta a Droga , Endocitose/efeitos dos fármacos , Endocitose/fisiologia , Inibidores Enzimáticos/farmacologia , Fosfolipases A2 do Grupo VI , Hipocampo/anatomia & histologia , Inibidores de Lipoxigenase , Masculino , Fosfolipases A/antagonistas & inibidores , Fosfolipases A2 , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Receptores de AMPA/efeitos dos fármacos , Serina/metabolismo , Membranas Sinápticas/enzimologia , Transmissão Sináptica/efeitos dos fármacos , Transmissão Sináptica/fisiologiaRESUMO
Oxidative stress has been recently considered as a mediator of nerve cell death in several neurodegenerative diseases. We studied the effect of the parkinsonism-inducing toxine 1-methyl-4-phenyl-pyridine (MPP+) on several parameters of cell distress using native and neuronal PC12 cells. Then, since estrogens have been reported to prevent neuronal degeneration caused by oxidative damage, we investigated the ability of 17beta- estradiol (E2); two Ginkgo biloba extracts, EGb 761 and Cp 202; as well as two flavonoids, quercetin and kaempferol, to rescue PC12 cells submitted to MPP+- induced oxidative stress. Our results consistently show that both Ginkgo biloba extracts could prevent cell death in native and neuronal PC12 cells, while in neuronal PC12 cells also quercetin and E2 could reverse MPP+ neurotoxic effet. Western blot analysis demonstrated that MPP+ injuries might modulate dopamine transporter (DAT) protein expression but not estrogen receptor beta (ERbeta) protein expression. EGb 761 and Cp 202 also modulate DAT and ERbeta protein expression in neuronal cells. From these studies, we outline the importance of testing estrogen-like plant-derived molecules as potent antioxidants and examine their effect on protein expression.