RESUMO
Sleep problems are a significant phenotype in children with fragile X syndrome. Our prior work assessed sleep-wake cycles in Fmr1KO male mice and wild type (WT) littermate controls in response to ketogenic diet therapy where mice were treated from weaning (postnatal day 18) through study completion (5-6 months of age). A potentially confounding issue with commencing treatment during an active period of growth is the significant reduction in weight gain in response to the ketogenic diet. The aim here was to employ sleep electroencephalography (EEG) to assess sleep-wake cycles in mice in response to the Fmr1 genotype and a ketogenic diet, with treatment starting at postnatal day 95. EEG results were compared with prior sleep outcomes to determine if the later intervention was efficacious, as well as with published rest-activity patterns to determine if actigraphy is a viable surrogate for sleep EEG. The data replicated findings that Fmr1KO mice exhibit sleep-wake patterns similar to wild type littermates during the dark cycle when maintained on a control purified-ingredient diet but revealed a genotype-specific difference during hours 4-6 of the light cycle of the increased wake (decreased sleep and NREM) state in Fmr1KO mice. Treatment with a high-fat, low-carbohydrate ketogenic diet increased the percentage of NREM sleep in both wild type and Fmr1KO mice during the dark cycle. Differences in sleep microstructure (length of wake bouts) supported the altered sleep states in response to ketogenic diet. Commencing ketogenic diet treatment in adulthood resulted in a 15% (WT) and 8.6% (Fmr1KO) decrease in body weight after 28 days of treatment, but not the severe reduction in body weight associated with starting treatment at weaning. We conclude that the lack of evidence for improved sleep during the light cycle (mouse sleep time) in Fmr1KO mice in response to ketogenic diet therapy in two studies suggests that ketogenic diet may not be beneficial in treating sleep problems associated with fragile X and that actigraphy is not a reliable surrogate for sleep EEG in mice.
Assuntos
Dieta Cetogênica , Proteína do X Frágil da Deficiência Intelectual , Síndrome do Cromossomo X Frágil , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sono , Animais , Camundongos , Síndrome do Cromossomo X Frágil/dietoterapia , Masculino , Sono/fisiologia , Proteína do X Frágil da Deficiência Intelectual/genética , Eletroencefalografia , Modelos Animais de DoençasRESUMO
Nearly half of children with fragile X syndrome experience sleep problems including trouble falling asleep and frequent nighttime awakenings. The goals here were to assess sleep-wake cycles in mice in response to Fmr1 genotype and a dietary intervention that reduces hyperactivity. Electroencephalography (EEG) results were compared with published rest-activity patterns to determine if actigraphy is a viable surrogate for sleep EEG. Specifically, sleep-wake patterns in adult wild type and Fmr1KO littermate mice were recorded after EEG electrode implantation and the recordings manually scored for vigilance states. The data indicated that Fmr1KO mice exhibited sleep-wake patterns similar to wild type littermates when maintained on a control purified ingredient diet. Treatment with a high-fat, low-carbohydrate ketogenic diet increased the percentage of non-rapid eye movement (NREM) sleep in both wild type and Fmr1KO mice during the dark cycle, which corresponded to decreased activity levels. Treatment with a ketogenic diet flattened diurnal sleep periodicity in both wild type and Fmr1KO mice. Differences in several sleep microstructure outcomes (number and length of sleep and wake bouts) supported the altered sleep states in response to a ketogenic diet and were correlated with altered rest-activity cycles. While actigraphy may be a less expensive, reduced labor surrogate for sleep EEG during the dark cycle, daytime resting in mice did not correlate with EEG sleep states.
Assuntos
Dieta Cetogênica , Humanos , Criança , Animais , Camundongos , Camundongos Endogâmicos C57BL , Sono/fisiologia , Vigília/fisiologia , Eletroencefalografia , Camundongos Knockout , Proteína do X Frágil da Deficiência Intelectual/genéticaRESUMO
OBJECTIVE: PS (protein S) is a plasma protein that directly inhibits the coagulation FIXa (factor IXa) in vitro. Because elevated FIXa is associated with increased risk of venous thromboembolism, it is important to establish how PS inhibits FIXa function in vivo. The goal of this study is to confirm direct binding of PS with FIXa in vivo, identify FIXa amino acid residues required for binding PS in vivo, and use an enzymatically active FIXa mutant that is unable to bind PS to measure the significance of PS-FIXa interaction in hemostasis. APPROACH AND RESULTS: We demonstrate that PS inhibits FIXa in vivo by associating with the FIXa heparin-binding exosite. We used fluorescence tagging, immunohistochemistry, and protein-protein crosslinking to show in vivo interaction between FIXa and PS. Importantly, platelet colocalization required a direct interaction between the 2 proteins. FIXa and PS also coimmunoprecipitated from plasma, substantiating their interaction in a physiological milieu. PS binding to FIXa and PS inhibition of the intrinsic Xase complex required residues K132, K126, and R170 in the FIXa heparin-binding exosite. A double mutant, K132A/R170A, retained full activity but could not bind to PS. Crucially, Hemophilia B mice infused with FIXa K132A/R170A displayed an accelerated rate of fibrin clot formation compared with wild-type FIXa. CONCLUSIONS: Our findings establish PS as an important in vivo inhibitor of FIXa. Disruption of the interaction between PS and FIXa causes an increased rate of thrombus formation in mice. This newly discovered function of PS implies an unexploited target for antithrombotic therapeutics.
Assuntos
Plaquetas/metabolismo , Fator IXa/metabolismo , Hemofilia B/sangue , Hemostasia , Heparina/metabolismo , Proteína S/metabolismo , Trombose Venosa/prevenção & controle , Animais , Sítios de Ligação , Ligação Competitiva , Coagulantes/administração & dosagem , Modelos Animais de Doenças , Fator IX/genética , Fator IX/metabolismo , Fator IXa/administração & dosagem , Fator IXa/genética , Hemofilia B/tratamento farmacológico , Hemofilia B/genética , Hemostasia/efeitos dos fármacos , Humanos , Infusões Intravenosas , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Trombose Venosa/sangue , Trombose Venosa/genéticaRESUMO
Metabotropic glutamate receptor 5 (mGluR5) is a drug target for central nervous system disorders such as fragile X syndrome that involve excessive glutamate-induced excitation. We tested the efficacy of a novel negative allosteric modulator of mGluR5 developed by Merz Pharmaceuticals, MRZ-8456, in comparison to MPEP and AFQ-056 (Novartis, a.k.a. mavoglurant) in both in vivo and in vitro assays in a mouse model of fragile X syndrome, Fmr1KO mice. The in vivo assays included susceptibility to audiogenic-induced seizures and pharmacokinetic measurements of drug availability. The in vitro assays included dose response assessments of biomarker expression and dendritic spine length and density in cultured primary neurons. Both MRZ-8456 and AFQ-056 attenuated wild running and audiogenic-induced seizures in Fmr1KO mice with similar pharmacokinetic profiles. Both drugs significantly reduced dendritic expression of amyloid-beta protein precursor (APP) and rescued the ratio of mature to immature dendritic spines. These findings demonstrate that MRZ-8456, a drug being developed for the treatment of motor complications of L-DOPA in Parkinson's disease and which completed a phase I clinical trial, is effective in attenuating both well-established (seizures and dendritic spine maturity) and exploratory biomarker (APP expression) phenotypes in a mouse model of fragile X syndrome.
Assuntos
Antagonistas de Aminoácidos Excitatórios/uso terapêutico , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Indóis/uso terapêutico , Isoquinolinas/uso terapêutico , Fenótipo , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Receptor de Glutamato Metabotrópico 5/antagonistas & inibidores , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Antagonistas de Aminoácidos Excitatórios/química , Antagonistas de Aminoácidos Excitatórios/farmacologia , Feminino , Síndrome do Cromossomo X Frágil/tratamento farmacológico , Indóis/química , Indóis/farmacologia , Isoquinolinas/química , Isoquinolinas/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Gravidez , Pirazóis/química , Pirazóis/farmacologia , Pirimidinas/química , Pirimidinas/farmacologia , Distribuição AleatóriaRESUMO
OBJECTIVE: Enhanced tissue factor (TF) expression in epithelial ovarian cancer (EOC) is associated with aggressive disease. Our objective was to evaluate the role of the TF-factor VIIa-protease-activated receptor-2 (PAR-2) pathway in human EOC. METHODS: TCGA RNAseq data from EOC databases were analyzed for PAR expression. Cell and microparticle (MP) associated TF protein expression (Western blot) and MP-associated coagulant activity were determined in human EOC (SKOV-3, OVCAR-3 and CaOV-3) and control cell lines. PAR-1 and PAR-2 protein expressions were similarly examined. The PAR dependence of VEGF-A release (ELISA) and chemotactic migration in response to FVIIa and cellular proliferation in response to thrombin was evaluated with small molecule antagonists. RESULTS: Relative mRNA expression consistently demonstrated PAR-2>PAR-1â«PAR-3/4 in multiple EOC datasets. Human EOC cell line lysates confirmed expression of TF, PAR-1 and PAR-2 proteins. MPs isolated from EOC cell lines demonstrated markedly enhanced (4-10 fold) TF coagulant activity relative to control cell lines. FVIIa induced a dose-dependent increase in VEGF-A release (2.5-3 fold) from EOC cell lines that was abrogated by the PAR-2 antagonist ENMD-1068. FVIIa treatment of CaOV-3 and OVCAR-3 cells resulted in increased chemotactic migration that was abolished by ENMD-1068. Thrombin induced dose-dependent EOC cell line proliferation was completely reversed by the PAR-1 antagonist vorapaxar. Small molecule antagonists had no effect on these phenotypes without protease present. CONCLUSIONS: Enhanced activity of the TF-FVIIa-PAR-2 axis may contribute to the EOC progression via PAR-2 dependent signaling that supports an angiogenic and invasive phenotype and local thrombin generation supporting PAR-1 dependent proliferation.
Assuntos
Movimento Celular , Fator VIIa/metabolismo , Neoplasias Epiteliais e Glandulares/genética , Neoplasias Ovarianas/genética , RNA Mensageiro/metabolismo , Receptor PAR-1/genética , Receptor PAR-2/genética , Tromboplastina/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Western Blotting , Carcinoma Epitelial do Ovário , Linhagem Celular Tumoral , Proliferação de Células , Quimiotaxia , Feminino , Humanos , Invasividade Neoplásica , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Neoplasias Ovarianas/metabolismo , Neoplasias Ovarianas/patologia , Receptor PAR-1/metabolismo , Receptor PAR-2/metabolismo , Receptores Ativados por Proteinase/genética , Receptores Ativados por Proteinase/metabolismo , Transdução de Sinais , Trombina/metabolismoRESUMO
Obesity is a pediatric epidemic that is more prevalent in children with developmental disabilities. We hypothesize that soy protein-based diets increase weight gain and alter neurobehavioral outcomes. Our objective herein was to test matched casein- and soy protein-based purified ingredient diets in a mouse model of fragile X syndrome, Fmr1KO mice. The experimental methods included assessment of growth; 24-7 activity levels; motor coordination; learning and memory; blood-based amino acid, phytoestrogen and glucose levels; and organ weights. The primary outcome measure was body weight. We find increased body weight in male Fmr1KO from postnatal day 6 (P6) to P224, male wild type (WT) from P32-P39, female Fmr1KO from P6-P18 and P168-P224, and female Fmr1HET from P9-P18 as a function of soy. Activity at the beginning of the light and dark cycles increased in female Fmr1HET and Fmr1KO mice fed soy. We did not find significant differences in rotarod or passive avoidance behavior as a function of genotype or diet. Several blood-based amino acids and phytoestrogens were significantly altered in response to soy. Liver weight was increased in WT and adipose tissue in Fmr1KO mice fed soy. Activity levels at the beginning of the light cycle and testes weight were greater in Fmr1KO versus WT males irrespective of diet. DEXA analysis at 8-months-old indicated increased fat mass and total body area in Fmr1KO females and lean mass and bone mineral density in Fmr1KO males fed soy. Overall, dietary consumption of soy protein isolate by C57BL/6J mice caused increased growth, which could be attributed to increased lean mass in males and fat mass in females. There were sex-specific differences with more pronounced effects in Fmr1KO versus WT and in males versus females.
Assuntos
Cetonas , Proteínas de Soja , Humanos , Criança , Animais , Camundongos , Feminino , Masculino , Lactente , Camundongos Endogâmicos C57BL , Proteínas de Soja/farmacologia , Fenótipo , Genótipo , Obesidade , Proteína do X Frágil da Deficiência Intelectual/genéticaRESUMO
Fragile X syndrome (FXS), the leading cause of inherited intellectual disability and autism, is caused by the transcriptional silencing of the FMR1 gene, which encodes the fragile X messenger ribonucleoprotein (FMRP). FMRP interacts with numerous brain mRNAs that are involved in synaptic plasticity and implicated in autism spectrum disorders. Our published studies indicate that single-source, soy-based diets are associated with increased seizures and autism. Thus, there is an acute need for an unbiased protein marker identification in FXS in response to soy consumption. Herein, we present a spatial proteomics approach integrating mass spectrometry imaging with label-free proteomics in the FXS mouse model to map the spatial distribution and quantify levels of proteins in the hippocampus and hypothalamus brain regions. In total, 1250 unique peptides were spatially resolved, demonstrating the diverse array of peptidomes present in the tissue slices and the broad coverage of the strategy. A group of proteins that are known to be involved in glycolysis, synaptic transmission, and coexpression network analysis suggest a significant association between soy proteins and metabolic and synaptic processes in the Fmr1KO brain. Ultimately, this spatial proteomics work represents a crucial step toward identifying potential candidate protein markers and novel therapeutic targets for FXS.
Assuntos
Síndrome do Cromossomo X Frágil , Proteínas de Soja , Camundongos , Animais , Proteínas de Soja/metabolismo , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Síndrome do Cromossomo X Frágil/metabolismo , Proteômica , Camundongos Knockout , Modelos Animais de DoençasRESUMO
Mice fed soy-based diets exhibit increased weight gain compared to mice fed casein-based diets, and the effects are more pronounced in a model of fragile X syndrome (FXS; Fmr1KO). FXS is a neurodevelopmental disability characterized by intellectual impairment, seizures, autistic behavior, anxiety, and obesity. Here, we analyzed body weight as a function of mouse age, diet, and genotype to determine the effect of diet (soy, casein, and grain-based) on weight gain. We also assessed plasma protein biomarker expression and behavior in response to diet. Juvenile Fmr1KO mice fed a soy protein-based rodent chow throughout gestation and postnatal development exhibit increased weight gain compared to mice fed a casein-based purified ingredient diet or grain-based, low phytoestrogen chow. Adolescent and adult Fmr1KO mice fed a soy-based infant formula diet exhibited increased weight gain compared to reference diets. Increased body mass was due to increased lean mass. Wild-type male mice fed soy-based infant formula exhibited increased learning in a passive avoidance paradigm, and Fmr1KO male mice had a deficit in nest building. Thus, at the systems level, consumption of soy-based diets increases weight gain and affects behavior. At the molecular level, a soy-based infant formula diet was associated with altered expression of numerous plasma proteins, including the adipose hormone leptin and the ß-amyloid degrading enzyme neprilysin. In conclusion, single-source, soy-based diets may contribute to the development of obesity and the exacerbation of neurological phenotypes in developmental disabilities, such as FXS.
Assuntos
Transtorno Autístico , Síndrome do Cromossomo X Frágil , Adolescente , Animais , Caseínas/metabolismo , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/genética , Humanos , Fórmulas Infantis , Masculino , Camundongos , Obesidade , Aumento de PesoRESUMO
Eosinophilic inflammation is a cornerstone of chronic asthma that often culminates in subepithelial fibrosis with variable airway obstruction. Pulmonary eosinophils (Eos) are a predominant source of TGF-beta1, which drives fibroblast proliferation and extracellular matrix deposition. We investigated the regulation of TGF-beta1 and show here that the peptidyl-prolyl isomerase (PPIase) Pin1 promoted the stability of TGF-beta1 mRNA in human Eos. In addition, Pin1 regulated cytokine production by both in vitro and in vivo activated human Eos. We found that Pin1 interacted with both PKC-alpha and protein phosphatase 2A, which together control Pin1 isomerase activity. Pharmacologic blockade of Pin1 in a rat asthma model selectively reduced eosinophilic pulmonary inflammation, TGF-beta1 and collagen expression, and airway remodeling. Furthermore, chronically challenged Pin1(-/-) mice showed reduced peribronchiolar collagen deposition compared with wild-type controls. These data suggest that pharmacologic suppression of Pin1 may be a novel therapeutic option to prevent airway fibrosis in individuals with chronic asthma.
Assuntos
Asma/imunologia , Eosinófilos/imunologia , Peptidilprolil Isomerase/metabolismo , Fibrose Pulmonar/imunologia , Hipersensibilidade Respiratória/imunologia , Fator de Crescimento Transformador beta1/metabolismo , Alérgenos/imunologia , Animais , Antígenos de Superfície/metabolismo , Asma/genética , Asma/patologia , Brônquios/química , Colágeno/análise , Proteínas ELAV , Proteína Semelhante a ELAV 1 , Ribonucleoproteína Nuclear Heterogênea D0 , Ribonucleoproteínas Nucleares Heterogêneas Grupo D/metabolismo , Humanos , Camundongos , Camundongos Mutantes , Peptidilprolil Isomerase de Interação com NIMA , Peptidilprolil Isomerase/antagonistas & inibidores , Peptidilprolil Isomerase/genética , Proteína Quinase C-alfa/metabolismo , Proteína Fosfatase 2/metabolismo , Fibrose Pulmonar/genética , Fibrose Pulmonar/patologia , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Ratos , Hipersensibilidade Respiratória/genética , Hipersensibilidade Respiratória/patologia , Fator de Crescimento Transformador beta1/genéticaRESUMO
Glycogen synthase kinase 3 (GSK3) is a proline-directed serine-threonine kinase that is associated with several neurological disorders, including Alzheimer's disease and fragile X syndrome (FXS). We tested the efficacy of a novel GSK3 inhibitor AFC03127, which was developed by Angelini Pharma, in comparison to the metabotropic glutamate receptor 5 inhibitor 2-Methyl-6-(phenylethynyl)pyridine hydrochloride (MPEP) and the GSK3 inhibitor SB216763 in in vivo and in vitro assays in Fmr1 KO mice, a mouse model useful for the study of FXS. The in vivo assay tested susceptibility to audiogenic-induced seizures (AGS) whereas the in vitro assays assessed biomarker expression and dendritic spine length and density in cultured primary neurons as a function of drug dose. MPEP and SB216763 attenuated AGS in Fmr1 KO mice, whereas AFC03127 did not. MPEP and AFC03127 significantly reduced dendritic expression of amyloid-beta protein precursor (APP). All drugs rescued spine length and the ratio of mature dendritic spines. Spine density was not statistically different between vehicle and GSK3 inhibitor-treated cells. The drugs were tested over a wide concentration range in the in vitro assays to determine dose responses. A bell-shaped dose response decrease in APP expression was observed in response to AFC03127, which was more effective than SB216763. These findings confirm previous studies demonstrating differential effects of various GSK3 inhibitors on AGS propensity in Fmr1 KO mice and confirm APP as a downstream biomarker that is responsive to GSK3 activity.
RESUMO
The ketogenic diet is highly effective at attenuating seizures in refractory epilepsy, and accumulating evidence in the literature suggests that it may be beneficial in autism. To our knowledge, no one has studied the ketogenic diet in any fragile X syndrome (FXS) model. FXS is the leading known genetic cause of autism. Herein, we tested the effects of chronic ketogenic diet treatment on seizures, body weight, ketone and glucose levels, diurnal activity levels, learning and memory, and anxiety behaviors in Fmr1KO and littermate control mice as a function of age. The ketogenic diet selectively attenuates seizures in male but not female Fmr1KO mice and differentially affects weight gain and diurnal activity levels dependent on Fmr1 genotype, sex and age.
Assuntos
Dieta Cetogênica , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Animais , Ansiedade/genética , Ansiedade/metabolismo , Modelos Animais de Doenças , Proteína do X Frágil da Deficiência Intelectual/efeitos dos fármacos , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Memória/fisiologia , Camundongos KnockoutRESUMO
The COVID-19 pandemic is a global health crisis that requires the application of interdisciplinary research to address numerous knowledge gaps including molecular strategies to prevent viral reproduction in affected individuals. In response to the Frontiers Research Topic, "Coronavirus disease (COVID-19): Pathophysiology, Epidemiology, Clinical Management, and Public Health Response," this Hypothesis article proposes a novel therapeutic strategy to repurpose metabotropic glutamate 5 receptor (mGluR5) inhibitors to interfere with viral hijacking of the host protein synthesis machinery. We review pertinent background on SARS-CoV-2, fragile X syndrome (FXS) and metabotropic glutamate receptor 5 (mGluR5) and provide a mechanistic-based hypothesis and preliminary data to support testing mGluR5 inhibitors in COVID-19 research.
RESUMO
STUDY OBJECTIVES: Accumulating evidence suggests a strong association between sleep, amyloid-beta (Aß) deposition, and Alzheimer's disease (AD). We sought to determine if (1) deficits in rest-activity rhythms and sleep are significant phenotypes in J20 AD mice, (2) metabotropic glutamate receptor 5 inhibitors (mGluR5) could rescue deficits in rest-activity rhythms and sleep, and (3) Aß levels are responsive to treatment with mGluR5 inhibitors. METHODS: Diurnal rest-activity levels were measured by actigraphy and sleep-wake patterns by electroencephalography, while animals were chronically treated with mGluR5 inhibitors. Behavioral tests were performed, and Aß levels measured in brain lysates. RESULTS: J20 mice exhibited a 4.5-h delay in the acrophase of activity levels compared to wild-type littermates and spent less time in rapid eye movement (REM) sleep during the second half of the light period. J20 mice also exhibited decreased non-rapid eye movement (NREM) delta power but increased NREM sigma power. The mGluR5 inhibitor CTEP rescued the REM sleep deficit and improved NREM delta and sigma power but did not correct rest-activity rhythms. No statistically significant differences were observed in Aß levels, rotarod performance, or the passive avoidance task following chronic mGluR5 inhibitor treatment. CONCLUSIONS: J20 mice have disruptions in rest-activity rhythms and reduced homeostatic sleep pressure (reduced NREM delta power). NREM delta power was increased following treatment with a mGluR5 inhibitor. Drug bioavailability was poor. Further work is necessary to determine if mGluR5 is a viable target for treating sleep phenotypes in AD.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Animais , Ritmo Circadiano , Eletroencefalografia , Camundongos , Sono , Sono REMRESUMO
The use of synaptoneurosomes (SN) enables the detection of synaptic activity including the assessment of glutamate receptor function. SN are normally prepared by filtration and centrifugation methods. Here we review the preparation of SN by Percoll density gradient methodology for downstream applications that assesses glutamate receptor function such as measuring de novo protein synthesis. Major procedural steps include preparation of discontinuous Percoll-sucrose density gradients, collection of brain tissue, preparation of brain homogenates, isolation of synaptoneurosome bands from the discontinuous Percoll-sucrose gradients, and radiolabeling SN proteins. De novo protein synthesis can be reproducibly measured in SN prepared by this method.
Assuntos
Encéfalo/metabolismo , Centrifugação com Gradiente de Concentração/métodos , Neurônios/metabolismo , Biossíntese de Proteínas , Receptores de Glutamato/metabolismo , Sinaptossomos/metabolismo , Animais , Encéfalo/citologia , Camundongos , Neurônios/citologia , Povidona , Dióxido de SilícioRESUMO
Overproduction of amyloid precursor protein (APP) and beta-amyloid likely contribute to neurodegeneration in Alzheimer's disease (AD). In an effort to understand neuronal APP gene regulation, we identified a 52 base element (52sce) immediately downstream from the stop codon that stabilizes APP mRNA. Deletion of this domain drastically destabilized APP mRNAs and reduced APP synthesis in vitro. Chimeric globin-APP mRNAs containing the globin coding sequence fused to the entire APP 3'-UTR, showed regulation similar to full-length APP mRNA. A variety of cytoplasmic lysates contain 52sce RNA binding activity, suggesting cis-trans interactions regulate the element's functionality. Finally, the overexpression of chimeric mRNAs, containing the GFP coding sequence and APP 3'-UTR, dramatically reduced endogenous APP steady-state levels in SH-SY5Y neuroblastoma cells and suggests a novel approach to reduce the amyloid burden in AD patients.
Assuntos
Precursor de Proteína beta-Amiloide/genética , Regulação da Expressão Gênica/fisiologia , Neurônios/fisiologia , RNA Mensageiro/metabolismo , Regiões 3' não Traduzidas/fisiologia , Precursor de Proteína beta-Amiloide/metabolismo , Sequência de Bases , Northern Blotting/métodos , Linhagem Celular , Ensaio de Desvio de Mobilidade Eletroforética/métodos , Proteínas de Fluorescência Verde/biossíntese , Humanos , Modelos Moleculares , Mutagênese/fisiologia , Biossíntese de Proteínas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo , Transcrição Gênica , Transfecção/métodosRESUMO
Amyloid-beta protein precursor (APP) and metabolite levels are altered in fragile X syndrome (FXS) patients and in the mouse model of the disorder, Fmr1KO mice. Normalization of APP levels in Fmr1KO mice (Fmr1KO /APPHET mice) rescues many disease phenotypes. Thus, APP is a potential biomarker as well as therapeutic target for FXS. Hyperexcitability is a key phenotype of FXS. Herein, we determine the effects of APP levels on hyperexcitability in Fmr1KO brain slices. Fmr1KO /APPHET slices exhibit complete rescue of UP states in a neocortical hyperexcitability model and reduced duration of ictal discharges in a CA3 hippocampal model. These data demonstrate that APP plays a pivotal role in maintaining an appropriate balance of excitation and inhibition (E/I) in neural circuits. A model is proposed whereby APP acts as a rheostat in a molecular circuit that modulates hyperexcitability through mGluR5 and FMRP. Both over- and under-expression of APP in the context of the Fmr1KO increases seizure propensity suggesting that an APP rheostat maintains appropriate E/I levels but is overloaded by mGluR5-mediated excitation in the absence of FMRP. These findings are discussed in relation to novel treatment approaches to restore APP homeostasis in FXS.
RESUMO
Seizures are a common phenotype in many neurological disorders including Alzheimer's disease, Down syndrome, and fragile X syndrome. Mouse models of these disorders overexpress amyloid-ß protein precursor (AßPP) and amyloid-ß (Aß) and are highly susceptible to audiogenic-induced seizures (AGS). We observed decreased AGS in these mice fed a casein-based, purified diet (D07030301) as opposed to a standard soy protein-containing, non-purified diet (Purina 5015). Our objective in this manuscript was to determine if soy protein, and in particular soy isoflavones, in the Purina 5015 were contributing to the seizure phenotype. Wild running, AGS, and death rates were assessed in juvenile mice fed Purina 5015, D07030301, D07030301 containing soy protein, or D07030301 supplemented with individual isoflavones (750 mg/kg daidzein or genistein). A short treatment (3 days) with Purina 5015 induced wild running and AGS in Alzheimer's disease mice. A 3-day treatment with daidzein-supplemented diet, but not genistein, induced wild running in wild type mice. To understand the mechanism underlying daidzein activity, we assessed dendritic AßPP expression in primary, cultured, wild type neurons treated with daidzein or genistein. In vitro, daidzein significantly increased dendritic AßPP. Thus, the soy isoflavone daidzein recapitulated seizure induction in vivo and altered AßPP expression in vitro. These results have important implications for individuals on soy-based diets as well as for rodent model research.
Assuntos
Dieta , Doenças do Sistema Nervoso/complicações , Convulsões/etiologia , Proteínas de Soja/efeitos adversos , Estimulação Acústica/efeitos adversos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animais , Células Cultivadas , Córtex Cerebral/citologia , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Proteína do X Frágil da Deficiência Intelectual/genética , Humanos , Locomoção/genética , Masculino , Camundongos , Camundongos Transgênicos , Doenças do Sistema Nervoso/genética , Doenças do Sistema Nervoso/mortalidade , Gravidez , Presenilina-1/genética , Convulsões/dietoterapia , Proteínas de Soja/químicaRESUMO
Synaptoneurosomes (SNs) are obtained after homogenization and fractionation of mouse brain cortex. They are resealed vesicles or isolated terminals that break away from axon terminals when the cortical tissue is homogenized. The SNs retain pre- and postsynaptic characteristics, which makes them useful in the study of synaptic transmission. They retain the molecular machinery used in neuronal signaling and are capable of uptake, storage, and release of neurotransmitters. The production and isolation of active SNs can be problematic using medias like Ficoll, which can be cytotoxic and require extended centrifugation due to high density, and filtration and centrifugation methods, which can result in low activity due to mechanical damage of the SNs. However, the use of discontinuous Percoll-sucrose density gradients to isolate SNs provides a rapid method to produce good yields of translationally active SNs. The Percoll-sucrose gradient method is quick and gentle as it employs isotonic conditions, has fewer and shorter centrifugation spins and avoids centrifugation steps that pellet SNs and cause mechanical damage.
Assuntos
Centrifugação com Gradiente de Concentração/métodos , Córtex Cerebral/citologia , Animais , Córtex Cerebral/química , Camundongos , Neurônios/química , Neurônios/citologia , Povidona/química , Dióxido de Silício/química , Sacarose/química , Transmissão Sináptica , Sinaptossomos/químicaRESUMO
Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading known genetic cause of autism. Fragile X mental retardation protein (FMRP), which is absent or expressed at substantially reduced levels in FXS, binds to and controls the postsynaptic translation of amyloid ß-protein precursor (AßPP) mRNA. Cleavage of AßPP can produce ß-amyloid (Aß), a 39-43 amino acid peptide mis-expressed in Alzheimer's disease (AD) and Down syndrome (DS). Aß is over-expressed in the brain of Fmr1(KO) mice, suggesting a pathogenic role in FXS. To determine if genetic reduction of AßPP/Aß rescues characteristic FXS phenotypes, we assessed audiogenic seizures (AGS), anxiety, the ratio of mature versus immature dendritic spines and metabotropic glutamate receptor (mGluR)-mediated long-term depression (LTD) in Fmr1(KO) mice after removal of one App allele. All of these phenotypes were partially or completely reverted to normal. Plasma Aß(1-42) was significantly reduced in full-mutation FXS males compared to age-matched controls while cortical and hippocampal levels were somewhat increased, suggesting that Aß is sequestered in the brain. Evolving therapies directed at reducing Aß in AD may be applicable to FXS and Aß may serve as a plasma-based biomarker to facilitate disease diagnosis or assess therapeutic efficacy.