RESUMEN
Neural wiring and activity are essential for proper brain function and behavioral outputs and rely on mechanisms that guide the formation, elimination, and remodeling of synapses. During development, it is therefore vital that synaptic densities and architecture are tightly regulated to allow for appropriate neural circuit formation and function. δ-Catenin, a component of the cadherin-catenin cell adhesion complex, has been demonstrated to be a critical regulator of synaptic density and function in the developing central neurons. In this study, we identified forms of δ-catenin that include only the N-terminal (DcatNT) or the C-terminal (DcatCT) regions. We found that these δ-catenin forms are differentially expressed in different regions of the male mouse brain. Our results also indicated that in rat primary cortical culture, these forms are generated in an activity-dependent manner by Ca2+-dependent and calpain-mediated cleavage of δ-catenin or in an activity-independent but lysosome-dependent manner. Functionally, loss of the domain containing the calpain-cleavage sites allowing for generation of DcatCT and DcatNT perturbed the density of a subpopulation of dendritic protrusions in rat hippocampal neurons. This subpopulation likely included protrusions that are either in transition toward becoming mature mushroom spines or in the process of being eliminated. By influencing this subpopulation of spines, proteolytic processing of δ-catenin can likely regulate the balance between mature and immature dendritic protrusions in coordination with neural activity. We conclude that by undergoing cleavage, δ-catenin differentially regulates the densities of subpopulations of dendritic spines and contributes to proper neural circuit wiring in the developing brain.
Asunto(s)
Encéfalo/metabolismo , Cateninas/metabolismo , Espinas Dendríticas/metabolismo , Animales , Calpaína/metabolismo , Células Cultivadas , Hipocampo/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Neuronas/metabolismo , Isoformas de Proteínas/metabolismo , Proteolisis , Ratas , Catenina deltaRESUMEN
Methamphetamine (METH) abuse in conjunction with human immunodeficiency virus (HIV) exacerbates neuropathogenesis and accelerates neurocognitive impairments in the central nervous system (CNS), collectively termed HIV Associated Neurocognitive Disorders (HAND). Since both HIV and METH have been implicated in altering the synaptic architecture, this study focused on investigating alterations in synaptic proteins. Employing a quantitative proteomics approach on synaptosomes isolated from the caudate nucleus from two groups of rhesus monkeys chronically infected with simian immunodeficiency virus (SIV) differing by one regimen, METH treatment, we identified the neuron specific Na(+)/K(+)-ATPase alpha 1 isoform 3 (ATP1A3) to be up regulated after METH treatment, and validated its up regulation by METH in vitro. Further studies on signaling mechanisms revealed that the activation of ATP1A3 involves the extracellular regulated kinase (ERK) pathway. Given its function in maintaining ionic gradients and emerging role as a signaling molecule, changes in ATP1A3 yields insights into the mechanisms associated with HAND and interactions with drugs of abuse.
Asunto(s)
Trastornos del Conocimiento/etiología , Trastornos del Conocimiento/metabolismo , Citocinas/farmacología , Infecciones por VIH/complicaciones , Metanfetamina/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Trastornos Relacionados con Sustancias/complicaciones , Animales , Supervivencia Celular/efectos de los fármacos , Trastornos del Conocimiento/tratamiento farmacológico , Activación Enzimática/efectos de los fármacos , Humanos , Macaca mulatta , Metanfetamina/uso terapéutico , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Ratas , Ratas Sprague-Dawley , Síndrome de Inmunodeficiencia Adquirida del Simio/complicacionesRESUMEN
OTK18 is a C2H2 type zinc finger protein involved in the regulation of HIV-1 replication in human mononuclear phagocytes. Previously, we reported OTK18 expression in brain perivascular macrophages but not in microglia in HIV encephalitis brain. We have cloned the OTK18 promoter region proximal to the transcriptional start site and determined the region responsible (-884/+1) for the basal transcriptional activity in a microglia cell line. Sequential deletion mutation analyses reveal three important response elements: Yingyang-1 (YY1; -805/-777), an HIV-1 response element for promoter activation; FoxD3 (-743/-725), a negative regulatory element; and Ets response element (-725/-707), a basal transcriptional activity response element. HIV-1 infection-induced upregulation of YY1 and c-Ets-1 protein, binding to the promoter region as determined by immunoblotting and chromatin immunoprecipitation and polymerase chain reaction (PCR) assays, and induction of YY1 was also observed in virus-infected monocyte-derived macrophages. Silencing of FoxD3 and YY1 in the cell line by small interfering RNA duplexes specific to these molecules significantly up- and downregulated basal OTK18 promoter activity in FoxD3 and YY1 response element-dependent manners, respectively. On the other hand, infection of primary cultured human microglia significantly reduced YY1 expression and induced FoxD3 as determined by immunoblotting and reverse transcription real-time PCR. These data suggest that HIV-1 induces OTK18 expression through a YY1-mediated manner in human macrophages, although its gene expression is suppressed by FoxD3 upregulation and YY1 downregulation in human microglia. This mechanism may explain the perivascular macrophage-specific expression of OTK18 in HIV encephalitis brains.
Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/genética , Factores de Transcripción Forkhead/fisiología , Infecciones por VIH/metabolismo , VIH-1 , Regiones Promotoras Genéticas/genética , Regiones Promotoras Genéticas/fisiología , Factores de Transcripción/biosíntesis , Factores de Transcripción/genética , Factor de Transcripción YY1/fisiología , Región de Flanqueo 5'/genética , Western Blotting , Inmunoprecipitación de Cromatina , Regulación de la Expresión Génica , Humanos , Luciferasas/genética , Macrófagos/metabolismo , Microglía/metabolismo , Proteína Proto-Oncogénica c-ets-1/fisiología , ARN Interferente Pequeño/genética , Elementos Reguladores de la Transcripción/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , TransfecciónRESUMEN
OTK18 is a C2H2 type zinc finger protein expressed by human macrophages following HIV infection. OTK18 possesses antiretroviral activity, and its processing products accumulate in the cytoplasm of perivascular brain macrophages in advanced HIV encephalitis cases. Since the regulation of OTK18 expression in living patients following human immunodeficiency virus-1 (HIV-1) infection is unknown, our objective is to investigate the first cohort study on OTK18 protein levels in living patients. We assessed OTK18 levels in plasma and cerebrospinal fluid (CSF) in 44 living patients with or without HIV-1 infection, with diverse demographic and clinical background. A novel high-sensitivity OTK18 ELISA system was developed to measure OTK18 levels in CSF and plasma using custom made biotinylated monoclonal antibodies against OTK18. The correlation of OTK18 levels with epidemiological parameters was statistically analyzed. Multiple linear regression modeling suggested that plasma OTK18 levels for HIV-1-positive subjects were only about one sixth of that for HIV-1-negative subjects. Higher CD8 T-cell counts were associated with higher levels of OTK18. Using proportional odds logistic regression, we showed that HIV-1-positive patients have significantly lower OTK18 in CSF samples, but we did not observe significant correlation between CD8 T-cell counts and CSF OTK18 levels. OTK18 levels in both plasma and CSF are significantly lower in HIV-1-positive subjects as compared to HIV-1-negative subjects. Plasma OTK18 levels are positively correlated to CD8 T-cell counts, independent of HIV-1 status.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/inmunología , Síndrome de Inmunodeficiencia Adquirida/metabolismo , Linfocitos T CD8-positivos/inmunología , Proteínas de Unión al ADN/análisis , Factores de Transcripción/análisis , Carga Viral , Adulto , Estudios de Cohortes , Ensayo de Inmunoadsorción Enzimática , Femenino , VIH-1/inmunología , Humanos , Recuento de Linfocitos , Masculino , Persona de Mediana EdadRESUMEN
Chronic administration of anorexigenic substances to experimental animals by injections or continuous infusion typically produces either no effect or a transient reduction in food intake and body weight. Our aim here was to identify an intermittent dosing strategy for intraperitoneal infusion of peptide YY(3-36) [PYY(3-36)] that produces a sustained reduction in daily food intake and adiposity in diet-induced obese rats. Rats (665+/-10 g body wt, 166+/-7 g body fat) with intraperitoneal catheters tethered to infusion swivels had free access to a high-fat diet. Vehicle-treated rats (n=23) had relatively stable food intake, body weight, and adiposity during the 9-wk test period. None of 15 PYY(3-36) dosing regimens administered in succession to a second group of rats (n=22) produced a sustained 15-25% reduction in daily food intake for >5 days, although body weight and adiposity were reduced across the 9-wk period by 12% (594+/-15 vs. 672+/-15 g) and 43% (96+/-7 vs. 169+/-9 g), respectively. The declining inhibitory effect of PYY(3-36) on daily food intake when the interinfusion interval was >or=3 h appeared to be due in part to an increase in food intake between infusions. The declining inhibitory effect of PYY(3-36) on daily food intake when the interinfusion interval was <3 h suggested possible receptor downregulation and tolerance to frequent PYY(3-36) administration; however, food intake significantly increased when PYY(3-36) treatments were discontinued for 1 day following apparent loss in treatment efficacies. Together, these results demonstrate the development of a potent homeostatic response to increase food intake when PYY(3-36) reduces food intake and energy reserves in diet-induced obese rats.
Asunto(s)
Adiposidad/efectos de los fármacos , Depresores del Apetito/administración & dosificación , Conducta Animal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Ingestión de Alimentos/efectos de los fármacos , Obesidad/tratamiento farmacológico , Péptido YY/administración & dosificación , Animales , Grasas de la Dieta , Modelos Animales de Enfermedad , Esquema de Medicación , Homeostasis , Infusiones Parenterales , Masculino , Obesidad/etiología , Obesidad/fisiopatología , Fragmentos de Péptidos , Ratas , Ratas Sprague-Dawley , Factores de TiempoRESUMEN
Reactive astrocytes and microglia in Alzheimer's disease surround amyloid plaques and secrete proinflammatory cytokines that affect neuronal function. Relationship between cytokine signaling and amyloid-beta peptide (Abeta) accumulation is poorly understood. Thus, we generated a novel Swedish beta-amyloid precursor protein mutant (APP) transgenic mouse in which the interferon (IFN)-gamma receptor type I was knocked out (APP/GRKO). IFN-gamma signaling loss in the APP/GRKO mice reduced gliosis and amyloid plaques at 14 months of age. Aggregated Abeta induced IFN-gamma production from co-culture of astrocytes and microglia, and IFN-gamma elicited tumor necrosis factor (TNF)-alpha secretion in wild type (WT) but not GRKO microglia co-cultured with astrocytes. Both IFN-gamma and TNF-alpha enhanced Abeta production from APP-expressing astrocytes and cortical neurons. TNF-alpha directly stimulated beta-site APP-cleaving enzyme (BACE1) expression and enhanced beta-processing of APP in astrocytes. The numbers of reactive astrocytes expressing BACE1 were increased in APP compared with APP/GRKO mice in both cortex and hippocampus. IFN-gamma and TNF-alpha activation of WT microglia suppressed Abeta degradation, whereas GRKO microglia had no changes. These results support the idea that glial IFN-gamma and TNF-alpha enhance Abeta deposition through BACE1 expression and suppression of Abeta clearance. Taken together, these observations suggest that proinflammatory cytokines are directly linked to Alzheimer's disease pathogenesis.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Péptidos beta-Amiloides/biosíntesis , Ácido Aspártico Endopeptidasas/biosíntesis , Interferón gamma/metabolismo , Placa Amiloide/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Animales , Astrocitos/metabolismo , Astrocitos/patología , Células Cultivadas , Corteza Cerebelosa/metabolismo , Corteza Cerebelosa/patología , Técnicas de Cocultivo , Activación Enzimática , Regulación Enzimológica de la Expresión Génica , Gliosis/genética , Gliosis/metabolismo , Gliosis/patología , Humanos , Ratones , Ratones Noqueados , Microglía/metabolismo , Microglía/patología , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/patología , Receptores de Interferón/deficiencia , Receptores de Interferón/metabolismo , Transducción de Señal/genética , Receptor de Interferón gammaRESUMEN
It has previously been shown by our laboratory that OTK18, a human immunodeficiency virus (HIV)-inducible zinc-finger protein, reduces progeny-virion production in infected human macrophages. OTK18 antiviral activity is mediated through suppression of Tat-induced HIV-1 long terminal repeat (LTR) promoter activity. Through the use of LTR-scanning mutant vectors, the specific regions responsible for OTK18-mediated LTR suppression have been defined. Two different LTR regions were identified as potential OTK18-binding sites by an enhanced DNA-transcription factor ELISA system; the negative-regulatory element (NRE) at -255/-238 and the Ets-binding site (EBS) at -150/-139 in the LTR. In addition, deletion of the EBS in the LTR blocked OTK18-mediated LTR suppression. These data indicate that OTK18 suppresses LTR activity through two distinct regulatory elements. Spontaneous mutations in these regions might enable HIV-1 to escape from OTK18 antiretroviral activity in human macrophages.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación Viral de la Expresión Génica/fisiología , Duplicado del Terminal Largo de VIH/fisiología , VIH-1/fisiología , Factores de Transcripción/fisiología , ADN Viral/genética , Proteínas de Unión al ADN/metabolismo , VIH-1/genética , VIH-1/inmunología , Humanos , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
Neurofibrillary tangles, which are major pathological hallmarks of Alzheimer's disease (AD), are composed of paired helical filaments (PHFs) containing hyperphosphorylated tau. Specific kinases regulate tau phosphorylation and are closely linked to the pathogenesis of AD. We have characterized a human tau-tubulin kinase 1 (TTBK1) gene located on chromosome 6p21.1. TTBK1 is a serine/threonine/tyrosine kinase that is conserved among species and belongs to the casein kinase 1 superfamily. It is specifically expressed in the brain, especially in the cytoplasm of cortical and hippocampal neurons. TTBK1 phosphorylates tau proteins in both a Mg2+- and a Mn2+-dependent manner. Phosphopeptide mapping and immunoblotting analysis confirmed a direct tau phosphorylation by TTBK1 at Ser198, Ser199, Ser202 and Ser422, which are also phosphorylated in PHFs. TTBK1 also induces tau aggregation in human neuronal cells in a dose-dependent manner. We conclude that TTBK1 is a neuron-specific dual kinase involved in tau phosphorylation at AD-related sites and is also associated with tau aggregation.
Asunto(s)
Proteínas Serina-Treonina Quinasas/fisiología , Proteínas tau/metabolismo , Animales , Línea Celular , Chlorocebus aethiops , Biología Computacional/métodos , Técnica del Anticuerpo Fluorescente/métodos , Expresión Génica/fisiología , Humanos , Inmunoprecipitación/métodos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Neuroblastoma , Fosforilación , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Alineación de Secuencia/métodos , Serina/metabolismo , Treonina/metabolismo , Distribución Tisular , Transfección/métodosRESUMEN
Microglia accumulation at the site of amyloid plaques is a strong indication that microglia play a major role in Alzheimer's disease pathogenesis. However, how microglia affect amyloid-beta peptide (Abeta) deposition remains poorly understood. To address this question, we developed a novel bigenic mouse that overexpresses both amyloid precursor protein (APP) and monocyte chemotactic protein-1 (MCP-1; CCL2 in systematic nomenclature). CCL2 expression, driven by the glial fibrillary acidic protein promoter, induced mononuclear phagocyte (MP; monocyte-derived macrophage and microglial) accumulation in the brain. When APP/CCL2 transgenic mice were compared to APP mice, a fivefold increase in Abeta deposition was present despite increased MP accumulation around hippocampal and cortical amyloid plaques. Levels of full-length APP, its C-terminal fragment, and Abeta-degrading enzymes (insulin-degrading enzyme and neprilysin) in APP/CCL2 and APP mice were indistinguishable. Sodium dodecyl sulfate-insoluble Abeta (an indicator of fibrillar Abeta) was increased in APP/CCL2 mice at 5 months of age. Apolipoprotein E, which enhances Abeta deposition, was also increased (2.2-fold) in aged APP/CCL2 as compared to APP mice. We propose that although CCL2 stimulates MP accumulation, it increases Abeta deposition by reducing Abeta clearance through increased apolipoprotein E expression. Understanding the mechanisms underlying these events could be used to modulate microglial function in Alzheimer's disease and positively affect disease outcomes.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Quimiocina CCL2/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/citología , Corteza Cerebral/metabolismo , Quimiocina CCL2/genética , Femenino , Hipocampo/metabolismo , Masculino , Ratones , Ratones Transgénicos/genética , Fagocitos/citología , Distribución TisularRESUMEN
Increasing evidence strongly supports the role of glial immunity in the pathogenesis of Alzheimer's disease (AD). To investigate such events we have developed cell systems mimicking the interactions between beta-amyloid precursor protein (APP)-expressing neurons and brain mononuclear phagocytes (MP; macrophages and microglia). MP were co-cultured with neuronal cells expressing wild type APP or familial AD-linked APP mutants. The latter was derived from recombinant adenoviral constructs. Neuronal APP processing products induced MP activation, reactive oxygen species, and neurotoxic activities. These occurred without the addition of pro-inflammatory cytokines and were reversed by depletion of amyloid beta-peptide (Abeta) and secreted APP (sAPP). Neurotoxic activities were diminished by superoxide dismutase mimetics and NMDA receptor inhibitors. Microglial glutamate secretion was suppressed by the cystine-glutamate antiporter inhibitor and its levels paralleled the depletion of sAPP and Abeta from conditioned media prepared from APP-expressing neurons. The excitotoxins from activated MP were potent enough to evoke recombinant NMDA receptor-mediated inward currents expressed in vitro in the Xenopus oocytes. These results demonstrate that neuronal APP-processing products can induce oxidative neurotoxicity through microglial activation.