RESUMEN
Neurotrophins, essential regulators of many aspects of neuronal differentiation and function, signal via four receptors, p75, TrkA, TrkB and TrkC. The three Trk paralogs are members of the LIG superfamily of membrane proteins, which share extracellular domains consisting of leucine-rich repeat and C2 Ig domains. Another LIG protein, LINGO-1 has been reported to bind and influence signaling of p75 as well as TrkA, TrkB and TrkC. Here we examine the manner in which LINGO-1 influences the function of TrkA, TrkB and TrkC. We report that Trk activation promotes Trk association with LINGO-1, and that this association promotes Trk degradation by a lysosomal mechanism. This mechanism resembles the mechanism by which another LIG protein, LRIG1, promotes lysosomal degradation of receptor tyrosine kinases such as the EGF receptor. We present evidence indicating that the Trk/LINGO-1 interaction occurs, in part, within recycling endosomes. We show that a mutant form of LINGO-1, with much of the extracellular domain deleted, has the capacity to enhance TrkA signaling in PC12 cells, possibly by acting as an inhibitor of Trk down-regulation by full length LINGO-1. We propose that LINGO-1 functions as a negative feedback regulator of signaling by cognate receptor tyrosine kinases including TrkA, TrkB and TrkC.
Asunto(s)
Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptor trkA/metabolismo , Receptor trkB/metabolismo , Receptor trkC/metabolismo , Transducción de Señal/genética , Animales , Citoplasma/metabolismo , Regulación hacia Abajo , Endosomas/metabolismo , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Células PC12 , Fosforilación , RatasRESUMEN
Axon outgrowth inhibition in response to trauma is thought to be mediated via the binding of myelin-associated inhibitory factors (e.g. Nogo-66, myelin-associated glycoprotein, oligodendrocyte myelin glycoprotein, and myelin basic protein) to a putative tripartite LINGO-1·p75(NTR)·Nogo-66 receptor (NgR) complex at the cell surface. We found that endogenous LINGO-1 expression in neurons in the cortex and cerebellum is intracellular. Mutation or truncation of the highly conserved LINGO-1 C terminus altered this intracellular localization, causing poor intracellular retention and increased plasma membrane expression. p75(NTR) associated predominantly with natively expressed LINGO-1 containing immature N-glycans, characteristic of protein that has not completed trans-Golgi-mediated processing, whereas mutant forms of LINGO-1 with enhanced plasma membrane expression did not associate with p75(NTR). Co-immunoprecipitation experiments demonstrated that LINGO-1 and NgR competed for binding to p75(NTR) in a manner that is difficult to reconcile with the existence of a LINGO-1·p75(NTR)·NgR ternary complex. These findings contradict models postulating functional LINGO-1·p75(NTR)·NgR complexes in the plasma membrane.
Asunto(s)
Membranas Intracelulares/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores de Factor de Crecimiento Nervioso/metabolismo , Animales , Animales Recién Nacidos , Unión Competitiva , Encéfalo/citología , Encéfalo/metabolismo , Membrana Celular/metabolismo , Proteínas Ligadas a GPI/genética , Proteínas Ligadas a GPI/metabolismo , Células HEK293 , Humanos , Immunoblotting , Inmunoprecipitación , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Microscopía Confocal , Mutación , Proteínas de la Mielina/genética , Proteínas de la Mielina/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Receptor Nogo 1 , Polisacáridos/metabolismo , Unión Proteica , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismo , Receptores de Factor de Crecimiento Nervioso/genéticaRESUMEN
INTRODUCTION: Alzheimer's disease (AD) is the predominant dementia globally, with heterogeneous presentation and penetrance of clinical symptoms, variable presence of mixed pathologies, potential disease subtypes, and numerous associated endophenotypes. Beyond the difficulty of designing treatments that address the core pathological characteristics of the disease, therapeutic development is challenged by the uncertainty of which endophenotypic areas and specific targets implicated by those endophenotypes to prioritize for further translational research. However, publicly funded consortia driving large-scale open science efforts have produced multiple omic analyses that address both disease risk relevance and biological process involvement of genes across the genome. METHODS: Here we report the development of an informatic pipeline that draws from genetic association studies, predicted variant impact, and linkage with dementia associated phenotypes to create a genetic risk score. This is paired with a multi-omic risk score utilizing extensive sets of both transcriptomic and proteomic studies to identify system-level changes in expression associated with AD. These two elements combined constitute our target risk score that ranks AD risk genome-wide. The ranked genes are organized into endophenotypic space through the development of 19 biological domains associated with AD in the described genetics and genomics studies and accompanying literature. The biological domains are constructed from exhaustive Gene Ontology (GO) term compilations, allowing automated assignment of genes into objectively defined disease-associated biology. This rank-and-organize approach, performed genome-wide, allows the characterization of aggregations of AD risk across biological domains. RESULTS: The top AD-risk-associated biological domains are Synapse, Immune Response, Lipid Metabolism, Mitochondrial Metabolism, Structural Stabilization, and Proteostasis, with slightly lower levels of risk enrichment present within the other 13 biological domains. DISCUSSION: This provides an objective methodology to localize risk within specific biological endophenotypes and drill down into the most significantly associated sets of GO terms and annotated genes for potential therapeutic targets.
RESUMEN
RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM, however, these compounds lack selectivity and this limits their utility as chemical tools.
Asunto(s)
Proteínas Tirosina Quinasas , Dominios Homologos src , Humanos , Proteínas Tirosina Quinasas/metabolismo , Motivo de Activación del Inmunorreceptor Basado en Tirosina , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Quinasa Syk/metabolismo , Fosforilación , Receptores Fc/metabolismo , Precursores Enzimáticos/metabolismoRESUMEN
RNA sequencing and genetic data support spleen tyrosine kinase (SYK) and high affinity immunoglobulin epsilon receptor subunit gamma (FCER1G) as putative targets to be modulated for Alzheimer's disease (AD) therapy. FCER1G is a component of Fc receptor complexes that contain an immunoreceptor tyrosine-based activation motif (ITAM). SYK interacts with the Fc receptor by binding to doubly phosphorylated ITAM (p-ITAM) via its two tandem SH2 domains (SYK-tSH2). Interaction of the FCER1G p-ITAM with SYK-tSH2 enables SYK activation via phosphorylation. Since SYK activation is reported to exacerbate AD pathology, we hypothesized that disruption of this interaction would be beneficial for AD patients. Herein, we developed biochemical and biophysical assays to enable the discovery of small molecules that perturb the interaction between the FCER1G p-ITAM and SYK-tSH2. We identified two distinct chemotypes using a high-throughput screen (HTS) and orthogonally assessed their binding. Both chemotypes covalently modify SYK-tSH2 and inhibit its interaction with FCER1G p-ITAM.
RESUMEN
BACKGROUND: Alzheimer's disease (AD) is an incurable neurodegenerative disease currently affecting 1.75% of the US population, with projected growth to 3.46% by 2050. Identifying common genetic variants driving differences in transcript expression that confer AD risk is necessary to elucidate AD mechanism and develop therapeutic interventions. We modify the FUSION transcriptome-wide association study (TWAS) pipeline to ingest gene expression values from multiple neocortical regions. METHODS: A combined dataset of 2003 genotypes clustered to 1000 Genomes individuals from Utah with Northern and Western European ancestry (CEU) was used to construct a training set of 790 genotypes paired to 888 RNASeq profiles from temporal cortex (TCX = 248), prefrontal cortex (FP = 50), inferior frontal gyrus (IFG = 41), superior temporal gyrus (STG = 34), parahippocampal cortex (PHG = 34), and dorsolateral prefrontal cortex (DLPFC = 461). Following within-tissue normalization and covariate adjustment, predictive weights to impute expression components based on a gene's surrounding cis-variants were trained. The FUSION pipeline was modified to support input of pre-scaled expression values and support cross validation with a repeated measure design arising from the presence of multiple transcriptome samples from the same individual across different tissues. RESULTS: Cis-variant architecture alone was informative to train weights and impute expression for 6780 (49.67%) autosomal genes, the majority of which significantly correlated with gene expression; FDR < 5%: N = 6775 (99.92%), Bonferroni: N = 6716 (99.06%). Validation of weights in 515 matched genotype to RNASeq profiles from the CommonMind Consortium (CMC) was (72.14%) in DLPFC profiles. Association of imputed expression components from all 2003 genotype profiles yielded 8 genes significantly associated with AD (FDR < 0.05): APOC1, EED, CD2AP, CEACAM19, CLPTM1, MTCH2, TREM2, and KNOP1. CONCLUSIONS: We provide evidence of cis-genetic variation conferring AD risk through 8 genes across six distinct genomic loci. Moreover, we provide expression weights for 6780 genes as a valuable resource to the community, which can be abstracted across the neocortex and a wide range of neuronal phenotypes.
Asunto(s)
Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Neocórtex/metabolismo , Sitios de Carácter Cuantitativo , Transcriptoma , Biología Computacional/métodos , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo/métodos , Humanos , Especificidad de Órganos/genéticaRESUMEN
The diagnosis of Parkinson's disease (PD) currently relies almost exclusively on the clinical judgment of an experienced neurologist, ideally a specialist in movement disorders. However, such clinical diagnosis is often incorrect in a large percentage of patients, particularly in the early stages of the disease. A commercially available, objective and quantitative marker of nigrostriatal neurodegeneration was recently provided by 123-iodine 123I-ioflupane SPECT imaging, which is however unable to differentiate PD from a variety of other parkinsonian syndromes associated with striatal dopamine deficiency. There is evidence to support an algorithm utilizing a dual neuroimaging strategy combining 123I-ioflupane SPECT and the noradrenergic receptor ligand 123I-metaiodobenzylguanidine (MIBG), which assesses the post-ganglion peripheral autonomic nervous system. Evolving concepts regarding the synucleinopathy affecting the central and peripheral autonomic nervous systems as part of a multisystem disease are reviewed to sustain such strategy. Data are presented to show how MIBG deficits are a common feature of multisystem Lewy body disease and can be used as a unique feature to distinguish PD from atypical parkinsonisms. We propose that the combination of cardiac (MIBG) and cerebral 123I-ioflupane SPECT could satisfy one of the most significant unmet needs of current PD diagnosis and management, namely the early and accurate diagnosis of patients with typical Lewy body PD. Exemplary case scenarios will be described, highlighting how dual neuroimaging strategy can maximize diagnostic accuracy for patient care, clinical trials, pre-symptomatic PD screening, and special cases provided by specific genetic mutations associated with PD.
RESUMEN
The Comparative Mouse Genomics Centers Consortium (CMGCC) is a branch of the Environmental Genome Project sponsored by the National Institute of Environmental Health Sciences (NIEHS) focusing upon the identification of human single nucleotide polymorphisms (SNPs) that may confer disease susceptibility within the human population. The goal of the CMGCC (http://www.niehs.nih.gov/cmgcc/) is to make genetic mouse models for human SNPs within cell cycle control, DNA replication and DNA repair genes that may be associated with human pathologies. In order to facilitate information sharing and analysis within the consortium a set of informatics resources have been generated to support the mouse model development efforts. The primary entry point for information about the mouse models developed by the consortium is through the CMGCC Genotype Database (http://mrages.niehs.nih.gov/genotype/), which maintains both a consortium specific and public access display of the available and developing mouse models.
Asunto(s)
Bases de Datos Genéticas , Genómica , Ratones/genética , Animales , Biología Computacional , Cruzamientos Genéticos , Femenino , Genotipo , Haplotipos/genética , Masculino , Modelos Animales , Polimorfismo Genético/genéticaRESUMEN
The 75 kDa neurotrophin receptor (p75NTR) and two neurotrophin receptor homologs (NRH1, NRH2) constitute a subfamily of the nerve growth factor/tumor necrosis factor receptor superfamily. NRH1 coexists with p75NTR in fish, amphibians, and birds but is absent in mammals, whereas NRH2 exists only in mammals. Unlike p75NTR and NRH1, NRH2 lacks a canonical extracellular ligand binding domain. The similarity of NRH2 to the product of metalloproteinase cleavage of p75NTR prompted us to examine the cleavage of p75NTR in greater detail. p75NTR, NRH1, and NRH2 undergo multiple proteolytic cleavages that ultimately release cytoplasmic fragments. For p75NTR, cleavage in the extracellular domain by a PMA-inducible membrane metalloproteinase is followed by cleavage within or near the transmembrane domain, releasing the intracellular domain into the cytoplasm. This processing resembles the alpha- and gamma-secretase-mediated processing of beta-amyloid precursor protein and the similar processing of Notch. Although neurotrophins did not regulate p75NTR processing, the alpha- and gamma-secretase-mediated cleavage of p75 is modulated by receptor tyrosine kinases (Trks) TrkA and TrkB but not TrkC. Surprisingly, although NRH1 and NRH2 also undergo proteolytic cytoplasmic release of intracellular domains, a different protease mediates the cleavage. Furthermore, whereas the p75NTR soluble intracellular domain accumulates only in the presence of proteasome inhibitors, the equivalent fragment of NRH2 is stable and localizes in the nucleus. Because soluble intracellular domains of p75NTR and NRH2 were found to activate NF-kappaB in concert with TNF receptor associated factor 6 (TRAF6), we propose that cleavage of these proteins may serve conserved cytoplasmic and nuclear signaling functions through distinct proteases.
Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de la Membrana/metabolismo , Fragmentos de Péptidos/fisiología , Procesamiento Proteico-Postraduccional/fisiología , Receptores de Factor de Crecimiento Nervioso/metabolismo , Transducción de Señal/fisiología , Secretasas de la Proteína Precursora del Amiloide , Animales , Proteínas Reguladoras de la Apoptosis , Ácido Aspártico Endopeptidasas , Proteínas Portadoras/genética , Línea Celular , Ecdisterona/análogos & derivados , Ecdisterona/farmacología , Endopeptidasas/metabolismo , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Humanos , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Fragmentos de Péptidos/biosíntesis , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Estructura Terciaria de Proteína/fisiología , Proteínas/metabolismo , Ratas , Receptor de Factor de Crecimiento Nervioso , Receptores de Factor de Crecimiento Nervioso/genética , Homología de Secuencia de Aminoácido , Factor 6 Asociado a Receptor de TNF , XenopusRESUMEN
Sequential proteolytic cleavages of amyloid-ß protein precursor (AßPP) by ß-secretase and γ-secretase generate amyloid ß (Aß) peptides, which are thought to contribute to Alzheimer's disease (AD). Much of this processing occurs in endosomes following endocytosis of AßPP from the plasma membrane. However, this pathogenic mode of processing AßPP may occur in competition with lysosomal degradation of AßPP, a common fate of membrane proteins trafficking through the endosomal system. Following up on published reports that LINGO-1 binds and promotes the amyloidogenic processing of AßPP we have examined the consequences of LINGO-1/AßPP interactions. We report that LINGO-1 and its paralogs, LINGO-2 and LINGO-3, decrease processing of AßPP in the amyloidogenic pathway by promoting lysosomal degradation of AßPP. We also report that LINGO-1 levels are reduced in AD brain, representing a possible pathogenic mechanism stimulating the generation of Aß peptides in AD.
RESUMEN
The length of time required for preinvasive adenoma to progress to carcinoma, the immunogenicity of colorectal cancer (CRC), and the identification of high-risk populations make development and testing of a prophylactic vaccine for the prevention of CRC possible. We hypothesized that genes upregulated in adenoma relative to normal tissue, which maintained increased expression in CRC, would encode proteins suitable as putative targets for immunoprevention. We evaluated existing adenoma and CRC microarray datasets and identified 160 genes that were ≥2-fold upregulated in both adenoma and CRC relative to normal colon tissue. We further identified 23 genes that showed protein overexpression in colon adenoma and CRC based on literature review. Silencing the most highly upregulated genes, CDH3, CLDN1, KRT23, and MMP7, in adenoma and CRC cell lines resulted in a significant decrease in viability (P < 0.0001) and proliferation (P < 0.0001) as compared to controls and an increase in cellular apoptosis (P < 0.05 for CDH3, KRT23). Results were duplicated across cell lines representing microsatellite instability, CpG island methylator, and chromosomal instability phenotypes, suggesting immunologic elimination of cells expressing these proteins could impact the progression of all CRC phenotypes. To determine whether these proteins were immunogens, we interrogated sera from early stage CRC patients and controls and found significantly elevated CDH3 (P = 0.006), KRT23 (P = 0.0007), and MMP7 (P < 0.0001) serum immunoglobulin G in cases as compared to controls. These data show a high throughput approach to the identification of biologically relevant putative immunologic targets for CRC and identified three candidates suitable for vaccine development.
Asunto(s)
Adenoma/diagnóstico , Biomarcadores de Tumor/metabolismo , Neoplasias Colorrectales/diagnóstico , Regulación Neoplásica de la Expresión Génica , Lesiones Precancerosas/diagnóstico , Adenoma/metabolismo , Adenoma/prevención & control , Adulto , Anciano , Anciano de 80 o más Años , Apoptosis , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Western Blotting , Cadherinas/antagonistas & inhibidores , Cadherinas/genética , Cadherinas/metabolismo , Estudios de Casos y Controles , Proliferación Celular , Claudina-1/antagonistas & inhibidores , Claudina-1/genética , Claudina-1/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/prevención & control , Metilación de ADN , Ensayo de Inmunoadsorción Enzimática , Femenino , Humanos , Queratinas Tipo I/antagonistas & inhibidores , Queratinas Tipo I/genética , Queratinas Tipo I/metabolismo , Masculino , Metaloproteinasa 7 de la Matriz/química , Metaloproteinasa 7 de la Matriz/genética , Metaloproteinasa 7 de la Matriz/metabolismo , Inestabilidad de Microsatélites , Persona de Mediana Edad , Estadificación de Neoplasias , Lesiones Precancerosas/metabolismo , Lesiones Precancerosas/prevención & control , Pronóstico , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Adulto JovenRESUMEN
There is currently no treatment and cure for age-related dementia and cognitive impairment in humans. Mice suffer from age-related cognitive decline just as people do, but assessment is challenging because of cumbersome and at times stressful performance tasks. We developed a novel radial water tread (RWT) maze and tested male C57BL/6 (B6) and C57BL/6 x Balb/c F1 (CB6F1) mice at ages 4, 12, 20, and 28 months. B6 mice showed a consistent learning experience and memory retention that gradually decreased with age. CB6F1 mice showed a moderate learning experience in the 4 and 12 month groups, which was not evident in the 20 and 28 month groups. In conclusion, CB6F1 mice showed more severe age-related cognitive impairment compared to B6 mice and might be a suitable model for intervention studies. In addition, the RWT maze has a number of operational advantages compared to currently accepted tasks and can be used to assess age-related cognition impairment in B6 and CB6F1 mice as early as 12 months of age.
RESUMEN
Trafficking through the secretory pathway is known to regulate the maturation of the APP-cleaving secretases and APP proteolysis. The coupling of stress signaling and pathological deterioration of the brain in Alzheimer's disease (AD) supports a mechanistic connection between endoplasmic reticulum (ER) stress and neurodegeneration. Consequently, small molecular chaperones, which promote protein folding and minimize ER stress, might be effective in delaying or attenuating the deleterious progression of AD. We tested this hypothesis by treating APPswePS1delta9 AD transgenic mice with the molecular chaperone phenylbutyric acid (PBA) for 14 months at a dose of 1 mg PBA g(-1) of body weight in the drinking water. Phenylbutyric acid treatment increased secretase-mediated APP cleavage, but was not associated with any increase in amyloid biosynthesis. The PBA-treated AD transgenic mice had significantly decreased incidence and size of amyloid plaques throughout the cortex and hippocampus. There was no change in total amyloid levels suggesting that PBA modifies amyloid aggregation or pathogenesis independently of biogenesis. The decrease in amyloid plaques was paralleled by increased memory retention, as PBA treatment facilitated cognitive performance in a spatial memory task in both wild-type and AD transgenic mice. The molecular mechanism underlying the cognitive facilitation of PBA is not clear; however, increased levels of both metabotropic and ionotropic glutamate receptors, as well as ADAM10 and TACE, were observed in the cortex and hippocampus of PBA-treated mice. The data suggest that PBA ameliorates the cognitive and pathological features of AD and supports the investigation of PBA as a therapeutic for AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Hipocampo/metabolismo , Memoria/efectos de los fármacos , Neuronas/metabolismo , Nootrópicos , Fenilbutiratos , Placa Amiloide/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Proteína ADAM17 , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Cognición/efectos de los fármacos , Modelos Animales de Enfermedad , Retículo Endoplásmico/metabolismo , Hipocampo/efectos de los fármacos , Hipocampo/patología , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/patología , Nootrópicos/administración & dosificación , Nootrópicos/uso terapéutico , Fenilbutiratos/administración & dosificación , Fenilbutiratos/uso terapéutico , Placa Amiloide/patología , Receptores Ionotrópicos de Glutamato/genética , Receptores Ionotrópicos de Glutamato/metabolismo , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismoRESUMEN
PURPOSE: The ratio of human epidermal growth factor receptor 2 (HER2) to CEP17 by fluorescent in situ hybridization (FISH) with the centromeric probe CEP17 is used to determine HER2 gene status in breast cancer. Increases in CEP17 copy number have been interpreted as representing polysomy 17. However, pangenomic studies have demonstrated that polysomy 17 is rare. This study tests the hypothesis that the use of alternative chromosome 17 reference genes might more accurately assess true HER2 gene status. PATIENTS AND METHODS: In all, 171 patients with breast cancer who had HER2 FISH that had increased mean CEP17 copy numbers (> 2.6) were selected for additional chromosome 17 studies that used probes for Smith-Magenis syndrome (SMS), retinoic acid receptor alpha (RARA), and tumor protein p53 (TP53) genes. A eusomic copy number exhibited in one or more of these loci was used to calculate a revised HER2-to-chromosome-17 ratio by using the eusomic gene locus as the reference. RESULTS: Of 132 cases classified as nonamplified on the basis of their HER2:CEP17 ratios, 58 (43.9%) were scored as amplified by using alternative chromosome 17 reference gene probes, and 13 (92.9%) of 14 cases scored as equivocal were reclassified as amplified. Among the cases with mean HER2 copy number of 4 to 6, 41 (47.7%) of 86 had their HER2 gene status upgraded from nonamplified to amplified, and four (4.7%) of 86 were upgraded from equivocal to amplified. CONCLUSION: Our results support the findings of recent pangenomic studies that true polysomy 17 is uncommon. Additional FISH studies that use probes to the SMS, RARA, and TP53 genes are an effective way to determine the true HER2 amplification status in patients with polysomy 17 and they have important potential implications for guiding HER2-targeted therapy in breast cancer.
Asunto(s)
Biomarcadores de Tumor/genética , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/genética , Cromosomas Humanos Par 17/genética , Genes erbB-2 , Terapia Molecular Dirigida , Fosfoproteínas/genética , Receptor ErbB-2/genética , Adulto , Anciano , Algoritmos , Antineoplásicos/uso terapéutico , Biomarcadores de Tumor/análisis , Neoplasias de la Mama/química , Femenino , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes erbB-2/efectos de los fármacos , Humanos , Inmunohistoquímica , Hibridación Fluorescente in Situ , Proteínas Asociadas a Microtúbulos , Persona de Mediana Edad , Fosfoproteínas/análisis , Receptor ErbB-2/análisis , Proteína p53 Supresora de Tumor/análisisRESUMEN
BACKGROUND: The familial and sporadic forms of Alzheimer's disease (AD) have an identical pathology with a severe disparity in the time of onset [1]. The pathological similarity suggests that epigenetic processes may phenocopy the Familial Alzheimer's disease (FAD) mutations within sporadic AD. Numerous groups have demonstrated that FAD mutations in presenilin result in 'loss of function' of gamma-secretase mediated APP cleavage [2], [3], [4], [5]. Accordingly, ER stress is prominent within the pathologically impacted brain regions in AD patients [6] and is reported to inhibit APP trafficking through the secretory pathway [7], [8]. As the maturation of APP and the cleaving secretases requires trafficking through the secretory pathway [9], [10], [11], we hypothesized that ER stress may block trafficking requisite for normal levels of APP cleavage and that the small molecular chaperone 4-phenylbutyrate (PBA) may rescue the proteolytic deficit. METHODOLOGY/PRINCIPAL FINDINGS: The APP-Gal4VP16/Gal4-reporter screen was stably incorporated into neuroblastoma cells in order to assay gamma-secretase mediated APP proteolysis under normal and pharmacologically induced ER stress conditions. Three unrelated pharmacological agents (tunicamycin, thapsigargin and brefeldin A) all repressed APP proteolysis in parallel with activation of unfolded protein response (UPR) signaling-a biochemical marker of ER stress. Co-treatment of the gamma-secretase reporter cells with PBA blocked the repressive effects of tunicamycin and thapsigargin upon APP proteolysis, UPR activation, and apoptosis. In unstressed cells, PBA stimulated gamma-secretase mediated cleavage of APP by 8-10 fold, in the absence of any significant effects upon amyloid production, by promoting APP trafficking through the secretory pathway and the stimulation of the non-pathogenic alpha/gamma-cleavage. CONCLUSIONS/SIGNIFICANCE: ER stress represses gamma-secretase mediated APP proteolysis, which replicates some of the proteolytic deficits associated with the FAD mutations. The small molecular chaperone PBA can reverse ER stress induced effects upon APP proteolysis, trafficking and cellular viability. Pharmaceutical agents, such as PBA, that stimulate alpha/gamma-cleavage of APP by modifying intracellular trafficking should be explored as AD therapeutics.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Apoptosis/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Fenilbutiratos/farmacología , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/genética , Animales , Antibacterianos/farmacología , Antineoplásicos/farmacología , Western Blotting , Brefeldino A/farmacología , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Retículo Endoplásmico/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Inmunohistoquímica , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Transporte de Proteínas/efectos de los fármacos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tapsigargina/farmacología , Transfección , Tunicamicina/farmacologíaRESUMEN
Presenilin 1 (PS1) and Presenilin 2 (PS2) are the enzymatic component of the γ-secretase complex that cleaves amyloid precursor protein (APP) to release amyloid beta (Aß) peptide. PS deficiency in mice results in neuroinflammation and neurodegeneration in the absence of accumulated Aß. We hypothesize that PS influences neuroinflammation through its γ-secretase action in CNS innate immune cells. We exposed primary murine microglia to a pharmacological γ-secretase inhibitor which resulted in exaggerated release of TNFα and IL-6 in response to lipopolysaccharide. To determine if this response was mediated by PS1, PS2 or both we used shRNA to knockdown each PS in a murine microglia cell line. Knockdown of PS1 did not lead to decreased γ-secretase activity while PS2 knockdown caused markedly decreased γ-secretase activity. Augmented proinflammatory cytokine release was observed after knockdown of PS2 but not PS1. Proinflammatory stimuli increased microglial PS2 gene transcription and protein in vitro. This is the first demonstration that PS2 regulates CNS innate immunity. Taken together, our findings suggest that PS2 is the predominant γ-secretase in microglia and modulates release of proinflammatory cytokines. We propose PS2 may participate in a negative feedback loop regulating inflammatory behavior in microglia.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Citocinas/metabolismo , Microglía/metabolismo , Presenilina-2/metabolismo , Animales , Línea Celular , Sistema Nervioso Central/metabolismo , Regulación Enzimológica de la Expresión Génica , Inmunidad Innata , Interleucina-6/metabolismo , Lipopolisacáridos/metabolismo , Ratones , Presenilina-1/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
We introduce Chalkboard, a prototype tool for representing and displaying cell-signaling pathway knowledge, for carrying out simple qualitative reasoning over these pathways, and for generating quantitative biosimulation code. The design of Chalkboard has been driven by the need to quickly model and visualize alternative hypotheses about uncertain pathway knowledge. Chalkboard allows the biologists to test in silico the implications of various hypotheses. To fulfill this need, chalkboard includes (1) a rich ontology of pathway entities and interactions, which is ultimately informed by the basic chemistry and physics among molecules, and (2) a form of qualitative reasoning that computes causal chains and feedback loops within the network of entities and reactions. We demonstrate Chalkboard's capabilities in the domain of APP proteolysis, a pathway that plays a key role in the pathogenesis of Alzheimer's disease. In this pathway (as is common), information is incomplete and parts of the pathways are conjectural, rather than experimentally verified. With Chalkboard, we can carry out in silico perturbation experiments and explore the consequences of different conjectural connections and relationships in the network. We believe that pathway reasoning capabilities and in silico experiments will become a critical component of the hypothesis generation phase of modern biological research.
Asunto(s)
Simulación por Computador , Modelos Biológicos , Programas Informáticos , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Biología Computacional , Gráficos por Computador , Retroalimentación , Humanos , Procesamiento Proteico-Postraduccional , Transducción de SeñalRESUMEN
The beta-amyloid precursor protein (APP)-binding protein Fe65 is involved in APP nuclear signaling and several steps in APP proteolytic processing. In this study, we show that Fe65 stimulates gamma-secretase-mediated liberation of the APP intracellular domain (AICD). The mechanism of Fe65-mediated stimulation of AICD formation appears to be through enhanced production of the carboxyl-terminal fragment substrates of gamma-secretase and direct stimulation of processing by gamma-secretase. The stimulatory capacity of Fe65 is isoform-dependent, as the non-neuronal and a2 isoforms promote APP processing more effectively than the exon 9 inclusive neuronal form of Fe65. Intriguingly, Fe65 stimulation of AICD production appears to be inversely related to pathogenic beta-amyloid production as the Fe65 isoforms profoundly stimulate AICD production and simultaneously decrease Abeta42 production. Despite the capacity of Fe65 to stimulate gamma-secretase-mediated APP proteolysis, it does not rescue the loss of proteolytic function associated with the presenilin-1 familial Alzheimer disease mutations. These data suggest that Fe65 regulation of APP proteolysis may be integrally associated with its nuclear signaling function, as all antecedent proteolytic steps prior to release of Fe65 from the membrane are fostered by the APP-Fe65 interaction.
Asunto(s)
Péptidos beta-Amiloides/química , Proteínas del Tejido Nervioso/química , Proteínas Nucleares/química , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Humanos , Modelos Biológicos , Mutación , Proteínas del Tejido Nervioso/metabolismo , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Unión Proteica , Isoformas de Proteínas , Transducción de SeñalRESUMEN
Modern genomic research has access to a plethora of knowledge sources. Often, it is imperative that researchers combine and integrate knowledge from multiple perspectives. Although some technology exists for connecting data and knowledge bases, these methods are only just beginning to be successfully applied to research in modem cell biology. In this paper, we argue that one way to integrate multiple knowledge sources is through anatomy--both generic cellular anatomy, as well as anatomic knowledge about the tissues and organs that may be studied via microarray gene expression experiments. We present two examples where we have combined a large ontology of human anatomy (the FMA) with other genomic knowledge sources: the gene ontology (GO) and the mouse genomic databases (MGD) of the Jackson Labs. These two initial examples of knowledge integration provide a proof of concept that anatomy can act as a hub through which we can usefully combine a variety of genomic knowledge and data.
Asunto(s)
Anatomía , Genómica , Animales , Fenómenos Fisiológicos Celulares , Células/citología , Biología Computacional , Humanos , Modelos Anatómicos , Modelos Genéticos , Vocabulario ControladoRESUMEN
Cleavage of the beta-secretase processed beta-amyloid precursor protein by gamma-secretase leads to the extracellular release of Abeta42, the more amyloidogenic form of the beta-amyloid peptide, which subsequently forms the amyloid-plaques diagnostic of Alzheimer's disease. Mutations in beta-amyloid precursor protein (APP), presenilin-1 and presenilin-2 associated with familial Alzheimer's disease (FAD) increase release of Abeta42, suggesting that FAD may directly result from increased gamma-secretase activity. Here, we show that familial Alzheimer's disease mutations clustered near the sites of gamma-secretase cleavage actually decrease gamma-secretase-mediated release of the intracellular fragment of APP (CTFgamma). Concordantly, presenilin-1 mutations that result in Alzheimer's disease also decrease the release of CTFgamma. Mutagenesis of the epsilon cleavage site in APP mimicked the effects of the FAD mutations, both decreasing CTFgamma release and increasing Abeta42 production, suggesting that perturbation of this site may account for the observed decrement in gamma-secretase-mediated proteolysis of APP. As CTFgamma has been implicated in transcriptional activation, these data indicate that decreased signaling and transcriptional regulation resulting from FAD mutations in beta-amyloid precursor protein and presenilin-1 may contribute to the pathology of Alzheimer's disease.