RESUMEN
2-Deoxy-2-[18F]fluoro-d-glucose positron emission tomography (FDG-PET) is widely used to study cerebral glucose metabolism. Here, we investigated whether the FDG-PET signal is directly influenced by microglial glucose uptake in mouse models and patients with neurodegenerative diseases. Using a recently developed approach for cell sorting after FDG injection, we found that, at cellular resolution, microglia displayed higher glucose uptake than neurons and astrocytes. Alterations in microglial glucose uptake were responsible for both the FDG-PET signal decrease in Trem2-deficient mice and the FDG-PET signal increase in mouse models for amyloidosis. Thus, opposite microglial activation states determine the differential FDG uptake. Consistently, 12 patients with Alzheimer's disease and 21 patients with four-repeat tauopathies also exhibited a positive association between glucose uptake and microglial activity as determined by 18F-GE-180 18-kDa translocator protein PET (TSPO-PET) in preserved brain regions, indicating that the cerebral glucose uptake in humans is also strongly influenced by microglial activity. Our findings suggest that microglia activation states are responsible for FDG-PET signal alterations in patients with neurodegenerative diseases and mouse models for amyloidosis. Microglial activation states should therefore be considered when performing FDG-PET.
Asunto(s)
Fluorodesoxiglucosa F18 , Enfermedades Neurodegenerativas , Humanos , Glucosa , Microglía , Enfermedades Neurodegenerativas/diagnóstico por imagen , Tomografía de Emisión de Positrones , Animales , RatonesRESUMEN
Alzheimer's disease (AD) is the most common age-related neurodegenerative disorder. Increased Aß production plays a fundamental role in the pathogenesis of the disease and BACE1, the protease that triggers the amyloidogenic processing of APP, is a key protein and a pharmacological target in AD. Changes in neuronal activity have been linked to BACE1 expression and Aß generation, but the underlying mechanisms are still unclear. We provide clear evidence for the role of Casein Kinase 2 in the control of activity-driven BACE1 expression in cultured primary neurons, organotypic brain slices, and murine AD models. More specifically, we demonstrate that neuronal activity promotes Casein Kinase 2 dependent phosphorylation of the translation initiation factor eIF4B and this, in turn, controls BACE1 expression and APP processing. Finally, we show that eIF4B expression and phosphorylation are increased in the brain of APPPS1 and APP-KI mice, as well as in AD patients. Overall, we provide a definition of a mechanism linking brain activity with amyloid production and deposition, opening new perspectives from the therapeutic standpoint.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Quinasa de la Caseína II/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Potenciales de Acción , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/metabolismo , Animales , Quinasa de la Caseína II/antagonistas & inhibidores , Modelos Animales de Enfermedad , Silenciador del Gen , Células HEK293 , Humanos , Ratones Endogámicos C57BL , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Presenilina-1/metabolismo , Biosíntesis de Proteínas/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Genetic variants in the triggering receptor expressed on myeloid cells 2 (TREM2) have been linked to Nasu-Hakola disease, Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis, frontotemporal dementia (FTD), and FTD-like syndrome without bone involvement. TREM2 is an innate immune receptor preferentially expressed by microglia and is involved in inflammation and phagocytosis. Whether and how TREM2 missense mutations affect TREM2 function is unclear. We report that missense mutations associated with FTD and FTD-like syndrome reduce TREM2 maturation, abolish shedding by ADAM proteases, and impair the phagocytic activity of TREM2-expressing cells. As a consequence of reduced shedding, TREM2 is virtually absent in the cerebrospinal fluid (CSF) and plasma of a patient with FTD-like syndrome. A decrease in soluble TREM2 was also observed in the CSF of patients with AD and FTD, further suggesting that reduced TREM2 function may contribute to increased risk for two neurodegenerative disorders.
Asunto(s)
Glicoproteínas de Membrana/genética , Enfermedades Neurodegenerativas/genética , Fagocitosis/fisiología , Receptores Inmunológicos/genética , Enfermedad de Alzheimer/genética , Transporte Biológico/genética , Transporte Biológico/fisiología , Línea Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Demencia Frontotemporal/genética , Humanos , Mutación , Fagocitosis/genéticaRESUMEN
The metalloproteinase ADAM10 is of importance for Notch-dependent cortical brain development. The protease is tightly linked with α-secretase activity toward the amyloid precursor protein (APP) substrate. Increasing ADAM10 activity is suggested as a therapy to prevent the production of the neurotoxic amyloid ß (Aß) peptide in Alzheimer's disease. To investigate the function of ADAM10 in postnatal brain, we generated Adam10 conditional knock-out (A10cKO) mice using a CaMKIIα-Cre deleter strain. The lack of ADAM10 protein expression was evident in the brain cortex leading to a reduced generation of sAPPα and increased levels of sAPPß and endogenous Aß peptides. The A10cKO mice are characterized by weight loss and increased mortality after weaning associated with seizures. Behavioral comparison of adult mice revealed that the loss of ADAM10 in the A10cKO mice resulted in decreased neuromotor abilities and reduced learning performance, which were associated with altered in vivo network activities in the hippocampal CA1 region and impaired synaptic function. Histological and ultrastructural analysis of ADAM10-depleted brain revealed astrogliosis, microglia activation, and impaired number and altered morphology of postsynaptic spine structures. A defect in spine morphology was further supported by a reduction of the expression of NMDA receptors subunit 2A and 2B. The reduced shedding of essential postsynaptic cell adhesion proteins such as N-Cadherin, Nectin-1, and APP may explain the postsynaptic defects and the impaired learning, altered network activity, and synaptic plasticity of the A10cKO mice. Our study reveals that ADAM10 is instrumental for synaptic and neuronal network function in the adult murine brain.
Asunto(s)
Proteínas ADAM/deficiencia , Secretasas de la Proteína Precursora del Amiloide/deficiencia , Encéfalo/ultraestructura , Espinas Dendríticas/patología , Epilepsia/genética , Epilepsia/patología , Discapacidades para el Aprendizaje/patología , Proteínas de la Membrana/deficiencia , Sinapsis/patología , Proteína ADAM10 , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Animales Recién Nacidos , Encéfalo/patología , Cadherinas/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Moléculas de Adhesión Celular/metabolismo , Espinas Dendríticas/metabolismo , Modelos Animales de Enfermedad , Regulación del Desarrollo de la Expresión Génica/genética , Gliosis/genética , Discapacidades para el Aprendizaje/genética , Ratones , Ratones Transgénicos , Nectinas , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Genetic analysis revealed the hexanucleotide repeat expansion GGGGCC within the regulatory region of the gene C9orf72 as the most common cause of familial amyotrophic lateral sclerosis and the second most common cause of frontotemporal lobar degeneration. Since repeat expansions might cause RNA toxicity via sequestration of RNA-binding proteins, we searched for proteins capable of binding to GGGGCC repeats. In vitro-transcribed biotinylated RNA containing hexanucleotide GGGGCC or, as control, AAAACC repeats were incubated with nuclear protein extracts. Using stringent filtering protocols 20 RNA-binding proteins with a variety of different functions in RNA metabolism, translation and transport were identified. A subset of these proteins was further investigated by immunohistochemistry in human autopsy brains. This revealed that hnRNP A3 formed neuronal cytoplasmic and intranuclear inclusions in the hippocampus of patients with C9orf72 repeat extensions. Confocal microcopy showed that these inclusions belong to the group of the so far enigmatic p62-positive/TDP-43 negative inclusions characteristically seen in autopsy cases of diseased C9orf72 repeat expansion carriers. Thus, we have identified one protein component of these pathognomonic inclusions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Esclerosis Amiotrófica Lateral , Degeneración Lobar Frontotemporal , Ribonucleoproteína Heterogénea-Nuclear Grupo A-B/metabolismo , Hipocampo/patología , Cuerpos de Inclusión/metabolismo , Mutación/genética , Proteínas/genética , Secuencias Repetitivas de Ácidos Nucleicos/fisiología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72 , Cromatografía Líquida de Alta Presión , Proteínas de Unión al ADN/metabolismo , Degeneración Lobar Frontotemporal/genética , Degeneración Lobar Frontotemporal/metabolismo , Degeneración Lobar Frontotemporal/patología , Regulación de la Expresión Génica/genética , Células HEK293 , Humanos , Cuerpos de Inclusión/patología , Espectrometría de Masas , ARN Interferente Pequeño/metabolismo , Proteína Sequestosoma-1 , TransfecciónRESUMEN
Mutations in presenilins (PS1 and PS2) account for the vast majority of early onset familial Alzheimer's disease cases. Beside the well investigated role of presenilins as the catalytic unit in γ-secretase complex, their involvement in regulation of intracellular calcium homeostasis has recently come into more focus of Alzheimer's disease research. Here we report that the overexpression of PS1 full-length holoprotein forms, in particular familial Alzheimer's disease-causing forms of PS1, result in significantly attenuated calcium release from thapsigargin- and bradykinin-sensitive stores. Interestingly, treatment of HEK293 cells with γ-secretase inhibitors also leads to decreased amount of calcium release from endoplasmic reticulum (ER) accompanying elevated PS1 holoprotein levels. Similarly, the knockdown of PEN-2 which is associated with deficient PS1 endoproteolysis and accumulation of its holoprotein form also leads to decreased ER calcium release. Notably, we detected enhanced PS1 holoprotein levels also in postmortem brains of patients carrying familial Alzheimer's disease PS1 mutations. Taken together, the conditions in which the amount of full length PS1 holoprotein is increased result in reduction of calcium release from ER. Based on these results, we propose that the disturbed ER calcium homeostasis mediated by the elevation of PS1 holoprotein levels may be a contributing factor to the pathogenesis of Alzheimer's disease.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas de la Membrana/metabolismo , Presenilina-1/metabolismo , Adulto , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/genética , Western Blotting , Estudios de Casos y Controles , Inhibidores Enzimáticos/farmacología , Femenino , Células HEK293 , Homeostasis , Humanos , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Persona de Mediana Edad , Mutación/genética , Presenilina-1/genética , Regulación hacia ArribaRESUMEN
Anti-amyloidogenic processing of the amyloid precursor protein APP by α-secretase prevents formation of the amyloid-ß peptide, which accumulates in senile plaques of Alzheimer disease patients. α-Secretase belongs to the family of a disintegrin and metalloproteases (ADAMs), and ADAM10 is the primary candidate for this anti-amyloidogenic activity. We recently demonstrated that ADAM10 translation is repressed by its 5'-UTR and that in particular the first half of ADAM10 5'-UTR is responsible for translational repression. Here, we asked whether specific sequence motifs exist in the ADAM10 5'-UTR that are able to form complex secondary structures and thus potentially inhibit ADAM10 translation. Using circular dichroism spectroscopy, we demonstrate that a G-rich region between nucleotides 66 and 94 of the ADAM10 5'-UTR forms a highly stable, intramolecular, parallel G-quadruplex secondary structure under physiological conditions. Mutation of guanines in this sequence abrogates the formation of the G-quadruplex structure. Although the G-quadruplex structure efficiently inhibits translation of a luciferase reporter in in vitro translation assays and in living cells, inhibition of G-quadruplex formation fails to do so. Moreover, expression of ADAM10 was similarly repressed by the G-quadruplex. Mutation of the G-quadruplex motif results in a significant increase of ADAM10 levels and consequently APPsα secretion. Thus, we identified a critical RNA secondary structure within the 5'-UTR, which contributes to the translational repression of ADAM10.
Asunto(s)
Regiones no Traducidas 5'/fisiología , Proteínas ADAM/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Proteínas de la Membrana/biosíntesis , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/fisiología , Proteínas ADAM/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide/genética , Células HEK293 , Humanos , Proteínas de la Membrana/genética , MutaciónRESUMEN
Numerous loss-of-function mutations in the progranulin (GRN) gene cause frontotemporal lobar degeneration with ubiquitin and TAR-DNA binding protein 43-positive inclusions by reduced production and secretion of GRN. Consistent with the observation that GRN has neurotrophic properties, pharmacological stimulation of GRN production is a promising approach to rescue GRN haploinsufficiency and prevent disease progression. We therefore searched for compounds capable of selectively increasing GRN levels. Here, we demonstrate that four independent and highly selective inhibitors of vacuolar ATPase (bafilomycin A1, concanamycin A, archazolid B, and apicularen A) significantly elevate intracellular and secreted GRN. Furthermore, clinically used alkalizing drugs, including chloroquine, bepridil, and amiodarone, similarly stimulate GRN production. Elevation of GRN levels occurs via a translational mechanism independent of lysosomal degradation, autophagy, or endocytosis. Importantly, alkalizing reagents rescue GRN deficiency in organotypic cortical slice cultures from a mouse model for GRN deficiency and in primary cells derived from human patients with GRN loss-of-function mutations. Thus, alkalizing reagents, specifically those already used in humans for other applications, and vacuolar ATPase inhibitors may be therapeutically used to prevent GRN-dependent neurodegeneration.
Asunto(s)
Álcalis/farmacología , Corteza Cerebral/metabolismo , Fibroblastos/metabolismo , Degeneración Lobar Frontotemporal/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Neuronas/metabolismo , ATPasas de Translocación de Protón Vacuolares/antagonistas & inhibidores , Amiodarona/farmacología , Animales , Animales Recién Nacidos , Proteína 5 Relacionada con la Autofagia , Bepridil/farmacología , Northern Blotting , Compuestos Bicíclicos Heterocíclicos con Puentes/farmacología , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Cloroquina/farmacología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fibroblastos/efectos de los fármacos , Degeneración Lobar Frontotemporal/tratamiento farmacológico , Degeneración Lobar Frontotemporal/genética , Granulinas , Células HEK293 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intercelular/deficiencia , Péptidos y Proteínas de Señalización Intercelular/genética , Macrólidos/farmacología , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/deficiencia , Proteínas Asociadas a Microtúbulos/genética , Mutación , Neuronas/efectos de los fármacos , Progranulinas , ARN Mensajero/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiazoles/farmacologíaRESUMEN
Proteolytic processing of the amyloid precursor protein by alpha-secretase prevents formation of the amyloid beta-peptide (Abeta), which is the main constituent of amyloid plaques in brains of Alzheimer disease (AD) patients. alpha-Secretase activity is decreased in AD, and overexpression of the alpha-secretase ADAM10 (a disintegrin and metalloprotease 10) in an AD animal model prevents amyloid pathology. ADAM10 has a 444-nucleotide-long, very GC-rich 5'-untranslated region (5'-UTR) with two upstream open reading frames. Because similar properties of 5'-UTRs are found in transcripts of many genes, which are regulated by translational control mechanisms, we asked whether ADAM10 expression is translationally controlled by its 5'-UTR. We demonstrate that the 5'-UTR of ADAM10 represses the rate of ADAM10 translation. In the absence of the 5'-UTR, we observed a significant increase of ADAM10 protein levels in HEK293 cells, whereas mRNA levels were not changed. Moreover, the 5'-UTR of ADAM10 inhibits translation of a luciferase reporter in an in vitro transcription/translation assay. Successive deletion of the first half of the ADAM10 5'-UTR revealed a striking increase in ADAM10 protein expression in HEK293 cells, suggesting that this part of the 5'-UTR contains inhibitory elements for translation. Moreover, we detect an enhanced alpha-secretase activity and consequently reduced Abeta levels in the conditioned medium of HEK293 cells expressing both amyloid precursor protein and a 5'-UTR-ADAM10 deletion construct lacking the first half of the 5'-UTR. Thus, we provide evidence that the 5'-UTR of ADAM10 may have an important role for post-transcriptional regulation of ADAM10 expression and consequently Abeta production.
Asunto(s)
Regiones no Traducidas 5' , Proteínas ADAM/biosíntesis , Secretasas de la Proteína Precursora del Amiloide/biosíntesis , Péptidos beta-Amiloides/biosíntesis , Regulación Enzimológica de la Expresión Génica , Proteínas de la Membrana/biosíntesis , Biosíntesis de Proteínas , Proteínas ADAM/genética , Proteína ADAM10 , Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Péptidos beta-Amiloides/genética , Animales , Secuencia de Bases , Encéfalo/enzimología , Células COS , Chlorocebus aethiops , Humanos , Proteínas de la Membrana/genética , Eliminación de SecuenciaRESUMEN
gamma-Secretase is a pivotal intramembrane-cleaving protease complex and important drug target for Alzheimer's disease. The protease not only releases small peptides, such as the amyloid-beta peptide, which drives Alzheimer's disease pathogenesis, but also intracellular domains, which can have critical functions in nuclear signaling. Unlike typical aspartyl proteases, gamma-secretase contains a non-classical GxGD active site motif in its catalytic subunit presenilin (PS) 1 or PS2. It is not known whether both glycines are of similar functional relevance and why the glycine residues are invariant elements of the motif. Here we identify the N-terminal glycine of the GxGD motif in PS1, G382, as a critical residue of the active site domain of gamma-secretase. Substitution of G382 by a number of different amino acids abrogated gamma-secretase activity. Only the smallest possible G382A substitution allowed substantial gamma-secretase activity. Depending on the substrate, however, the presence of G382 could become even an absolute functional requirement of gamma-secretase. Very similar results were obtained for the C-terminal glycine residue (G384) of the GxGD motif. Our data thus identify a requirement for small side chain residues in the active site domain of gamma-secretase and suggest that the glycines of the GxGD motif could be evolutionary conserved to allow cleavage of all possible gamma-secretase substrates, including those, which are highly sensitive to minimal alteration of the PS active site domain. These findings broaden our understanding of gamma-secretase substrate recognition and cleavage, which may prove crucial for therapeutic targeting of the enzyme.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Glicina/genética , Presenilina-1/química , Presenilina-2/química , Alanina/genética , Secuencias de Aminoácidos , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Células Cultivadas , Embrión de Mamíferos , Humanos , Receptores de Hialuranos/genética , Receptores de Hialuranos/metabolismo , Inmunoprecipitación , Ratones , Ratones Noqueados , Mutación/genética , Presenilina-1/genética , Presenilina-2/genética , Estructura Terciaria de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Transfección/métodosRESUMEN
beta-Secretase (beta-site amyloid precursor protein cleaving enzyme 1; BACE1) has been identified as the rate limiting enzyme for amyloid-beta-peptide (Abeta) production. Abeta is the major component of amyloid plaques and vascular deposits in Alzheimer's disease (AD) brains and believed to initiate the deadly amyloid cascade. BACE1 is the principle beta-secretase, since its knock-out completely prevents Abeta generation. BACE1 is likely to process a number of different substrates and consequently several independent physiological functions may be exerted by BACE1. Currently the function of BACE1 in myelination is best understood. BACE1 cleaves and activates Neuregulin-1 and is thus directly involved in myelination of the peripheral nervous system during early postnatal development. However, additional physiological functions specifically within the central nervous system are so far less understood. BACE1 is upregulated in at least some AD brains. Multiple cellular mechanisms for BACE1 regulation are known including post-transcriptional regulation via its 5'-untranslated region, microRNA and non-coding anti-sense RNA. BACE1 is a primary target for Abeta lowering therapies, however the development of high affinity bio-available inhibitors has been a major challenge so far.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Péptidos beta-Amiloides , Humanos , Procesamiento Proteico-PostraduccionalRESUMEN
beta-site APP cleaving enzyme-1 (BACE1), the rate-limiting enzyme for beta-amyloid (Abeta) production, is elevated in Alzheimer's disease (AD). Here, we show that energy deprivation induces phosphorylation of the translation initiation factor eIF2alpha (eIF2alpha-P), which increases the translation of BACE1. Salubrinal, an inhibitor of eIF2alpha-P phosphatase PP1c, directly increases BACE1 and elevates Abeta production in primary neurons. Preventing eIF2alpha phosphorylation by transfection with constitutively active PP1c regulatory subunit, dominant-negative eIF2alpha kinase PERK, or PERK inhibitor P58(IPK) blocks the energy-deprivation-induced BACE1 increase. Furthermore, chronic treatment of aged Tg2576 mice with energy inhibitors increases levels of eIF2alpha-P, BACE1, Abeta, and amyloid plaques. Importantly, eIF2alpha-P and BACE1 are elevated in aggressive plaque-forming 5XFAD transgenic mice, and BACE1, eIF2alpha-P, and amyloid load are correlated in humans with AD. These results strongly suggest that eIF2alpha phosphorylation increases BACE1 levels and causes Abeta overproduction, which could be an early, initiating molecular mechanism in sporadic AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Factores de Transcripción/metabolismo , Factores de Edad , Anciano de 80 o más Años , Secretasas de la Proteína Precursora del Amiloide/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Antimetabolitos/farmacología , Ácido Aspártico Endopeptidasas/genética , Células Cultivadas , Corteza Cerebral/citología , Convulsivantes/farmacología , Dactinomicina/farmacología , Desoxiglucosa/farmacología , Modelos Animales de Enfermedad , Embrión de Mamíferos , Activación Enzimática/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Glucosa/farmacología , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Nitrocompuestos/farmacología , Fragmentos de Péptidos/metabolismo , Fosforilación , Placa Amiloide/patología , Propionatos/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Factores de Tiempo , Transfección/métodos , eIF-2 Quinasa/metabolismoRESUMEN
Several receptor protein tyrosine phosphatases (RPTPs) are cell adhesion molecules involved in homophilic interactions, suggesting that RPTP outside-in signaling is coupled to cell contact formation. However, little is known about the mechanisms by which cell density regulates RPTP function. We show that the MAM family prototype RPTPkappa is cleaved by three proteases: furin, ADAM 10, and gamma-secretase. Cell density promotes ADAM 10-mediated cleavage and shedding of RPTPkappa. This is followed by gamma-secretase-dependent intramembrane proteolysis of the remaining transmembrane part to release the phosphatase intracellular portion (PIC) from the membrane, thereby allowing its translocation to the nucleus. When cells were treated with leptomycin B, a nuclear export inhibitor, PIC accumulated in nuclear bodies. PIC is an active protein tyrosine phosphatase that binds to and dephosphorylates beta-catenin, an RPTPkappa substrate. The expression of RPTPkappa suppresses beta-catenin's transcriptional activity, whereas the expression of PIC increases it. Notably, this increase required the phosphatase activity of PIC. Thus, both isoforms have acquired opposing roles in the regulation of beta-catenin signaling. We also found that RPTPmu, another MAM family member, undergoes gamma-secretase-dependent processing. Our results identify intramembrane proteolysis as a regulatory switch in RPTPkappa signaling and implicate PIC in the activation of beta-catenin-mediated transcription.
Asunto(s)
Proteínas ADAM/metabolismo , Endopeptidasas/metabolismo , Furina/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , beta Catenina/genética , Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas , Biotinilación , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Células COS , Carcinoma/genética , Carcinoma/patología , Línea Celular Tumoral , Chlorocebus aethiops , Inhibidores de Cisteína Proteinasa/farmacología , Densitometría , Relación Dosis-Respuesta a Droga , Femenino , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Células HCT116 , Humanos , Cinética , Leupeptinas/farmacología , Luciferasas/análisis , Luciferasas/metabolismo , Ratones , Modelos Biológicos , Células 3T3 NIH , Plásmidos/genética , Pruebas de Precipitina , Interferencia de ARN , Proteínas Tirosina Fosfatasas Clase 2 Similares a Receptores , Transcripción Genética , Trifluoperazina/farmacologíaRESUMEN
The aspartyl protease BACE1 has a pivotal role in the pathogenesis of Alzheimer's disease. Recently, it was shown that in Alzheimer's disease patients, BACE1 levels were elevated although mRNA levels were not changed compared with controls. Here, we demonstrate that the 5'-untranslated region (5'UTR) of BACE1 controls the rate of BACE1 translation. In the presence of the 5'UTR, we observed more than 90% reduction of BACE1 protein levels in HEK293, COS7 and H4 cells, and a similar reduction of BACE1 activity in vitro. mRNA levels were not affected, demonstrating that the 5'UTR repressed the translation but not the transcription of BACE1. The 3'UTR did not affect BACE1 expression. An extensive mutagenesis analysis predicts that the GC-rich region of the 5'UTR forms a constitutive translation barrier, which may prevent the ribosome from efficiently translating the BACE1 mRNA. Our data therefore demonstrate translational repression as a new mechanism controlling BACE1 expression.
Asunto(s)
Regiones no Traducidas 5'/genética , Enfermedad de Alzheimer/enzimología , Ácido Aspártico Endopeptidasas/genética , Regulación Enzimológica de la Expresión Génica , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide , Animales , Ácido Aspártico Endopeptidasas/análisis , Ácido Aspártico Endopeptidasas/biosíntesis , Secuencia de Bases , Regulación hacia Abajo , Endopeptidasas , Humanos , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta/genética , Biosíntesis de Proteínas/genética , ARN Mensajero/análisis , ARN Mensajero/metabolismoRESUMEN
The beta-amyloid precursor protein (beta APP) is proteolytically processed by two secretase activities to produce the pathogenic amyloid beta-peptide (A beta). N-terminal cleavage is mediated by beta-secretase (BACE) whereas C-terminal intramembraneous cleavage is exerted by the presenilin (PS) gamma-secretase complex. The A beta-generating gamma-secretase cleavage principally occurs after amino acid 40 or 42 and results in secretion of A beta-(1-40) or A beta-(1-42). Upon overexpression of BACE in cultured cells we unexpectedly noticed a reduction of secreted A beta-(1-40/42). However, mass spectrometry revealed a truncated A beta species, which terminates at amino acid 34 (A beta-(1-34)) suggesting an alternative gamma-secretase cut. Indeed, expression of a loss-of-function variant of PS1 inhibited not only the production of A beta-(1-40) and A beta-(1-42) but also that of A beta-(1-34). However, expression levels of BACE correlate with the amount of A beta-(1-34), and A beta-(1-34) is produced at the expense of A beta-(1-40) and A beta-(1-42). Since this suggested that BACE is involved in a C-terminal truncation of A beta, we incubated purified BACE with A beta-(1-40) in vitro. Under these conditions A beta-(1-34) was generated. Moreover, when conditioned media containing Abeta-(1-40) and A beta-(1-42) were incubated with cells expressing a loss-of-function PS1 variant together with BACE, A beta-(1-34) was efficiently produced in vivo. These data demonstrate that an apparently gamma-secretase-dependent A beta derivative is produced after the generation of the non-truncated A beta via an additional and unexpected activity of BACE.
Asunto(s)
Precursor de Proteína beta-Amiloide/biosíntesis , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Proteínas de la Membrana/metabolismo , Enfermedad de Alzheimer/genética , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Ácido Aspártico Endopeptidasas/metabolismo , Línea Celular , Membrana Celular/fisiología , Humanos , Riñón , Datos de Secuencia Molecular , Mutación , Presenilina-1 , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , TransfecciónRESUMEN
Alzheimer's disease (AD)-associated gamma-secretase is a presenilin (PS)- dependent proteolytic activity involved in the intramembraneous cleavage of the beta-amyloid precursor protein, Notch, LDL receptor-related protein, E-cadherin, and ErbB-4. This cut produces the corresponding intracellular domains (ICD), which are required for nuclear signaling of Notch and probably ErbB-4, the beta-amyloid precursor protein, E-cadherin, and the LDL receptor-related protein as well. We have now investigated CD44, a cell surface adhesion molecule, which also undergoes an intramembraneous cleavage to liberate its ICD. We demonstrate that this cleavage requires a PS-dependent gamma-secretase activity. A loss-of-function PS1 mutation, a PS1/PS2 knockout, as well as two independent and highly specific gamma-secretase inhibitors, abolish this cleavage. Surprisingly, small peptides similar to the amyloid beta-peptide (Abeta) are generated by an additional cut in the middle of the transmembrane region of CD44. Like Abeta, these CD44 beta-peptides are generated in a PS-dependent manner. These findings therefore suggest a dual intramembraneous cleavage mechanism mediated by PS proteins. The dual cleavage mechanism is required for nuclear signaling as well as removal of remaining transmembrane domains, a general function of PS in membrane protein metabolism.
Asunto(s)
Membrana Celular/metabolismo , Endopeptidasas/metabolismo , Receptores de Hialuranos/metabolismo , Proteínas de la Membrana/metabolismo , Péptidos/metabolismo , Ácido gamma-Aminobutírico/análogos & derivados , Enfermedad de Alzheimer/metabolismo , Secuencia de Aminoácidos , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Ácido Aspártico Endopeptidasas/metabolismo , Carbamatos/farmacología , Línea Celular , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Humanos , Receptores de Hialuranos/genética , Proteínas de la Membrana/genética , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Péptidos/genética , Presenilina-1 , Presenilina-2 , Estructura Terciaria de Proteína , Alineación de Secuencia , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Triglicéridos/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
Amyloid beta-peptide (Abeta) is generated by the consecutive cleavages of beta- and gamma-secretase. The intramembraneous gamma-secretase cleavage critically depends on the activity of presenilins (PS1 and PS2). Although there is evidence that PSs are aspartyl proteases with gamma-secretase activity, it remains controversial whether their subcellular localization overlaps with the cellular sites of Abeta production. We now demonstrate that biologically active GFP-tagged PS1 as well as endogenous PS1 are targeted to the plasma membrane (PM) of living cells. On the way to the PM, PS1 binds to nicastrin (Nct), an essential component of the gamma-secretase complex. This complex is targeted through the secretory pathway where PS1-bound Nct becomes endoglycosidase H resistant. Moreover, surface-biotinylated Nct can be coimmunoprecipitated with PS1 antibodies, demonstrating that this complex is located to cellular sites with gamma-secretase activity. Inactivating PS1 or PS2 function by mutagenesis of one of the critical aspartate residues or by gamma-secretase inhibitors results in delayed reinternalization of the beta-amyloid precursor protein and its accumulation at the cell surface. Our data suggest that PS is targeted as a biologically active complex with Nct through the secretory pathway to the cell surface and suggest a dual function of PS in gamma-secretase processing and in trafficking.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Membrana Celular/metabolismo , Células Eucariotas/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , Transporte de Proteínas/fisiología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/fisiopatología , Secretasas de la Proteína Precursora del Amiloide , Péptidos beta-Amiloides/biosíntesis , Péptidos beta-Amiloides/genética , Ácido Aspártico/genética , Ácido Aspártico Endopeptidasas , Compartimento Celular/fisiología , Membrana Celular/ultraestructura , Células Cultivadas , Endocitosis/genética , Endopeptidasas/efectos de los fármacos , Endopeptidasas/metabolismo , Células Eucariotas/ultraestructura , Proteínas Fluorescentes Verdes , Humanos , Indicadores y Reactivos , Proteínas Luminiscentes , Sustancias Macromoleculares , Proteínas de la Membrana/genética , Mutagénesis Sitio-Dirigida/genética , Mutación/fisiología , Presenilina-1 , Unión Proteica/fisiología , Receptores Notch , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , TransfecciónRESUMEN
The intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein (APP) is dependent on biologically active presenilins (PS). Notch also undergoes a similar PS-dependent gamma-secretase-like cleavage, resulting in the liberation of the Notch intracellular domain (NICD), which is critically required for developmental signal transduction. gamma-Secretase processing of APP results in the production of a similar fragment called AICD (APP intracellular domain), which may function in nuclear signaling as well. AICD, like NICD, is rapidly removed. By using a battery of protease inhibitors we demonstrate that AICD, in contrast to NICD, is degraded by a cytoplasmic metalloprotease. In vitro degradation of AICD can be reconstituted with cytoplasmic fractions obtained from neuronal and non-neuronal cells. Taking into account the inhibition profile and the cytoplasmic localization, we identified three candidate enzymes (neurolysin, thimet oligopeptidase, and insulin-degrading enzyme (IDE), also known as insulysin), which all are involved in the degradation of bioactive peptides in the brain. When insulin, a well characterized substrate of IDE, was added to the in vitro degradation assay, removal of AICD was efficiently blocked. Moreover, overexpression of IDE resulted in enhanced degradation of AICD, whereas overexpression of the inactive IDE E111Q mutant did not affect AICD degradation. Finally, immunodepletion of IDE significantly reduced the AICD degrading activity. Therefore our data demonstrate that IDE, which is one of the proteases implicated in the removal of extracellular Abeta, also removes the cytoplasmic product of gamma-secretase cleaved APP.