RESUMEN
Accumulation of amyloid-ß peptide (Aß) aggregates in synapses may contribute to the profound synaptic loss characteristic of Alzheimer's disease (AD). The origin of synaptic Aß aggregates remains elusive, but loss of endosomal proteostasis may trigger their formation. In this study, we identified the synaptic compartments where Aß accumulates, and performed a longitudinal analysis of synaptosomes isolated from brains of TgCRND8 APP transgenic mice of either sex. To evaluate the specific contribution of Aß-degrading protease endothelin-converting enzyme (ECE-1) to synaptic/endosomal Aß homeostasis, we analyzed the effect of partial Ece1 KO in brain and complete ECE1 KO in SH-SY5Y cells. Global inhibition of ECE family members was used to further assess their role in preventing synaptic Aß accumulation. Results showed that, before extracellular amyloid deposition, synapses were burdened with detergent-soluble Aß monomers, oligomers, and fibrils. Levels of all soluble Aß species declined thereafter, as Aß42 turned progressively insoluble and accumulated in Aß-producing synaptic endosomal vesicles with characteristics of multivesicular bodies. Accordingly, fibrillar Aß was detected in brain exosomes. ECE-1-deficient mice had significantly increased endogenous synaptosomal Aß42 levels, and protease inhibitor experiments showed that, in TgCRND8 mice, synaptic Aß42 became nearly resistant to degradation by ECE-related proteases. Our study supports that Aß accumulating in synapses is produced locally, within endosomes, and does not require the presence of amyloid plaques. ECE-1 is a determinant factor controlling the accumulation and fibrillization of nascent Aß in endosomes and, in TgCRND8 mice, Aß overproduction causes rapid loss of Aß42 solubility that curtails ECE-mediated degradation.SIGNIFICANCE STATEMENT Deposition of aggregated Aß in extracellular plaques is a defining feature of AD. Aß aggregates also accumulate in synapses and may contribute to the profound synaptic loss and cognitive dysfunction typical of the disease. However, it is not clear whether synaptotoxic Aß is mainly derived from plaques or if it is produced and aggregated locally, within affected synaptic compartments. Filling this knowledge gap is important for the development of an effective treatment for AD, as extracellular and intrasynaptic pools of Aß may not be equally modulated by immunotherapies or other therapeutic approaches. In this manuscript, we provide evidence that Aß aggregates building up in synapses are formed locally, within synaptic endosomes, because of disruptions in nascent Aß proteostasis.
Asunto(s)
Enfermedad de Alzheimer , Amiloidosis , Neuroblastoma , Humanos , Ratones , Animales , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Neuronas/metabolismo , Neuroblastoma/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ratones Transgénicos , Endosomas/metabolismo , Placa Amiloide/metabolismoRESUMEN
BACKGROUND: Demonstration of intrathecal production of Borrelia-specific antibodies (ITAb) is considered the most specific diagnostic marker of Lyme neuroborreliosis (LNB). Limitations include delayed detectability in early infection and continued presence long after successful treatment. Markers of active inflammation-increased cerebrospinal fluid (CSF) leukocytes, protein, and CXCL13-provide nonspecific markers of active infection. To assess the utility of CSF CXCL13, we measured its concentration in 132 patients with a broad spectrum of neuroinflammatory disorders, including LNB. METHODS: CSF CXCL13 was measured by immunoassay. Spearman rank correlation test was performed to explore its relationship to conventional markers of neuroinflammation and Borrelia-specific ITAb production. RESULTS: In non-LNB neuroinflammatory disorders, CSF CXCL13 elevation correlated with CSF immunoglobulin G (IgG) synthesis and leukocyte count. In LNB, CXCL13 concentration was far greater than expected from overall CSF IgG synthesis, and correlated with Borrelia-specific ITAb synthesis. Median CSF CXCL13 concentration in ITAb-positive LNB patients was > 500 times greater than in any other group. CONCLUSIONS: Intrathecal CXCL13 and IgG production are closely interrelated. CXCL13 is disproportionately increased in "definite LNB," defined as having demonstrable Borrelia-specific ITAb, but not "probable LNB," without ITAb. This disproportionate increase may help identify patients with very early infection or those with active vs treated LNB, or may help to differentiate ITAb-defined active LNB from other neuroinflammatory disorders. However, its reported specificity is closely related to the diagnostic requirement for ITAb. It may add little specificity to the demonstration of a pleocytosis or increased overall or specific IgG production in the CSF.
Asunto(s)
Quimiocina CXCL13/líquido cefalorraquídeo , Neuroborreliosis de Lyme , Biomarcadores , Borrelia , Humanos , Inmunoensayo , Pruebas Inmunológicas , Neuroborreliosis de Lyme/diagnósticoRESUMEN
Pleiotropic roles are proposed for brain extracellular vesicles (EVs) in the development of Alzheimer's disease (AD). Our previous studies have suggested a beneficial role for EVs in AD, where the endosomal system in vulnerable neurons is compromised, contributing to the removal of accumulated material from neurons. However, the involvement of EVs in propagating AD amyloidosis throughout the brain has been considered because the amyloid-ß precursor protein (APP), APP metabolites, and key APP cleaving enzymes were identified in association with EVs. Here, we undertook to determine whether the secretase machinery is actively processing APP in EVs isolated from the brains of wild-type and APP overexpressing Tg2576 mice. We found that full-length APP is cleaved in EVs incubated in the absence of cells. The resulting metabolites, both α- and ß-APP carboxyl-terminal fragments and APP intracellular domain accumulate in EVs over time and amyloid-ß dimerizes. Thus, EVs contribute to the removal from neurons and transport of APP-derived neurotoxic peptides. While this is potentially a venue for propagation of the pathology throughout the brain, it may contribute to efficient removal of neurotoxic peptides from the brain.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo , Vesículas Extracelulares/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Encéfalo/metabolismo , Encéfalo/patología , Femenino , Masculino , Ratones , Ratones Transgénicos , Agregación Patológica de ProteínasRESUMEN
Accumulating evidence suggests that the abnormal aggregation of amyloid-ß (Αß) peptide in Alzheimer's disease (AD) begins intraneuronally, within vesicles of the endosomal-lysosomal pathway where Aß is both generated and degraded. Metalloproteases, including endothelin-converting enzyme (ECE)-1 and -2, reside within these vesicles and normally limit the accumulation of intraneuronally produced Aß. In this study, we determined whether disruption of Aß catabolism could trigger Aß aggregation within neurons and increase the amount of Aß associated with exosomes, small extracellular vesicles derived from endosomal multivesicular bodies. Using cultured cell lines, primary neurons, and organotypic brain slices from an AD mouse model, we found that pharmacological inhibition of the ECE family of metalloproteases increased intracellular and extracellular Aß levels and promoted the intracellular formation of Aß oligomers, a process that did not require internalization of secreted Aß. In vivo, the accumulation of intraneuronal Aß aggregates was accompanied by increased levels of both extracellular and exosome-associated Aß, including oligomeric species. Neuronal exosomes were found to contain both ECE-1 and -2 activities, suggesting that multivesicular bodies are intracellular sites of Aß degradation by these enzymes. ECE dysfunction could lead to the accumulation of intraneuronal Aß aggregates and their subsequent release into the extracellular space via exosomes.-Pacheco-Quinto, J., Clausen, D., Pérez-González, R., Peng, H., Meszaros, A., Eckman, C. B., Levy, E., Eckman, E. A. Intracellular metalloprotease activity controls intraneuronal Aß aggregation and limits secretion of Aß via exosomes.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Exosomas/metabolismo , Metaloendopeptidasas/metabolismo , Agregación Patológica de Proteínas/metabolismo , Enfermedad de Alzheimer/metabolismo , Animales , Encéfalo/metabolismo , Línea Celular Tumoral , Endosomas/metabolismo , Enzimas Convertidoras de Endotelina/metabolismo , Espacio Extracelular/metabolismo , Femenino , Humanos , Lisosomas/metabolismo , Masculino , Ratones , Cuerpos Multivesiculares/metabolismo , Neuronas/metabolismo , ProteolisisRESUMEN
Background: Lyme encephalopathy, characterized by nonspecific neurobehavioral symptoms including mild cognitive difficulties, may occur in patients with systemic Lyme disease and is often mistakenly attributed to central nervous system (CNS) infection. Identical symptoms occur in many inflammatory states, possibly reflecting the effect of systemic immune mediators on the CNS. Methods: Multiplex immunoassays were used to measure serum and cerebrospinal fluid (CSF) cytokines in patients with or without Lyme disease to determine if there are specific markers of active CNS infection (neuroborreliosis), or systemic inflammatory mediators associated with neurobehavioral syndromes. Results: CSF CXCL13 levels were elevated dramatically in confirmed neuroborreliosis (n = 8), less so in possible neuroborreliosis (n = 11) and other neuroinflammatory conditions (n = 44). Patients with Lyme (n = 63) or non-Lyme (n = 8) encephalopathy had normal CSF findings, but had elevated serum levels of interleukins 7, 17A, and 17F, thymic stromal lymphopoietin and macrophage inflammatory protein-α. Conclusions: CSF CXCL13 is a sensitive and specific marker of neuroborreliosis in individuals with Borrelia-specific intrathecal antibody production. However, it does not distinguish individuals strongly suspected of having neuroborreliosis, but lacking confirmatory intrathecal antibodies, from those with other neuroinflammatory conditions. Patients with mild cognitive symptoms occurring during acute Lyme disease, and/or after appropriate treatment, have normal CSF but elevated serum levels of T-helper 17 markers and T-cell growth factors, which are also elevated in patients without Lyme disease but with similar symptoms. In the absence of CSF abnormalities, neurobehavioral symptoms appear to be associated with systemic inflammation, not CNS infection or inflammation, and are not specific to Lyme disease.
Asunto(s)
Encefalopatías/inmunología , Encefalopatías/microbiología , Quimiocina CXCL13/líquido cefalorraquídeo , Factores Inmunológicos , Neuroborreliosis de Lyme/inmunología , Adulto , Anticuerpos Antibacterianos/sangre , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Borrelia , Quimiocina CCL3/sangre , Citocinas/sangre , Citocinas/líquido cefalorraquídeo , Femenino , Humanos , Técnicas para Inmunoenzimas , Interleucina-17/sangre , Interleucina-7/sangre , Neuroborreliosis de Lyme/diagnóstico , Masculino , Persona de Mediana Edad , Linfopoyetina del Estroma TímicoRESUMEN
Impairments in Aß removal are increasingly being considered as a possible cause for the abnormal Aß build-up typical of Alzheimer disease. Of particular interest is a pool of Aß that accumulates intraneuronally and may contribute to neuronal toxicity. The mechanism for intraneuronal accumulation, however, is not well understood and is commonly attributed to impaired removal of extracellular Aß by neurons. Based on the intracellular distribution of the well established Aß degrading enzymes, ECE-1 and ECE-2, we tested whether impairments in their catalytic activity could lead to intracellular Aß accumulation. Using SH-SY5Y cells overexpressing wild-type amyloid precursor protein and pharmacological inhibition of endogenous ECE activity, we found that ECEs participate in the degradation of at least two distinct pools of Aß; one destined for secretion and the other being produced and degraded within the endosomal-autophagic-lysosomal pathways. Although ECE-1 regulates both pools of Aß, ECE-2 regulates mainly the intracellular pool of the peptide. Consistent with this result, ECE-2 was found to co-localize with markers of the endosomal/lysosomal pathway but not with a trans-Golgi network marker. Furthermore, ECE-2 was detected in autophagic vesicles in cells treated with chloroquine. Under these conditions, ECE inhibition produced significantly higher elevations in intracellular Aß than chloroquine treatment alone. This study highlights the existence of Aß clearance mechanisms by ECEs at intracellular sites of production. Alterations in ECE activity may be considered as a cause for increased intraneuronal Aß in Alzheimer disease.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Endosomas/metabolismo , Lisosomas/metabolismo , Metaloendopeptidasas/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/química , Autofagia , Línea Celular Tumoral , ADN Complementario/metabolismo , Progresión de la Enfermedad , Enzimas Convertidoras de Endotelina , Endotelinas/metabolismo , Ensayo de Inmunoadsorción Enzimática/métodos , Humanos , Neuronas/metabolismo , Red trans-Golgi/metabolismoRESUMEN
Krabbe disease (KD) is a progressive and devasting neurological disorder that leads to the toxic accumulation of psychosine in the white matter of the central nervous system (CNS). The condition is inherited via biallelic, loss-of-function mutations in the galactosylceramidase (GALC) gene. To rescue GALC gene function in the CNS of the twitcher mouse model of KD, an adeno-associated virus serotype 1 vector expressing murine GALC under control of a chicken ß-actin promoter (AAV1-GALC) was administered to newborn mice by unilateral intracerebroventricular injection. AAV1-GALC treatment significantly improved body weight gain and survival of the twitcher mice (n = 8) when compared with untreated controls (n = 5). The maximum weight gain after postnatal day 10 was significantly increased from 81% to 217%. The median lifespan was extended from 43 days to 78 days (range: 74-88 days) in the AAV1-GALC-treated group. Widespread expression of GALC protein and alleviation of KD neuropathology were detected in the CNS of the treated mice when examined at the moribund stage. Functionally, elevated levels of psychosine were completely normalized in the forebrain region of the treated mice. In the posterior region, which includes the mid- and the hindbrain, psychosine was reduced by an average of 77% (range: 53-93%) compared to the controls. Notably, psychosine levels in this region were inversely correlated with body weight and lifespan of AAV1-GALC-treated mice, suggesting that the degree of viral transduction of posterior brain regions following ventricular injection determined treatment efficacy on growth and survivability, respectively. Overall, our results suggest that viral vector delivery via the cerebroventricular system can partially correct psychosine accumulation in brain that leads to slower disease progression in KD.
Asunto(s)
Leucodistrofia de Células Globoides , Sustancia Blanca , Animales , Ratones , Galactosilceramidasa , Leucodistrofia de Células Globoides/genética , Leucodistrofia de Células Globoides/terapia , Psicosina , Longevidad/genética , Hidrolasas , Prosencéfalo , Peso CorporalRESUMEN
Globoid cell leukodystrophy (GLD) (Krabbe disease) is an autosomal recessive, degenerative, lysosomal storage disease caused by a severe loss of galactocerebrosidase (GALC) enzymatic activity. Of the >70 disease-causing mutations in the GALC gene, most are located outside of the catalytic domain of the enzyme. To determine how GALC mutations impair enzymatic activity, we investigated the impact of multiple disease-causing mutations on GALC processing, localization, and enzymatic activity. Studies in mammalian cells revealed dramatic decreases in GALC activity and a lack of appropriate protein processing into an N-terminal GALC fragment for each of the mutants examined. Consistent with this, we observed significantly less GALC localized to the lysosome and impairment in either the secretion or reuptake of mutant GALC. Notably, the D528N mutation was found to induce hyperglycosylation and protein misfolding. Reversal of these conditions resulted in an increase in proper processing and GALC activity, suggesting that glycosylation may play a critical role in the disease process in patients with this mutation. Recent studies have shown that enzyme inhibitors can sometimes "chaperone" misfolded polypeptides to their appropriate target organelle, bypassing the normal cellular quality control machinery and resulting in enhanced activity. To determine whether this may also work for GLD, we examined the effect of alpha-lobeline, an inhibitor of GALC, on D528N mutant cells. After treatment, GALC activity was significantly increased. This study suggests that mutations in GALC can cause GLD by impairing protein processing and/or folding and that pharmacological chaperones may be potential therapeutic agents for patients carrying certain mutations.
Asunto(s)
Galactosilceramidasa/genética , Leucodistrofia de Células Globoides/tratamiento farmacológico , Leucodistrofia de Células Globoides/genética , Chaperonas Moleculares/genética , Chaperonas Moleculares/uso terapéutico , Animales , Células COS , Chlorocebus aethiops , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Galactosilceramidasa/antagonistas & inhibidores , Galactosilceramidasa/metabolismo , Humanos , Leucodistrofia de Células Globoides/enzimología , Chaperonas Moleculares/farmacología , Mutagénesis Sitio-Dirigida , Pliegue de Proteína/efectos de los fármacos , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Procesamiento Proteico-Postraduccional/genéticaRESUMEN
Amyloid precursor protein (APP) family members and their proteolytic products are implicated in normal nervous system function and Alzheimer's disease pathogenesis. APP processing and Aß secretion are regulated by neuronal activity. Various data suggest that NMDA receptor (NMDAR) activity plays a role in both non-amyloidogenic and amyloidogenic APP processing depending on whether synaptic or extrasynaptic NMDARs are activated, respectively. The APP-interacting FE65 proteins modulate APP trafficking and processing in cell lines, but little is known about their contribution to APP trafficking and processing in neurons, either in vivo or in vitro. In this study, we examined the contribution of the FE65 protein family to APP trafficking and processing in WT and FE65/FE65L1 double knockout neurons under basal conditions and following NMDAR activation. We report that FE65 proteins facilitate neuronal Aß secretion without affecting APP fast axonal transport to pre-synaptic terminals. In addition, FE65 proteins facilitate an NMDAR-dependent non-amyloidogenic APP processing pathway. Generation of high-molecular weight (HMW) species bearing an APP C-terminal epitope was also observed following NMDAR activation. These HMW species require proteasomal and calpain activities for their accumulation. Recovery of APP polypeptide fragments from electroeluted HMW species having molecular weights consistent with calpain I cleavage of APP suggests that HMW species are complexes formed from APP metabolic products. Our results indicate that the FE65 proteins contribute to physiological APP processing and accumulation of APP metabolic products resulting from NMDAR activation.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Proteínas del Tejido Nervioso/fisiología , Proteínas Nucleares/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Péptidos beta-Amiloides/metabolismo , Animales , Transporte Axonal/fisiología , Western Blotting , Calpaína/farmacología , Células Cultivadas , Electroforesis en Gel de Poliacrilamida , Glicosilación , Ratones , Ratones Endogámicos ICR , Ratones Noqueados , Peso Molecular , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas Nucleares/genética , Fragmentos de Péptidos/metabolismo , Fosforilación , Polisacáridos/química , Complejo de la Endopetidasa Proteasomal/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Receptores de N-Metil-D-Aspartato/efectos de los fármacos , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
Proteolytic processing of the amyloid precursor protein (APP) by beta-secretase, beta-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid beta (Abeta) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP(C)), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, remains enigmatic. Because both APP and PrP(C) are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP(C) in the proteolytic processing of APP. Cellular overexpression of PrP(C) inhibited the beta-secretase cleavage of APP and reduced Abeta formation. Conversely, depletion of PrP(C) in mouse N2a cells by siRNA led to an increase in Abeta peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Abeta levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the beta-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP(C) on the beta-secretase cleavage of APP required the localization of PrP(C) to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP(C) via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Abeta is regulated by PrP(C) and may have implications for both Alzheimer's and prion diseases.
Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Priones/fisiología , Enfermedad de Alzheimer/etiología , Péptidos beta-Amiloides/metabolismo , Animales , Sitios de Unión , Línea Celular , Humanos , Microdominios de Membrana , Ratones , Mutación , Enfermedades por Prión/etiología , Priones/genética , Priones/metabolismoRESUMEN
For millennia, ginseng and some of its components have been used to treat a wide variety of medical conditions, including age-related memory impairment. Because of its purported effects and apparently low rate of side effects, ginseng remains one of the top selling natural product remedies in the United States. Given its potential role for improving age-related memory impairments and its common use in China for the treatment of Alzheimer's disease-like symptoms, we analyzed the effects of commercially available preparations of ginseng on the accumulation of the Alzheimer's amyloid beta peptide (Abeta) in a cell-based model system. In this model system, ginseng treatment resulted in a significant reduction in the levels of Abeta in the conditioned medium. We next examined the effects of several compounds isolated from ginseng and found that certain ginsenosides lowered Abeta concentration in a dose-dependent manner with ginsenoside Rg3 having an approximate IC50 of under 25 microM against Abeta42. Furthermore, we found that three of these isolated components, ginsenoside Rg1, Rg3, and RE, resulted in significant reductions in the amount of Abeta detected in the brains of animals after single oral doses of these agents. The results indicate that ginseng itself, or purified ginsenosides, may have similarly useful effects in human disease.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Ginsenósidos/farmacología , Administración Oral , Precursor de Proteína beta-Amiloide/genética , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Células CHO , Cricetinae , Cricetulus , Femenino , Ginsenósidos/administración & dosificación , Humanos , Ratones , Ratones Transgénicos , Panax/química , Fragmentos de Péptidos/metabolismo , Extractos Vegetales/administración & dosificación , Extractos Vegetales/farmacologíaRESUMEN
Alzheimer's disease (AD) is a devastating neurodegenerative disease. To rationally develop novel therapeutic and/or preventative agents for AD, an understanding of the etiology and pathogenesis of this complex disease is necessary. This article examines the evidence for the amyloid hypothesis of AD pathogenesis and discusses how it relates to the neurological and neuropathological features of AD, the known genetic risk factors and causative mutations, and the heightened risk associated with advanced age.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/genética , Amiloidosis/genética , Amiloidosis/patología , Anciano , Animales , HumanosRESUMEN
Impaired clearance of amyloid-ß peptide (Aß) has been postulated to significantly contribute to the amyloid accumulation typical of Alzheimer's disease. Among the enzymes known to degrade Aß in vivo are endothelin-converting enzyme (ECE)-1, ECE-2, and neprilysin (NEP), and evidence suggests that they regulate independent pools of Aß that may be functionally significant. To better understand the differential regulation of Aß concentration by its physiological degrading enzymes, we characterized the cell and region-specific expression pattern of ECE-1, ECE-2, and NEP by in situ hybridization and immunohistochemistry in brain areas relevant to Alzheimer's disease. In contrast to the broader distribution of ECE-1, ECE-2 and NEP were found enriched in GABAergic neurons. ECE-2 was majorly expressed by somatostatin-expressing interneurons and was active in isolated synaptosomes. NEP messenger RNA was found mainly in parvalbumin-expressing interneurons, with NEP protein localized to perisomatic parvalbuminergic synapses. The identification of somatostatinergic and parvalbuminergic synapses as hubs for Aß degradation is consistent with the possibility that Aß may have a physiological function related to the regulation of inhibitory signaling.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Enzimas Convertidoras de Endotelina/metabolismo , Neuronas GABAérgicas/enzimología , Hipocampo/citología , Hipocampo/enzimología , Neocórtex/citología , Neocórtex/enzimología , Neprilisina/metabolismo , Enfermedad de Alzheimer/etiología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/fisiología , Animales , Enzimas Convertidoras de Endotelina/genética , Enzimas Convertidoras de Endotelina/fisiología , Expresión Génica , Ratones Transgénicos , Neprilisina/genética , Neprilisina/fisiología , ARN Mensajero/metabolismo , Sinapsis/enzimologíaRESUMEN
Alzheimer's disease (AD) is the most common form of dementia in the elderly. Without a treatment that significantly delays the progression of the disease over 14 million Americans are likely to be affected with AD by the middle of the 21st Century, presenting an enormous economic and social burden. Evidence gathered over the last two decades has implicated the abnormal accumulation of A beta, in particular the longer more amyloidogenic form A beta42, as a potential causative agent in the disease. To screen for compounds that reduce A beta accumulation we have established several high throughput, cell based screens capable of the sensitive and selective detection of A beta40 and A beta42. Using these screens we have analyzed a proprietary library of natural product extracts for their ability to influence A beta accumulation. Using this approach, we have identified several agents capable of influencing total A beta concentration. In addition, we have identified one extract that selectively reduces A beta42. Intracerebroventricular administration of this agent to mice results in a selective reduction in A beta42 in the brain.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Fármacos Neuroprotectores/farmacología , Péptidos beta-Amiloides/efectos de los fármacos , Animales , Western Blotting , Supervivencia Celular , Células Cultivadas , Evaluación Preclínica de Medicamentos , Ensayo de Inmunoadsorción Enzimática , Humanos , Ratones , Fragmentos de Péptidos/metabolismoRESUMEN
The efficient clearance of amyloid-ß (Aß) is essential to modulate levels of the peptide in the brain and to prevent it from accumulating in senile plaques, a hallmark of Alzheimer's disease (AD) pathology.We and others have shown that failure in Aß catabolism can produce elevations in Aß concentration similar to those observed in familial forms of AD. Based on the available evidence, it remains plausible that in late-onset AD, disturbances in the activity of Aß degrading enzymes could induce Aß accumulation, and that this increase could result in AD pathology. The following review presents a historical perspective of the parallel discovery of three vasopeptidases (neprilysin and endothelin-converting enzymes-1 and -2) as important Aß degrading enzymes. The recognition of the role of these vasopeptidases in Aß degradation, beyond bringing to light a possible explanation of how cardiovascular risk factors may influence AD risk, highlights a possible risk of the use of inhibitors of these enzymes for other clinical indications such as hypertension. We will discuss in detail the experiments conducted to assess the impact of vasopeptidase deficiency (through pharmacological inhibition or genetic mutation) on Aß accumulation, as well as the cooperative effect of multiple Aß degrading enzymes to regulate the concentration of the peptide at multiple sites, both intracellular and extracellular, throughout the brain.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/enzimología , Metaloendopeptidasas/metabolismo , Metaloproteasas/metabolismo , Neprilisina/metabolismo , Peptidil-Dipeptidasa A/metabolismo , Animales , Enzimas Convertidoras de Endotelina , Humanos , RatonesRESUMEN
Targeted deletion of two members of the FE65 family of adaptor proteins, FE65 and FE65L1, results in cortical dysplasia. Heterotopias resembling those found in cobblestone lissencephalies in which neuroepithelial cells migrate into superficial layers of the developing cortex, aberrant cortical projections and loss of infrapyramidal mossy fibers arise in FE65/FE65L1 compound null animals, but not in single gene knockouts. The disruption of pial basal membranes underlying the heterotopias and poor organization of fibrillar laminin by isolated meningeal fibroblasts from double knockouts suggests that FE65 proteins are involved in basement membrane assembly. A similar phenotype is observed in triple mutant mice lacking the APP family members APP, APLP1 and APLP2, all of which interact with FE65 proteins, suggesting that this phenotype may be caused by decreased transmission of an APP-dependent signal through the FE65 proteins. The defects observed in the double knockout may also involve the family of Ena/Vasp proteins, which participate in actin cytoskeleton remodeling and interact with the WW domains of FE65 proteins.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Basal/crecimiento & desarrollo , Corteza Cerebral/crecimiento & desarrollo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Axones/patología , Membrana Basal/patología , Corteza Cerebral/patología , Fibroblastos/citología , Inmunohistoquímica , Hibridación in Situ , Meninges/citología , Ratones , Ratones Noqueados , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/genéticaRESUMEN
The deposition of beta-amyloid in the brain is a pathological hallmark of Alzheimer disease (AD). Normally, the accumulation of beta-amyloid is prevented in part by the activities of several degradative enzymes, including the endothelin-converting enzymes, neprilysin, insulin-degrading enzyme, and plasmin. Recent reports indicate that another metalloprotease, angiotensin-converting enzyme (ACE), can degrade beta-amyloid in vitro and in cellular overexpression experiments. In addition, ACE gene variants are linked to AD risk in several populations. Angiotensin-converting enzyme, neprilysin and endothelin-converting enzyme function as vasopeptidases and are the targets of drugs designed to treat cardiovascular disorders, and ACE inhibitors are commonly prescribed. We investigated the potential physiological role of ACE in regulating endogenous brain beta-amyloid levels for two reasons: first, to determine whether beta-amyloid degradation might be the mechanism by which ACE is associated with AD, and second, to determine whether ACE inhibitor drugs might block beta-amyloid degradation in the brain and potentially increase the risk for AD. We analyzed beta-amyloid accumulation in brains from ACE-deficient mice and in mice treated with ACE inhibitors and found that ACE deficiency did not alter steady-state beta-amyloid concentration. In contrast, beta-amyloid levels are significantly elevated in endothelin-converting enzyme and neprilysin knock-out mice, and inhibitors of these enzymes cause a rapid increase in beta-amyloid concentration in the brain. The results of these studies do not support a physiological role for ACE in the degradation of beta-amyloid in the brain but confirm roles for endothelin-converting enzyme and neprilysin and indicate that reductions in these enzymes result in additive increases in brain amyloid beta-peptide levels.
Asunto(s)
Péptidos beta-Amiloides/química , Ácido Aspártico Endopeptidasas/metabolismo , Regulación Enzimológica de la Expresión Génica , Metaloendopeptidasas/metabolismo , Neprilisina/fisiología , Peptidil-Dipeptidasa A/metabolismo , Administración Oral , Péptidos beta-Amiloides/metabolismo , Animales , Encéfalo/metabolismo , Enzimas Convertidoras de Endotelina , Inhibidores Enzimáticos/farmacología , Humanos , Ratones , Ratones NoqueadosRESUMEN
The abnormal accumulation of beta-amyloid (Abeta) in the brain is an early and invariant feature in Alzheimer's disease (AD) and is believed to play a pivotal role in the etiology and pathogenesis of the disease. As such, a major focus of AD research has been the elucidation of the mechanisms responsible for the generation of Abeta. As with any peptide, however, the degree of Abeta accumulation is dependent not only on its production but also on its removal. In cell-based and in vitro models we have previously characterized endothelin-converting enzyme-1 (ECE-1) as an Abeta-degrading enzyme that appears to act intracellularly, thus limiting the amount of Abeta available for secretion. To determine the physiological significance of this activity, we analyzed Abeta levels in the brains of mice deficient for ECE-1 and a closely related enzyme, ECE-2. Significant increases in the levels of both Abeta40 and Abeta42 were found in the brains of these animals when compared with age-matched littermate controls. The increase in Abeta levels in the ECE-deficient mice provides the first direct evidence for a physiological role for both ECE-1 and ECE-2 in limiting Abeta accumulation in the brain and also provides further insight into the factors involved in Abeta clearance in vivo.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/biosíntesis , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/fisiología , Precursor de Proteína beta-Amiloide/biosíntesis , Animales , Western Blotting , Enzimas Convertidoras de Endotelina , Ensayo de Inmunoadsorción Enzimática , Metaloendopeptidasas , Ratones , Ratones Noqueados , Estructura Terciaria de ProteínaRESUMEN
The causes of cerebral accumulation of amyloid beta-protein (Abeta) in most cases of Alzheimer's disease (AD) remain unknown. We recently found that homozygous deletion of the insulin-degrading enzyme (IDE) gene in mice results in an early and marked elevation of cerebral Abeta. Both genetic linkage and allelic association in the IDE region of chromosome 10 have been reported in families with late-onset AD. For IDE to remain a valid candidate gene for late-onset AD on functional grounds, it must be shown that partial loss of function of IDE can still alter Abeta degradation, but without causing early, severe elevation of brain Abeta. Here, we show that naturally occurring IDE missense mutations in a well-characterized rat model of type 2 diabetes mellitus (DM2) result in decreased catalytic efficiency and a significant approximately 15 to 30% deficit in the degradation of both insulin and Abeta. Endogenously secreted Abeta(40) and Abeta(42) are significantly elevated in primary neuronal cultures from animals with the IDE mutations, but there is no increase in steady-state levels of rodent Abeta in the brain up to age 14 months. We conclude that naturally occurring, partial loss-of-function mutations in IDE sufficient to cause DM2 also impair neuronal regulation of Abeta levels, but the brain can apparently compensate for the partial deficit during the life span of the rat. Our findings have relevance for the emerging genetic evidence suggesting that IDE may be a late-onset AD-risk gene, and for the epidemiological relationships among hyperinsulinemia, DM2, and AD.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Diabetes Mellitus Experimental/genética , Insulisina/genética , Neuronas/metabolismo , Enfermedad de Alzheimer/genética , Animales , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Diabetes Mellitus Tipo 2/genética , Humanos , Immunoblotting , Insulina/metabolismo , Mutación Missense , Ratas , TransfecciónRESUMEN
Factors that elevate amyloid-beta (Abeta) peptide levels are associated with an increased risk for Alzheimer's disease. Insulysin has been identified as one of several proteases potentially involved in Abeta degradation based on its hydrolysis of Abeta peptides in vitro. In this study, in vivo levels of brain Abeta40 and Abeta42 peptides were found to be increased significantly (1.6- and 1.4-fold, respectively) in an insulysin-deficient gene-trap mouse model. A 6-fold increase in the level of the gamma-secretase-generated C-terminal fragment of the Abeta precursor protein in the insulysin-deficient mouse also was found. In mice heterozygous for the insulysin gene trap, in which insulysin activity levels were decreased approximately 50%, brain Abeta peptides were increased to levels intermediate between those in wild-type mice and homozygous insulysin gene-trap mice that had no detectable insulysin activity. These findings indicate that there is an inverse correlation between in vivo insulysin activity levels and brain Abeta peptide levels and suggest that modulation of insulysin activity may alter the risk for Alzheimer's disease.