RESUMEN
Strong evidence supports the hypothesis that synapse damage and memory impairment in early Alzheimer Disease (AD) might be due to synaptic failure caused by amyloid beta oligomers (AßOs). The preclinical efficacy of a single-chain variable fragment (scFv) antibody NUsc1 that selectively targets a subpopulation of AßOs has been demonstrated; NUsc1 prevented inhibition of AßO-inducedlong-term potentiation in hippocampal slices and short-term memory impairment in mice. Since specific targeting of AßOs by NUsc1 can be a substantial improvement in target engagement and efficacy of AD therapy, an adeno-associated virus (AAV) vector was developed to drive neuronal expression of NUsc1 within the brain. AAV-NUsc1 rescued Short-Term Memory (STM) for objectand conspecific interaction in mouse models of AD. In the McGill-R-Thy1-APP (Tg+/) heterozygous transgenic McGill-R-Thy1-APP (Tg+/) rat model of AD, progressive amyloid pathology is ac-companied by cognitive impairment involving long-term memory (LTM) decline. LTM in a Novel-Object-Recognition (NOR) task was impaired in 4-month-old (Tg+/) male rats, suggesting that they are unable to form/evoke such discriminative memories. Hence, it was investigated if AAV-NUsc1 treatment could rescued this memory. 10-12 weeks-old either Tg or wild type male rats were i.c.v. infused with AAV-NUsc1. Two months later, short-term exploratory behavior, habituation to an Open Field (OF), object discrimination and LTM for objects were assessed. AAV-NUsc1 treated Tg rats were able to successfully perform the task 24 h after training, denoting recovery of LT discrimination capacity and LTM formation. Wild type rats successfully performed the task either treated or not with AAV-NUsc1. In addition, exploration and short-term habituation to an open field was preserved in Tg+/ rats either treated or not. Our present and previous results suggest that AAV-NUsc1 represents a significant advance in gene therapy, supporting the feasibility of immunotherapy using viral vector-mediated NUsc1 gene delivery as a potential therapeutic approach in AD.
Evidencias conspicuas respaldan la hipótesis de que la presencia de oligómeros beta-amiloides (AßO) ocasiona un deterioro sináptico y de la memoria en etapas tempranas de la Enfermedad de Alzheimer (AD). Se ha demostrado que el anticuerpo de cadena única y Fragmento variable (scFv) NUsc1, que une selectivamente una subpoblación de AßO, evitó el deterioro de la memoria a corto plazo, inducido por AßO en ratones. Dado que la selectividad de NUsc1 mejora sustancialmente la detección del AßO, y consecuentemente su eficacia terapéutica para AD, se ha desarrollado un vector derivado de Virus Adenoasociado para expresar NUsc1 (AAV-NUsc1) en el cerebro. AAV-NUsc1 rescató la Memoria de Corto Plazo (STM) para el reconocimiento de objetos e interacción con congéneres en ratones modelo de AD. La rata McGill-R-Thy1-APP transgénica heterocigota (Tg+/) modelo de AD, sufre una patología amiloide progresiva acompañada de deterioro cognitivo, que afecta la Memoria de Largo Plazo (LTM) de Reconocimiento de Objetos (NOR) evitando su formación/evocación. Evaluando si el tratamiento con AAV-NUsc1 podría rescatar la LTM de reconocimien-to en ratas macho (Tg+/) de 4 meses. Ratas macho Tg y de genotipo salvaje (Wt) de 10-12 semanas fueron infundidas i.c.v con AAV-NUsc1. Dos meses más tarde, se evaluaron: el comportamiento exploratorio a corto plazo, la habituación a un Campo Abierto (OF), la discriminación y LTM para objetos. 24 h después del entrenamiento se observó que las ratas Tg tratadas con AAV-NUsc1 recuperaron la capacidad de expresar una LTM y de reconocer objetos novedosos. De manera similar, las ratas Wt tratadas con AAV-NUsc1 y las del grupo control, realizaron la tarea con éxito.La exploración y habituación al OF fueron similares para ratas Tg+/ y Wt, tratadas y control. Nuestros resultados sugieren que AAV-NUsc1 representa un avance significativo en terapia génica, respaldando la viabilidad de la inmunoterapia mediada por vectores virales aportando el gen de NUsc1 como posible enfoque terapéutico para AD.
Asunto(s)
Humanos , Enfermedad de AlzheimerRESUMEN
The accumulation of amyloid protein aggregates in tissues is the basis for the onset of diseases known as amyloidoses. Intriguingly, many amyloidoses impact the central nervous system (CNS) and usually are devastating diseases. It is increasingly apparent that neurotoxic soluble oligomers formed by amyloidogenic proteins are the primary molecular drivers of these diseases, making them lucrative diagnostic and therapeutic targets. One promising diagnostic/therapeutic strategy has been the development of antibody fragments against amyloid oligomers. Antibody fragments, such as fragment antigen-binding (Fab), scFv (single chain variable fragments), and VHH (heavy chain variable domain or single-domain antibodies) are an alternative to full-length IgGs as diagnostics and therapeutics for a variety of diseases, mainly because of their increased tissue penetration (lower MW compared to IgG), decreased inflammatory potential (lack of Fc domain), and facile production (low structural complexity). Furthermore, through the use of in vitro-based ligand selection, it has been possible to identify antibody fragments presenting marked conformational selectivity. In this review, we summarize significant reports on antibody fragments selective for oligomers associated with prevalent CNS amyloidoses. We discuss promising results obtained using antibody fragments as both diagnostic and therapeutic agents against these diseases. In addition, the use of antibody fragments, particularly scFv and VHH, in the isolation of unique oligomeric assemblies is discussed as a strategy to unravel conformational moieties responsible for neurotoxicity. We envision that advances in this field may lead to the development of novel oligomer-selective antibody fragments with superior selectivity and, hopefully, good clinical outcomes.
Asunto(s)
Amiloide/inmunología , Amiloidosis/diagnóstico , Síndromes de Neurotoxicidad/diagnóstico , Agregación Patológica de Proteínas/diagnóstico , Amiloide/antagonistas & inhibidores , Amiloidosis/inmunología , Amiloidosis/patología , Animales , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/patología , Humanos , Fragmentos Fab de Inmunoglobulinas/inmunología , Fragmentos de Inmunoglobulinas/inmunología , Síndromes de Neurotoxicidad/inmunología , Síndromes de Neurotoxicidad/patología , Fragmentos de Péptidos/inmunología , Agregación Patológica de Proteínas/inmunología , Anticuerpos de Dominio Único , Relación Estructura-ActividadRESUMEN
BACKGROUND: Slice cultures have been prepared from several organs. With respect to the brain, advantages of slice cultures over dissociated cell cultures include maintenance of the cytoarchitecture and neuronal connectivity. Slice cultures from adult human brain have been reported and constitute a promising method to study neurological diseases. Despite this potential, few studies have characterized in detail cell survival and function along time in short-term, free-floating cultures. NEW METHOD: We used tissue from adult human brain cortex from patients undergoing temporal lobectomy to prepare 200 µm-thick slices. Along the period in culture, we evaluated neuronal survival, histological modifications, and neurotransmitter release. The toxicity of Alzheimer's-associated Aß oligomers (AßOs) to cultured slices was also analyzed. RESULTS: Neurons in human brain slices remain viable and neurochemically active for at least four days in vitro, which allowed detection of binding of AßOs. We further found that slices exposed to AßOs presented elevated levels of hyperphosphorylated Tau, a hallmark of Alzheimer's disease. COMPARISON WITH EXISTING METHOD(S): Although slice cultures from adult human brain have been previously prepared, this is the first report to analyze cell viability and neuronal activity in short-term free-floating cultures as a function of days in vitro. CONCLUSIONS: Once surgical tissue is available, the current protocol is easy to perform and produces functional slices from adult human brain. These slice cultures may represent a preferred model for translational studies of neurodegenerative disorders when long term culturing in not required, as in investigations on AßO neurotoxicity.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Adulto , Análisis de Varianza , Epilepsia del Lóbulo Temporal/patología , Femenino , Humanos , Técnicas In Vitro , Masculino , Persona de Mediana Edad , Técnicas de Cultivo de Órganos , Fosfopiruvato Hidratasa/metabolismo , Cloruro de Potasio/farmacología , Proteínas tau/metabolismoRESUMEN
Alzheimer's disease (AD) is a devastating neurological disorder that still lacks an effective treatment, and this has stimulated an intense pursuit of disease-modifying therapeutics. Given the increasingly recognized link between AD and defective brain insulin signaling, we investigated the actions of liraglutide, a glucagon-like peptide-1 (GLP-1) analog marketed for treatment of type 2 diabetes, in experimental models of AD. Insulin receptor pathology is an important feature of AD brains that impairs the neuroprotective actions of central insulin signaling. Here, we show that liraglutide prevented the loss of brain insulin receptors and synapses, and reversed memory impairment induced by AD-linked amyloid-ß oligomers (AßOs) in mice. Using hippocampal neuronal cultures, we determined that the mechanism of neuroprotection by liraglutide involves activation of the PKA signaling pathway. Infusion of AßOs into the lateral cerebral ventricle of non-human primates (NHPs) led to marked loss of insulin receptors and synapses in brain regions related to memory. Systemic treatment of NHPs with liraglutide provided partial protection, decreasing AD-related insulin receptor, synaptic, and tau pathology in specific brain regions. Synapse damage and elimination are amongst the earliest known pathological changes and the best correlates of memory impairment in AD. The results illuminate mechanisms of neuroprotection by liraglutide, and indicate that GLP-1 receptor activation may be harnessed to protect brain insulin receptors and synapses in AD. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.
Asunto(s)
Disfunción Cognitiva/tratamiento farmacológico , Liraglutida/farmacología , Memoria/efectos de los fármacos , Receptor de Insulina/efectos de los fármacos , Sinapsis/patología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Animales , Modelos Animales de Enfermedad , Hipocampo/efectos de los fármacos , Hipoglucemiantes/farmacología , Masculino , Ratones , Receptor de Insulina/metabolismo , Sinapsis/efectos de los fármacosRESUMEN
Alzheimer's disease (AD) is associated with peripheral metabolic disorders. Clinical/epidemiological data indicate increased risk of diabetes in AD patients. Here, we show that intracerebroventricular infusion of AD-associated Aß oligomers (AßOs) in mice triggered peripheral glucose intolerance, a phenomenon further verified in two transgenic mouse models of AD. Systemically injected AßOs failed to induce glucose intolerance, suggesting AßOs target brain regions involved in peripheral metabolic control. Accordingly, we show that AßOs affected hypothalamic neurons in culture, inducing eukaryotic translation initiation factor 2α phosphorylation (eIF2α-P). AßOs further induced eIF2α-P and activated pro-inflammatory IKKß/NF-κB signaling in the hypothalamus of mice and macaques. AßOs failed to trigger peripheral glucose intolerance in tumor necrosis factor-α (TNF-α) receptor 1 knockout mice. Pharmacological inhibition of brain inflammation and endoplasmic reticulum stress prevented glucose intolerance in mice, indicating that AßOs act via a central route to affect peripheral glucose homeostasis. While the hypothalamus has been largely ignored in the AD field, our findings indicate that AßOs affect this brain region and reveal novel shared molecular mechanisms between hypothalamic dysfunction in metabolic disorders and AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Hipotálamo/metabolismo , Oligonucleótidos/metabolismo , Nervios Periféricos/metabolismo , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/genética , Animales , Femenino , Glucosa/metabolismo , Humanos , Macaca , Masculino , Ratones , Ratones Endogámicos C57BL , FN-kappa B/genética , FN-kappa B/metabolismo , Neuronas/metabolismo , Oligonucleótidos/genética , Ratas , Transducción de Señal , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Alzheimer's disease (AD) is a devastating neurodegenerative disorder and a major medical problem. Here, we have investigated the impact of amyloid-ß (Aß) oligomers, AD-related neurotoxins, in the brains of rats and adult nonhuman primates (cynomolgus macaques). Soluble Aß oligomers are known to accumulate in the brains of AD patients and correlate with disease-associated cognitive dysfunction. When injected into the lateral ventricle of rats and macaques, Aß oligomers diffused into the brain and accumulated in several regions associated with memory and cognitive functions. Cardinal features of AD pathology, including synapse loss, tau hyperphosphorylation, astrocyte and microglial activation, were observed in regions of the macaque brain where Aß oligomers were abundantly detected. Most importantly, oligomer injections induced AD-type neurofibrillary tangle formation in the macaque brain. These outcomes were specifically associated with Aß oligomers, as fibrillar amyloid deposits were not detected in oligomer-injected brains. Human and macaque brains share significant similarities in terms of overall architecture and functional networks. Thus, generation of a macaque model of AD that links Aß oligomers to tau and synaptic pathology has the potential to greatly advance our understanding of mechanisms centrally implicated in AD pathogenesis. Furthermore, development of disease-modifying therapeutics for AD has been hampered by the difficulty in translating therapies that work in rodents to humans. This new approach may be a highly relevant nonhuman primate model for testing therapeutic interventions for AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/toxicidad , Fragmentos de Péptidos/toxicidad , Enfermedad de Alzheimer/inducido químicamente , Péptidos beta-Amiloides/administración & dosificación , Animales , Apoptosis/efectos de los fármacos , Astrocitos/patología , Inyecciones Intraventriculares , Macaca fascicularis , Masculino , Microglía/patología , Microinyecciones , Ovillos Neurofibrilares/patología , Fragmentos de Péptidos/administración & dosificación , Ratas , Ratas Wistar , Sinapsis/patología , Sinapsis/fisiología , Sinapsis/ultraestructuraRESUMEN
Alzheimer's disease (AD) and type 2 diabetes appear to share similar pathogenic mechanisms. dsRNA-dependent protein kinase (PKR) underlies peripheral insulin resistance in metabolic disorders. PKR phosphorylates eukaryotic translation initiation factor 2α (eIF2α-P), and AD brains exhibit elevated phospho-PKR and eIF2α-P levels. Whether and how PKR and eIF2α-P participate in defective brain insulin signaling and cognitive impairment in AD are unknown. We report that ß-amyloid oligomers, AD-associated toxins, activate PKR in a tumor necrosis factor α (TNF-α)-dependent manner, resulting in eIF2α-P, neuronal insulin receptor substrate (IRS-1) inhibition, synapse loss, and memory impairment. Brain phospho-PKR and eIF2α-P were elevated in AD animal models, including monkeys given intracerebroventricular oligomer infusions. Oligomers failed to trigger eIF2α-P and cognitive impairment in PKR(-/-) and TNFR1(-/-) mice. Bolstering insulin signaling rescued phospho-PKR and eIF2α-P. Results reveal pathogenic mechanisms shared by AD and diabetes and establish that proinflammatory signaling mediates oligomer-induced IRS-1 inhibition and PKR-dependent synapse and memory loss.
Asunto(s)
Péptidos beta-Amiloides/toxicidad , Encéfalo/efectos de los fármacos , Proteínas Sustrato del Receptor de Insulina/metabolismo , Polímeros/toxicidad , Factor de Necrosis Tumoral alfa/metabolismo , eIF-2 Quinasa/metabolismo , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Animales , Encéfalo/metabolismo , Modelos Animales de Enfermedad , Haplorrinos/metabolismo , Hipoglucemiantes/farmacología , Proteínas Sustrato del Receptor de Insulina/antagonistas & inhibidores , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/patología , Ratones , Ratones Noqueados , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Fosforilación/efectos de los fármacos , Polímeros/química , Receptores Tipo I de Factores de Necrosis Tumoral/deficiencia , Receptores Tipo I de Factores de Necrosis Tumoral/genética , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores , eIF-2 Quinasa/deficiencia , eIF-2 Quinasa/genéticaRESUMEN
Brain accumulation of soluble amyloid-ß oligomers (AßOs) has been implicated in synapse failure and cognitive impairment in Alzheimer's disease (AD). However, whether and how oligomers of different sizes induce synapse dysfunction is a matter of controversy. Here, we report that low-molecular-weight (LMW) and high-molecular-weight (HMW) Aß oligomers differentially impact synapses and memory. A single intracerebroventricular injection of LMW AßOs (10 pmol) induced rapid and persistent cognitive impairment in mice. On the other hand, memory deficit induced by HMW AßOs (10 pmol) was found to be reversible. While memory impairment in LMW oligomer-injected mice was associated with decreased hippocampal synaptophysin and GluN2B immunoreactivities, synaptic pathology was not detected in the hippocampi of HMW oligomer-injected mice. On the other hand, HMW oligomers, but not LMW oligomers, induced oxidative stress in hippocampal neurons. Memantine rescued both neuronal oxidative stress and the transient memory impairment caused by HMW oligomers, but did not prevent the persistent cognitive deficit induced by LMW oligomers. Results establish that different Aß oligomer assemblies act in an orchestrated manner, inducing different pathologies and leading to synapse dysfunction. Furthermore, results suggest a mechanistic explanation for the limited efficacy of memantine in preventing memory loss in AD.
Asunto(s)
Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Trastornos del Conocimiento/inducido químicamente , Trastornos del Conocimiento/tratamiento farmacológico , Memantina/farmacología , Fragmentos de Péptidos/farmacología , Péptidos beta-Amiloides/antagonistas & inhibidores , Animales , Células Cultivadas , Trastornos del Conocimiento/metabolismo , Masculino , Ratones , Peso Molecular , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , RatasRESUMEN
Defective brain insulin signaling has been suggested to contribute to the cognitive deficits in patients with Alzheimer's disease (AD). Although a connection between AD and diabetes has been suggested, a major unknown is the mechanism(s) by which insulin resistance in the brain arises in individuals with AD. Here, we show that serine phosphorylation of IRS-1 (IRS-1pSer) is common to both diseases. Brain tissue from humans with AD had elevated levels of IRS-1pSer and activated JNK, analogous to what occurs in peripheral tissue in patients with diabetes. We found that amyloid-ß peptide (Aß) oligomers, synaptotoxins that accumulate in the brains of AD patients, activated the JNK/TNF-α pathway, induced IRS-1 phosphorylation at multiple serine residues, and inhibited physiological IRS-1pTyr in mature cultured hippocampal neurons. Impaired IRS-1 signaling was also present in the hippocampi of Tg mice with a brain condition that models AD. Importantly, intracerebroventricular injection of Aß oligomers triggered hippocampal IRS-1pSer and JNK activation in cynomolgus monkeys. The oligomer-induced neuronal pathologies observed in vitro, including impaired axonal transport, were prevented by exposure to exendin-4 (exenatide), an anti-diabetes agent. In Tg mice, exendin-4 decreased levels of hippocampal IRS-1pSer and activated JNK and improved behavioral measures of cognition. By establishing molecular links between the dysregulated insulin signaling in AD and diabetes, our results open avenues for the investigation of new therapeutics in AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/toxicidad , Hipocampo/efectos de los fármacos , Hipoglucemiantes/uso terapéutico , Proteínas Sustrato del Receptor de Insulina/metabolismo , Resistencia a la Insulina , Insulina/fisiología , Péptidos/uso terapéutico , Ponzoñas/uso terapéutico , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/prevención & control , Enfermedad de Alzheimer/psicología , Animales , Anticuerpos Monoclonales/farmacología , Células Cultivadas/efectos de los fármacos , Células Cultivadas/metabolismo , Exenatida , Femenino , Hipocampo/citología , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Hipoglucemiantes/farmacología , Infliximab , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Macaca fascicularis , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Trastornos de la Memoria/etiología , Trastornos de la Memoria/metabolismo , Trastornos de la Memoria/prevención & control , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Persona de Mediana Edad , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Péptidos/farmacología , Fosforilación , Procesamiento Proteico-Postraduccional , Ratas , Ponzoñas/farmacologíaRESUMEN
Dysregulated cholinergic signaling is an early hallmark of Alzheimer disease (AD), usually ascribed to degeneration of cholinergic neurons induced by the amyloid-ß peptide (Aß). It is now generally accepted that neuronal dysfunction and memory deficits in the early stages of AD are caused by the neuronal impact of soluble Aß oligomers (AßOs). AßOs build up in AD brain and specifically attach to excitatory synapses, leading to synapse dysfunction. Here, we have investigated the possibility that AßOs could impact cholinergic signaling. The activity of choline acetyltransferase (ChAT, the enzyme that carries out ACh production) was inhibited by ~50% in cultured cholinergic neurons exposed to low nanomolar concentrations of AßOs. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction, lactate dehydrogenase release, and [(3)H]choline uptake assays showed no evidence of neuronal damage or loss of viability that could account for reduced ChAT activity under these conditions. Glutamate receptor antagonists fully blocked ChAT inhibition and oxidative stress induced by AßOs. Antioxidant polyunsaturated fatty acids had similar effects, indicating that oxidative damage may be involved in ChAT inhibition. Treatment with insulin, previously shown to down-regulate neuronal AßO binding sites, fully prevented AßO-induced inhibition of ChAT. Interestingly, we found that AßOs selectively bind to ~50% of cultured cholinergic neurons, suggesting that ChAT is fully inhibited in AßO-targeted neurons. Reduction in ChAT activity instigated by AßOs may thus be a relevant event in early stage AD pathology, preceding the loss of cholinergic neurons commonly observed in AD brains.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Proteínas Aviares/metabolismo , Colina O-Acetiltransferasa/metabolismo , Neuronas/metabolismo , Estrés Oxidativo , Enfermedad de Alzheimer/patología , Animales , Antioxidantes/metabolismo , Proteínas Aviares/genética , Técnicas de Cultivo de Célula , Células Cultivadas , Pollos , Antagonistas de Aminoácidos Excitadores/farmacología , Neuronas/patologíaRESUMEN
Alzheimer's disease (AD) is the 3rd most costly disease and the leading cause of dementia. It can linger for many years, but ultimately is fatal, the 6th leading cause of death. Alzheimer's disease (AD) is fatal and affected individuals can sometimes linger many years. Current treatments are palliative and transient, not disease modifying. This article reviews progress in the search to identify the primary AD-causing toxins. We summarize the shift from an initial focus on amyloid plaques to the contemporary concept that AD memory failure is caused by small soluble oligomers of the Aß peptide, toxins that target and disrupt particular synapses. Evidence is presented that links Aß oligomers to pathogenesis in animal models and humans, with reference to seminal discoveries from cell biology and new ideas concerning pathogenic mechanisms, including relationships to diabetes and Fragile X. These findings have established the oligomer hypothesis as a new molecular basis for the cause, diagnosis, and treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Trastornos de la Memoria/metabolismo , Sinapsis/metabolismo , Enfermedad de Alzheimer/patología , Encéfalo/patología , Humanos , Trastornos de la Memoria/patología , Neuronas/metabolismo , Neuronas/patología , Placa Amiloide/metabolismo , Placa Amiloide/patología , Sinapsis/patologíaRESUMEN
Brain accumulation of the amyloid-ß peptide (Aß) and oxidative stress underlie neuronal dysfunction and memory loss in Alzheimer's disease (AD). Hexokinase (HK), a key glycolytic enzyme, plays important pro-survival roles, reducing mitochondrial reactive oxygen species (ROS) generation and preventing apoptosis in neurons and other cell types. Brain isozyme HKI is mainly associated with mitochondria and HK release from mitochondria causes a significant decrease in enzyme activity and triggers oxidative damage. We here investigated the relationship between Aß-induced oxidative stress and HK activity. We found that Aß triggered HKI detachment from mitochondria decreasing HKI activity in cortical neurons. Aß oligomers further impair energy metabolism by decreasing neuronal ATP levels. Aß-induced HKI cellular redistribution was accompanied by excessive ROS generation and neuronal death. 2-deoxyglucose blocked Aß-induced oxidative stress and neuronal death. Results suggest that Aß-induced cellular redistribution and inactivation of neuronal HKI play important roles in oxidative stress and neurodegeneration in AD.
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Péptidos beta-Amiloides/fisiología , Hexoquinasa/metabolismo , Mitocondrias/metabolismo , Neuronas/enzimología , Animales , Supervivencia Celular , Citosol/metabolismo , Desoxiglucosa/farmacología , Humanos , Isoenzimas/química , Neuronas/citología , Estrés Oxidativo , Ratas , Especies Reactivas de Oxígeno , Sales de Tetrazolio/farmacología , Tiazoles/farmacologíaRESUMEN
Soluble amyloid-ß peptide (Aß) oligomers, known to accumulate in Alzheimer's disease brains, target excitatory post-synaptic terminals. This is thought to trigger synapse deterioration, a mechanism possibly underlying memory loss in early stage Alzheimer's disease. A major unknown is the identity of the receptor(s) targeted by oligomers at synapses. Because oligomers have been shown to interfere with N-methyl-d-aspartate receptor (NMDAR) function and trafficking, we hypothesized that NMDARs might be required for oligomer binding to synapses. An amplicon vector was used to knock-down NMDARs in mature hippocampal neurons in culture, yielding 90% reduction in dendritic NMDAR expression and blocking neuronal oxidative stress induced by Aß oligomers, a pathological response that has been shown to be mediated by NMDARs. Remarkably, NMDAR knock-down abolished oligomer binding to dendrites, indicating that NMDARs are required for synaptic targeting of oligomers. Nevertheless, oligomers do not appear to bind directly to NMDARs as indicated by the fact that both oligomer-attacked and non-attacked neurons exhibit similar surface levels of NMDARs. Furthermore, pre-treatment of neurons with insulin down-regulates oligomer-binding sites in the absence of a parallel reduction in surface levels of NMDARs. Establishing that NMDARs are key components of the synaptic oligomer binding complex may illuminate the development of novel approaches to prevent synapse failure triggered by Aß oligomers.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de N-Metil-D-Aspartato/fisiología , Sinapsis/metabolismo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/toxicidad , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Hipocampo/metabolismo , Hipocampo/patología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/toxicidad , Unión Proteica/fisiología , Ratas , Receptores de N-Metil-D-Aspartato/deficiencia , Sinapsis/patologíaRESUMEN
One of the earliest manifestations of Alzheimer's disease (AD) is the characteristic inability of affected individuals to form new memories. Memory impairment appears to significantly predate the death of nerve cells, implying that neuronal dysfunction is responsible for the pathophysiology of early stage AD. Mounting evidence now indicates that soluble oligomers of the amyloid-beta peptide (Abeta) are the main neurotoxins that lead to early neuronal dysfunction and memory deficits in AD. Cyclic AMP (cAMP) is a central component of intracellular signaling pathways that regulate a wide range of biological functions, including memory. Among other actions, cAMP triggers the phosphorylation and activation of the cAMP responsive element binding protein (CREB), a transcription factor that regulates the expression of genes that are important for long-term memory. Here, we discuss recent evidence suggesting that cAMP enhancing compounds may find applications as neurocognitive enhancers in AD and in other neurological disorders, as well as possible roles of cAMP in the regulation of neuronal regeneration. In particular, we review recent results showing that low concentrations of 2,4-dinitrophenol (DNP) upregulate neuronal cAMP and tau levels, promote neurite outgrowth and neuronal differentiation and block the oligomerization and neurotoxicity of Abeta. Possible implications of these findings in the development of novel therapeutic approaches in AD are discussed.
Asunto(s)
2,4-Dinitrofenol/uso terapéutico , Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/metabolismo , 2,4-Dinitrofenol/farmacología , Secretasas de la Proteína Precursora del Amiloide , Animales , AMP Cíclico/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Proteínas tau/metabolismoRESUMEN
Este estudo traz experiências dos autores, durante mais de dois anos, em trabalho de equipe no Curso de Projeciologia e Conscienciologia (CPC). São apresentadas as principais renovações conscienciais verificadas com estes autores e demais colegas que compuseram as equipes de professores neste laboratório de convivência(AU)