RESUMEN
Microglia are the brain immune cells and their function is highly dependent on cell motility. It was hypothesised that morphological variability leads to differences in motility, ultimately impacting on the microglial function. Here, we assessed microglial morphology in 32 controls, 44 Alzheimer's disease (AD) cases and 16 AD cases from patients immunised against Aß42 (iAD) using 2D and 3D approaches. Our 2D assessment showed an increased number of microglia in iAD vs. AD (P = 0.032) and controls (P = 0.018). Ramified microglia were fewer in AD vs. controls (P = 0.041) but increased in iAD compared to AD (P < 0.001) and controls (P = 0.006). 3D reconstructions highlighted larger cell bodies in AD vs. controls (P = 0.049) and increased total process length in iAD vs. AD (P = 0.032), with negative correlations detected for pan-Aß load with total process length (P < 0.001) in AD and number of primary processes (P = 0.043) in iAD. In summary, reactive/amoeboid microglia are the most represented population in the aged human brain. AD does not affect the number of microglia, but the ramified population is decreased adopting a more reactive morphology. Aß removal by immunotherapy leads to increased ramified microglia, implying that the cells retain plasticity in an aged disease brain meriting further investigation.
Asunto(s)
Enfermedad de Alzheimer/fisiopatología , Microglía/patología , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Femenino , Humanos , Inmunoterapia/métodos , Masculino , Microglía/metabolismo , Ovillos Neurofibrilares/metabolismo , Bancos de TejidosRESUMEN
Peripheral infections followed by systemic inflammation may contribute to the onset of Alzheimer`s disease (AD) and accelerate the disease progression later in life. Yet, the impact of systemic inflammation on the plasma and brain tissue metabolome and lipidome in AD has not been investigated. In this study, targeted metabolomic and untargeted lipidomic profiling experiments were performed on the plasma, cortices, and hippocampi of wild-type (WT) mice and transgenic APdE9 mice after chronic lipopolysaccharide (LPS) treatment, as well as saline-treated APdE9 mice. The lipidome and the metabolome of these mice were compared to saline-treated WT animals. In the brain tissue of all three models, the lipidome was more influenced than the metabolome. The LPS-treated APdE9 mice had the highest number of changes in brain metabolic pathways with significant alterations in levels of lysine, myo-inositol, spermine, phosphocreatine, acylcarnitines and diacylglycerols, which were not observed in the saline-treated APdE9 mice. In the WT mice, the effect of the LPS administration on metabolome and lipidome was negligible. The study provided exciting information about the biochemical perturbations due to LPS-induced inflammation in the transgenic AD model, which can significantly enhance our understanding of the role of systemic inflammation in AD pathogenesis.
Asunto(s)
Precursor de Proteína beta-Amiloide/inmunología , Encéfalo/metabolismo , Inflamación/metabolismo , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Hipocampo/metabolismo , Lipidómica/métodos , Masculino , Metaboloma , Metabolómica/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Presenilina-1/metabolismoRESUMEN
Alzheimer's disease is a common cause of dementia, for which no disease-modifying therapy is yet available. Aß3-10-KLH, a vaccine for active immunization, has been shown to prevent pathological changes in young transgenic models of AD, but the effects of treatment with it and its effects on mitochondrial dysfunction remain unclear. We immunized 6-month-old Tg-APPswe/PSEN1dE9 mice with Aß3-10-KLH to analyze whether it is capable of eliminating amyloid-ß after its appearance. The vaccine effectively decreased amyloid-ß deposits, improved cognitive function and ameliorated mitochondrial dysfunction. These results indicate the potential of Aß3-10-KLH as a vaccine to treat AD.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/prevención & control , Vacunas contra el Alzheimer/uso terapéutico , Precursor de Proteína beta-Amiloide/genética , Trastornos del Conocimiento/prevención & control , Trastornos del Conocimiento/psicología , Enfermedades Mitocondriales/prevención & control , Presenilina-1/genética , Vacunas/uso terapéutico , Enfermedad de Alzheimer/psicología , Precursor de Proteína beta-Amiloide/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Cognición , Humanos , Aprendizaje por Laberinto , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Enfermedades Mitocondriales/metabolismoRESUMEN
The mechanisms of amyloid-ß (Aß)-degradation and clearance in Alzheimer's disease (AD) pathogenesis have been relatively little studied. Short Aß-fragments form by enzymatic cleavage and alternate amyloid-beta precursor protein (APP)-processing. Here we characterized a novel polyclonal Aß-antibody raised against an Aß mid-domain and used it to investigate microglial Aß-uptake in situ by microscopy at the light- and ultrastructural levels. The rabbit Aß-mid-domain antibody (ab338), raised against the mid-domain amino acids 21-34 (Aß21-34), was characterized with biochemical and histological techniques. To identify the epitope in Aß recognized by ab338, solid phase and solution binding data were compared with peptide folding scores as calculated with the Tango software. The ab338 antibody displayed high average affinity (KD: 6.2 × 10-10 M) and showed preference for C-terminal truncated Aß-peptides ending at amino acid 34 and Aß-mid domain peptides with high scores of ß-turn structure. In transgenic APP-mouse brain, ab338 labelled amyloid plaques and detected Aß-fragments in microglia at the ultra- and light microscopic levels. This reinforces a role of microglia/macrophages in Aß-clearance in vivo. The ab338 antibody might be a valuable tool to study Aß-clearance by microglial uptake and Aß-mid-domain peptides generated by enzymatic degradation and alternate production.
Asunto(s)
Precursor de Proteína beta-Amiloide/inmunología , Microglía/fisiología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/patología , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Anticuerpos/inmunología , Modelos Animales de Enfermedad , Humanos , Dominios de Inmunoglobulinas/inmunología , Ratones , Ratones Transgénicos , Microglía/inmunología , Placa Amiloide/metabolismoRESUMEN
Alzheimer's disease (AD) is the most common cause of dementia worldwide with 10-30% prevalence in aging population and a high socioeconomic impact. Because AD definitive diagnostic requires post-mortem verification, new approaches to study the disease are necessary. Here, we analyze the humoral immune response in AD to survey whether APP+1 or UBB+1 frameshift proteins, produced as a consequence of the "molecular misreading" alteration in AD occurring in the APP (amyloid precursor protein) and UBB (ubiquitin-B protein) proteins' mRNA, elicit the production of autoantibodies specific of AD. To this end, APP+1 and UBB+1 peptides were expressed in bacteria as 6xHisHalo fusion proteins and after purification to homogeneity their seroreactivity was analyzed using 81 individual sera from AD patients and 43 individual sera from healthy individuals by luminescence beads immunoassay. We found that as a result of the molecular misreading, APP+1 and UBB+1 frameshift peptides produced a humoral immune response in AD patients, whose autoantibody levels are significantly higher in comparison with healthy controls. Their combination with a previously reported panel of four autoantigens specific of AD (ANTXR1, OR8J1, PYGB, and NUPR1) increased their diagnostic ability assessed by receiver operating characteristic (ROC) curves up to an area under the curve (AUC) of 73.5%. Collectively, our results demonstrate that APP+1 and UBB+1 frameshift proteins, non-previously described as AD-specific autoantigens, elicit the production of autoantibodies which might be useful as blood-based biomarkers to aid in the detection of the disease.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Ubiquitina/metabolismo , Anciano , Anciano de 80 o más Años , Enfermedad de Alzheimer/diagnóstico , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/inmunología , Femenino , Humanos , Inmunidad Humoral/inmunología , Masculino , Persona de Mediana Edad , Proteínas de Neoplasias/inmunología , Proteínas de Neoplasias/metabolismo , Complejo de la Endopetidasa Proteasomal/inmunología , Complejo de la Endopetidasa Proteasomal/metabolismo , Receptores de Superficie Celular/metabolismo , Transcripción Genética/genéticaRESUMEN
Geraniin has been reported to possess potent anti-inflammatory properties and to modulate the macrophage polarization. This study sought to evaluate the protective effects and underlying mechanisms of geraniin on lipopolysaccharide (LPS)-induced neuroinflammation and neurobiological alternations as well as cognitive impairment. Daily intragastrical administration with geraniin (20 mg kg-1 day-1) for 14 days significantly prolonged the duration in the target quadrant (26.53 ± 2.03 versus 37.09 ± 3.27%; p < 0.05) and increased crossing-target number (1.93 ± 0.22 versus 3.08 ± 0.17; p < 0.01) in the probe test of LPS-treated mice. Geraniin also ameliorated LPS-elicited neural/synaptic impairments and decreased levels of LPS-induced Aß generation (p < 0.05), amyloid precursor protein (APP) (p < 0.05) and ß-site amyloid precursor protein cleavage enzyme 1 (BACE1) (p < 0.05). Furthermore, geraniin suppressed the production of pro-inflammatory cytokines, including tumor necrosis factor α (TNF-α) (9.85 ± 0.58 versus 5.20 ± 0.52 pg/mg of protein; p < 0.01), interleukin (IL)-1ß (16.31 ± 0.67 versus 8.62 ± 0.46 pg/mg of protein; p < 0.01), and IL-6 (12.12 ± 0.45 versus 7.43 ± 0.32 pg/mg of protein; p < 0.05), and inhibited glial cell activation. Moreover, geraniin effectively polarized the microglia toward an anti-inflammatory M2 phenotype. Further study revealed that geraniin targeted toll-like receptor 4 (TLR4)-mediated signaling and decreased the production of pro-inflammatory cytokines in BV-2 microglial cells. These results indicate that geraniin mitigates LPS-elicited neural/synaptic neurodegeneration, amyloidogenesis, neuroinflammation, and cognitive impairment and suggest geraniin as a therapeutic option for neuroinflammation-associated neurological disorders, such as Alzheimer's disease.
Asunto(s)
Disfunción Cognitiva/tratamiento farmacológico , Glucósidos/administración & dosificación , Taninos Hidrolizables/administración & dosificación , Receptor Toll-Like 4/inmunología , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Animales , Ácido Aspártico Endopeptidasas , Disfunción Cognitiva/inducido químicamente , Disfunción Cognitiva/genética , Disfunción Cognitiva/inmunología , Humanos , Interleucina-1beta/genética , Interleucina-1beta/inmunología , Lipopolisacáridos/efectos adversos , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Microglía/inmunología , FN-kappa B/genética , FN-kappa B/inmunología , Neuroglía/efectos de los fármacos , Neuroglía/inmunología , Receptor Toll-Like 4/genética , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Prenatal and early childhood infections have been implicated in autism. Many autism susceptibility genes (206 Autworks genes) are localised in the immune system and are related to immune/infection pathways. They are enriched in the host/pathogen interactomes of 18 separate microbes (bacteria/viruses and fungi) and to the genes regulated by bacterial toxins, mycotoxins and Toll-like receptor ligands. This enrichment was also observed for misregulated genes from a microarray study of leukocytes from autistic toddlers. The upregulated genes from this leukocyte study also matched the expression profiles in response to numerous infectious agents from the Broad Institute molecular signatures database. They also matched genes related to sudden infant death syndrome and autism comorbid conditions (autoimmune disease, systemic lupus erythematosus, diabetes, epilepsy and cardiomyopathy) as well as to estrogen and thyrotropin responses and to those upregulated by different types of stressors including oxidative stress, hypoxia, endoplasmic reticulum stress, ultraviolet radiation or 2,4-dinitrofluorobenzene, a hapten used to develop allergic skin reactions in animal models. The oxidative/integrated stress response is also upregulated in the autism brain and may contribute to myelination problems. There was also a marked similarity between the expression signatures of autism and Alzheimer's disease, and 44 shared autism/Alzheimer's disease genes are almost exclusively expressed in the blood-brain barrier. However, in contrast to Alzheimer's disease, levels of the antimicrobial peptide beta-amyloid are decreased and the levels of the neurotrophic/myelinotrophic soluble APP alpha are increased in autism, together with an increased activity of α-secretase. sAPPα induces an increase in glutamatergic and a decrease in GABA-ergic synapses creating and excitatory/inhibitory imbalance that has also been observed in autism. A literature survey showed that multiple autism genes converge on APP processing and that many are able to increase sAPPalpha at the expense of beta-amyloid production. A genetically programmed tilt of this axis towards an overproduction of neurotrophic/gliotrophic sAPPalpha and underproduction of antimicrobial beta-amyloid may explain the brain overgrowth and myelination dysfunction, as well as the involvement of pathogens in autism.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Precursor de Proteína beta-Amiloide/inmunología , Trastorno Autístico/inmunología , Enfermedades Transmisibles/inmunología , Leucocitos/inmunología , Transcriptoma/fisiología , Péptidos beta-Amiloides/genética , Precursor de Proteína beta-Amiloide/genética , Trastorno Autístico/epidemiología , Trastorno Autístico/genética , Enfermedades Transmisibles/epidemiología , Enfermedades Transmisibles/genética , Predisposición Genética a la Enfermedad/genética , HumanosRESUMEN
One of the hallmarks of Alzheimer's disease is the presence of extracellular diffuse and fibrillar plaques predominantly consisting of the amyloid-ß (Aß) peptide. Apolipoprotein E (ApoE) influences the deposition of amyloid pathology through affecting the clearance and aggregation of monomeric Aß in the brain. In addition to influencing Aß metabolism, increasing evidence suggests that apoE influences microglial function in neurodegenerative diseases. Here, we characterize the impact that apoE has on amyloid pathology and the innate immune response in APPPS1ΔE9 and APPPS1-21 transgenic mice. We report that Apoe deficiency reduced fibrillar plaque deposition, consistent with previous studies. However, fibrillar plaques in Apoe-deficient mice exhibited a striking reduction in plaque compaction. Hyperspectral fluorescent imaging using luminescent conjugated oligothiophenes identified distinct Aß morphotypes in Apoe-deficient mice. We also observed a significant reduction in fibrillar plaque-associated microgliosis and activated microglial gene expression in Apoe-deficient mice, along with significant increases in dystrophic neurites around fibrillar plaques. Our results suggest that apoE is critical in stimulating the innate immune response to amyloid pathology.
Asunto(s)
Amiloide/metabolismo , Apolipoproteínas E/metabolismo , Microglía/metabolismo , Placa Amiloide/metabolismo , Placa Amiloide/patología , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Amiloide/inmunología , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Apolipoproteínas E/inmunología , Encéfalo/inmunología , Encéfalo/metabolismo , Encéfalo/patología , Modelos Animales de Enfermedad , Humanos , Inmunidad Innata/inmunología , Ratones , Ratones Transgénicos , Microglía/inmunología , Microglía/patología , Placa Amiloide/inmunologíaRESUMEN
In the process of drug development, in vitro studies do not always adequately predict human-specific drug responsiveness in clinical trials. Here, we applied the advantage of human iPSC-derived neurons, which offer human-specific drug responsiveness, to screen and evaluate therapeutic candidates for Alzheimer's disease (AD). Using AD patient neurons with nearly 100% purity from iPSCs, we established a robust and reproducible assay for amyloid ß peptide (Aß), a pathogenic molecule in AD, and screened a pharmaceutical compound library. We acquired 27 Aß-lowering screen hits, prioritized hits by chemical structure-based clustering, and selected 6 leading compounds. Next, to maximize the anti-Aß effect, we selected a synergistic combination of bromocriptine, cromolyn, and topiramate as an anti-Aß cocktail. Finally, using neurons from familial and sporadic AD patients, we found that the cocktail showed a significant and potent anti-Aß effect on patient cells. This human iPSC-based platform promises to be useful for AD drug development.
Asunto(s)
Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/inmunología , Células Madre Pluripotentes Inducidas/citología , Neuronas/patología , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/inmunología , Evaluación Preclínica de Medicamentos/métodos , HumanosRESUMEN
It is well known that mutations in the gene coding for amyloid precursor protein are responsible for autosomal dominant forms of Alzheimer's disease. Proteolytic processing of the protein leads to a number of metabolites including the amyloid beta peptide. Although brain amyloid precursor protein expression and amyloid beta production are associated with the pathophysiology of Alzheimer's disease, it is clear that amyloid precursor protein is expressed in numerous cell types and tissues. Here we demonstrate that amyloid precursor protein is involved in regulating the phenotype of both adipocytes and peripheral macrophages and is required for high fat diet-dependent weight gain in mice. These data suggest that functions of this protein include modulation of the peripheral immune system and lipid metabolism. This biology may have relevance not only to the pathophysiology of Alzheimer's disease but also diet-associated obesity.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Macrófagos/metabolismo , Fenotipo , Aumento de Peso , Tejido Adiposo/metabolismo , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Animales , Biomarcadores , Dieta , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Masculino , Ratones , Ratones Noqueados , Aumento de Peso/genéticaRESUMEN
Antibody drug conjugates (ADCs) provide an efficacious and relatively safe means by which chemotherapeutic agents can be specifically targeted to cancer cells. In addition to the selection of antibody targets, ADCs offer a modular design that allows selection of ADC characteristics through the choice of linker chemistries, toxins, and conjugation sites. Many studies have indicated that release of toxins bound to antibodies via noncleavable linker chemistries relies on the internalization and intracellular trafficking of the ADC. While this can make noncleavable ADCs more stable in the serum, it can also result in lower efficacy when their respective targets are not internalized efficiently or are recycled back to the cell surface following internalization. Here, we show that a lysosomally targeted ADC against the protein APLP2 mediates cell killing, both in vitro and in vivo, more effectively than an ADC against Trop2, a protein with less efficient lysosomal targeting. We also engineered a bispecific ADC with one arm targeting HER2 for the purpose of directing the ADC to tumors, and the other arm targeting APLP2, whose purpose is to direct the ADC to lysosomes for toxin release. This proof-of-concept bispecific ADC demonstrates that this technology can be used to shift the intracellular trafficking of a constitutively recycled target by directing one arm of the antibody against a lysosomally delivered protein. Our data also show limitations of this approach and potential future directions for development.
Asunto(s)
Sistemas de Liberación de Medicamentos , Inmunoconjugados/farmacología , Lisosomas/metabolismo , Transcitosis , Precursor de Proteína beta-Amiloide/inmunología , Precursor de Proteína beta-Amiloide/uso terapéutico , Animales , Anticuerpos Biespecíficos/uso terapéutico , Antineoplásicos/química , Línea Celular Tumoral , Humanos , Inmunoconjugados/metabolismo , Ratones Desnudos , Proteínas del Tejido Nervioso/inmunología , Proteínas del Tejido Nervioso/uso terapéutico , Receptor ErbB-2/inmunología , Receptor ErbB-2/uso terapéuticoRESUMEN
Nicotinic acetylcholine receptors of α7 subtype (α7 nAChRs) attenuate the inflammatory cytokines production by macrophages and are involved in pathogenesis of Alzheimer disease by directly influencing the processing of amyloid-beta (Aß) precursor protein in the brain. Previously we found that regular injections of bacterial lipopolysaccharide (LPS) decreased the level of α7 nAChRs and stimulated accumulation of Aß peptide (1-42) in the brain of mice resulting in memory impairment. Similar effects were observed in mice immunized with recombinant extracellular domain (1-208) of α7 nAChR subunit. However, the mechanism of inflammation-like effect of α7-specific antibodies remained unclear. The aim of the present study was to reveal the impact of carbohydrate component of recombinant α7(1-208) produced in yeast in the functional effect of resulting antibodies. For this purpose, C57Bl/6 mice were immunized with either initial α7(1-208) or with that pre-treated with endoglycosidase. Control groups of mice obtained injections of either LPS or complete Freund's adjuvant. Mice were tested for memory performance, their blood sera were examined for the presence and fine specificity of α7(1-208)-specific antibodies and the brain preparations were studied for the levels of α7 nAChR, Aß(1-42) and interleukin-6. It was found that the original α7(1-208) was more immunogenic than the deglycosylated one, and their epitopes were recognized with different efficiency. In contrast to LPS and original α7(1-208), deglycosylated α7(1-208) did not stimulate interleukin-6 elevation in the brain, i.e. had no pro-inflammatory effect. Nevertheless, immunizations with either the original or deglycosylated α7(1-208) resulted in similar decrease of α7 nAChRs, accumulation of Aß(1-42) in the brain and significant episodic memory decline, comparable to those exerted by LPS injections. We conclude that the decrease of α7 nAChR density, caused by α7(1-208)-specific antibody, is critical for Aß(1-42) accumulation and episodic memory impairment, while pro-inflammatory capacity of α7(1-208)-specific antibody plays a secondary role for the development of Alzheimer-like symptoms.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/inmunología , Encéfalo/inmunología , Carbohidratos/inmunología , Macrófagos/inmunología , Péptidos/inmunología , Receptor Nicotínico de Acetilcolina alfa 7/inmunología , Animales , Anticuerpos/sangre , Carbohidratos/genética , Células Cultivadas , Epítopos de Linfocito B/inmunología , Espacio Extracelular , Femenino , Glicósido Hidrolasas/química , Humanos , Interleucina-6/metabolismo , Pruebas de Memoria y Aprendizaje , Ratones , Ratones Endogámicos C57BL , Péptidos/genética , Dominios Proteicos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Receptor Nicotínico de Acetilcolina alfa 7/química , Receptor Nicotínico de Acetilcolina alfa 7/genéticaRESUMEN
Immunization against amyloid-ß (Aß) peptides deposited in Alzheimer's disease (AD) has shown considerable therapeutic effect in animal models however, the translation into human Alzheimer's patients is challenging. In recent years, a number of promising Aß immunotherapy trials failed to reach primary study endpoints. Aside from uncertainties in the selection of patients and the start and duration of treatment, these results also suggest that the mechanisms underlying AD are still not fully understood. Thorough characterizations of protein aggregates in AD brain have revealed a conspicuous heterogeneity of Aß peptides enabling the study of the toxic potential of each of the major forms. One such form, amino-terminally truncated and modified pyroglutamate (pGlu)-3 Aß peptide appears to play a seminal role for disease initiation, qualifying it as novel target for immunotherapy approaches.
Asunto(s)
Enfermedad de Alzheimer/patología , Enfermedad de Alzheimer/terapia , Vacunas contra el Alzheimer , Precursor de Proteína beta-Amiloide , Ácido Pirrolidona Carboxílico , Vacunas contra el Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/inmunología , Animales , Humanos , Ácido Pirrolidona Carboxílico/inmunologíaRESUMEN
Alzheimer's disease (AD) is histopathologically characterized by neurodegeneration, the formation of intracellular neurofibrillary tangles and extracellular Aß deposits that derive from proteolytic processing of the amyloid precursor protein (APP). As rodents do not normally develop Aß pathology, various transgenic animal models of AD were designed to overexpress human APP with mutations favouring its amyloidogenic processing. However, these mouse models display tremendous differences in the spatial and temporal appearance of Aß deposits, synaptic dysfunction, neurodegeneration and the manifestation of learning deficits which may be caused by age-related and brain region-specific differences in APP transgene levels. Consequentially, a comparative temporal and regional analysis of the pathological effects of Aß in mouse brains is difficult complicating the validation of therapeutic AD treatment strategies in different mouse models. To date, no antibodies are available that properly discriminate endogenous rodent and transgenic human APP in brains of APP-transgenic animals. Here, we developed and characterized rat monoclonal antibodies by immunohistochemistry and Western blot that detect human but not murine APP in brains of three APP-transgenic mouse and one APP-transgenic rat model. We observed remarkable differences in expression levels and brain region-specific expression of human APP among the investigated transgenic mouse lines. This may explain the differences between APP-transgenic models mentioned above. Furthermore, we provide compelling evidence that our new antibodies specifically detect endogenous human APP in immunocytochemistry, FACS and immunoprecipitation. Hence, we propose these antibodies as standard tool for monitoring expression of endogenous or transfected APP in human cells and APP expression in transgenic animals.
Asunto(s)
Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/inmunología , Anticuerpos/inmunología , Especificidad de Anticuerpos/inmunología , Expresión Génica , Transgenes/genética , Animales , Modelos Animales de Enfermedad , Humanos , Inmunohistoquímica , Ratones Endogámicos C57BL , Ratones Transgénicos , Ratas , Reproducibilidad de los ResultadosRESUMEN
Characterized by hypertension and proteinuria after the 20th week of gestation, pre-eclampsia (PE) is a major cause of maternal, fetal, and neonatal morbidity and mortality. Despite being recognized for centuries, PE still lacks a reliable, early means of diagnosis or prediction, and a safe and effective therapy. We have recently reported that the event of toxic protein misfolding and aggregation is a critical etiological manifestation in PE. Using comparative proteomic analysis of gestational age-matched sera from PE and normal pregnancy, we identified several proteins that appeared to be dysregulated in PE. Our efforts so far have focused on transthyretin (TTR), a transporter of thyroxine and retinol, and amyloid precursor protein whose aggregates were detected in the PE placenta. Based on these results and detection of TTR aggregates in sera from PE patients, we proposed that PE could be a disease of protein misfolding and aggregation. Protein misfolding and aggregation have long been linked with many neurodegenerative diseases such as Alzheimer's disease. However, linkage of protein misfolding and aggregation with the PE pathogenesis is a new and novel concept. This review aims to understand the roles of aggregated proteins in PE using the cues from the Alzheimer's etiology.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/inmunología , Preeclampsia/inmunología , Prealbúmina/inmunología , Agregación Patológica de Proteínas/inmunología , Enfermedad de Alzheimer/patología , Animales , Femenino , Preeclampsia/patología , Embarazo , Agregación Patológica de Proteínas/patologíaRESUMEN
The toxic effect of Ðß-oligomers accompanies chronic inflammation, with cytokines as main mediators. Therefore, the cytokine link of inflammation becomes a new target on the way to restrain amyloidosis. The aim of the study was the effect of aggregated Ðß42 on the dynamics of expression and formation of endogenous Ðß40 and cytokines (IL-1ß, TNFα, IL-6, IL-10) by peripheral blood mononuclear cells in vitro and its correction by curcumin. A suspension of mononuclear cells isolated ex tempore using ficoll-urografin gradient from venous blood samples of healthy volunteers were used to study the effects of Ðß42 (15 nM), curcumin (54 pM), and their combined action (at similar concentrations) in time dynamics: 0, 1, 3, 6 and 24 h incubation at 37 °C. Polymerase chain reaction with appropriate primers was used to determine the relative expression of mRNA for AßPP, TNFα, IL-1ß, IL-6, IL-10 and enzyme-linked immunosorbent assay to determine the content of Ðß40 and cytokines in mononuclear suspension during all periods of incubation. The individual dynamics AßPP and cytokine expression was shown under the action of the Aß42, which had influence on the content of Aß40, TNFα, IL-1ß, IL-6 and IL-10 in mononuclear suspension. Curcumin displayed the inhibitory effect on gene expression of AßPP, TNFα and IL6, which resulted in the decrease of the level of these two cytokines and Aß40. Thus, the dynamics of anti-inflammatory effect of curcumin in vitro for transcriptional and translational levels of cytokine's formation by mononuclear cells was shown in the work. Direct inhibitory effect of curcumin on the concentration of endogenous Aß40 during the 24 h incubation in conditions of toxic action of Aß42 aggregates was established.
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Péptidos beta-Amiloides/genética , Péptidos beta-Amiloides/farmacología , Precursor de Proteína beta-Amiloide/genética , Interleucina-10/genética , Interleucina-1beta/genética , Interleucina-6/genética , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/farmacología , Factor de Necrosis Tumoral alfa/genética , Péptidos beta-Amiloides/antagonistas & inhibidores , Péptidos beta-Amiloides/inmunología , Precursor de Proteína beta-Amiloide/inmunología , Antiinflamatorios no Esteroideos/farmacología , Curcumina/farmacología , Regulación de la Expresión Génica , Humanos , Interleucina-10/inmunología , Interleucina-1beta/inmunología , Interleucina-6/inmunología , Leucocitos Mononucleares/citología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/inmunología , Fragmentos de Péptidos/antagonistas & inhibidores , Fragmentos de Péptidos/inmunología , Cultivo Primario de Células , Transducción de Señal , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
INTRODUCTION: Alzheimer's disease brains are characterized by extracellular plaques containing the aggregated amyloid ß42 (Aß42) peptide and intraneuronal tangles containing hyperphosphorylated tau. Aß42 is produced by sequential processing of the amyloid precursor protein (APP) by ß-secretase followed by γ-secretase. Substantial efforts have been put into developing pharmaceuticals preventing the production or increasing the clearance of Aß42. However, treatments inhibiting γ-secretase have proven disappointing due to off-target effects. To circumvent these effects, γ-secretase modulators (GSMs) have been developed, which rather than inhibiting γ-secretase shift its preference into producing less aggregation-prone shorter Aß peptides. Belonging to the same family of proteins as APP, amyloid-like protein 1 (APLP1) is also a substrate for γ-secretase. Herein we investigated whether the GSM E2012 affects APLP1 processing in the central nervous system by measuring APLP1 peptide levels in cerebrospinal fluid (CSF) before and after E2012 treatment in dogs. METHODS: An in-house monoclonal APLP1 antibody, AP1, was produced and utilized for immunopurification of APLP1 from human and dog CSF in a hybrid immuno-affinity mass spectrometric method. Seven dogs received a single dose of 20 or 80 mg/kg of E2012 in a randomized cross-over design and CSF was collected prior to and 4, 8 and 24 hours after dosing. RESULTS: We have identified 14 CSF APLP1 peptides in humans and 12 CSF APLP1 peptides in dogs. Of these, seven were reproducibly detectable in dogs who received E2012. We found a dose-dependent relative increase of the CSF peptides APLP1ß17, 1ß18 and 1ß28 accompanied with a decrease of 1ß25 and 1ß27 in response to E2012 treatment. All peptides reverted to baseline over the time of sample collection. CONCLUSION: We show an in vivo effect of the GSM E2012 on the processing of APLP1 which is measurable in CSF. These data suggest that APLP1 peptides may be used as biomarkers to monitor drug effects of GSMs on γ-secretase processing in clinical trials. However, this requires further investigation in larger cohorts, including studies in man.
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Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/líquido cefalorraquídeo , Imidazoles/farmacología , Piperidinas/farmacología , Secuencia de Aminoácidos , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Biomarcadores Farmacológicos/líquido cefalorraquídeo , Estudios Cruzados , Perros , Relación Dosis-Respuesta a Droga , Femenino , Humanos , Inmunoprecipitación , Ratones Endogámicos BALB C , Distribución Aleatoria , Espectrometría de Masas en TándemRESUMEN
OBJECTIVE: To construct and express a single chain fragment of variety region (scFv) against ß-site of amyloid precursor protein (APP), and evaluate the effect of scFv on α- and ß-processing of APP. METHODS: The ß-site specific scFv 2H10 was amplified and fused with signal sequence Igκ and myc tag by PCR to create the expression cassette, which was then subcloned into expression vector pcDNA3.1hygâº. The plasmid was transferred into CHO cells over-expressing Swedish mutation type human APP695 (APP695sw/CHO). The positive clones were identified by hygromycin B. The concentrations of Aß40 and soluble APPα (sAPPα) in the conditioned media supernatant were measured by sandwich ELISA. RESULTS: The recombinant eukaryotic expression plasmids were identified by PCR, restriction endonuclease digestion and DNA sequencing. The expression of scFv was confirmed by Western blotting. When scFv 2H10 was stably expressed in APP695sw/CHO, the extracellular level of Aß40 decreased by 30.4%, while sAPPα increased by 23.1%. CONCLUSION: The APP ß-site scFv 2H10 could inhibit the secretion of Aß. Meanwhile, it might be favorable for the α-processing of APP. Hence it can be explored as an inhibitor of ß-site APP-cleaving enzyme (BACE).
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Péptidos beta-Amiloides/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/inmunología , Anticuerpos de Cadena Única/farmacología , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Péptidos beta-Amiloides/biosíntesis , Animales , Ácido Aspártico Endopeptidasas/antagonistas & inhibidores , Células CHO , Cricetulus , PlásmidosRESUMEN
Amyloid precursor protein (APP) and its cleaved products have been reported to have important functions in CNS health, including in memory and synapse formation, cell survival and neuroprotection. Furthermore APP and its cleaved products have been shown to be transiently increased in response to various CNS stressors, suggesting a role in response to acute cellular injury. In an attempt to further understand the function of APP in response to CNS injury, we have used intracranial LPS injection as an inflammatory injury model in APP knock out mice (APPKO). Our data show that innate immune responses to LPS injection is significantly blunted in APPKO mice compared to APP sufficient wild type (BL6) mice. Morphologically, glial cells in APPKO mice appear less reactive, with shorter ramified processes and smaller cell bodies in response to LPS. Additionally, quantitative RT-PCR analysis for several glia markers and innate immune cytokine levels (e.g. TNFα, IL-6, IL-1ß and IL-10) showed significantly reduced expression levels in LPS injected APPKO mice. In vitro cell culture assays confirmed this attenuated response to LPS stimulation by primary microglial cells isolated from APPKO mice. Our data suggests that APP full length protein and/or its cleaved products are necessary to mount a complete and effective innate immune cell response to inflammatory injury.