RESUMEN
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
Alzheimer's disease (AD) is characterized by the presence of amyloid-ß (Aß)-containing plaques, neurofibrillary tangles, and neuronal loss in the brain. Inflammatory changes, typified by activated microglia, particularly adjacent to Aß plaques, are also a characteristic of the disease, but it is unclear whether these contribute to the pathogenesis of AD or are a consequence of the progressive neurodegenerative processes. Furthermore, the factors that drive the inflammation and neurodegeneration remain poorly understood. CNS-infiltrating T cells play a pivotal role in the pathogenesis of multiple sclerosis, but their role in the progression of AD is still unclear. In this study, we examined the role of Aß-specific T cells on Aß accumulation in transgenic mice that overexpress amyloid precursor protein and presenilin 1 (APP/PS1). We found significant infiltration of T cells in the brains of APP/PS1 mice, and a proportion of these cells secreted IFN-γ or IL-17. Aß-specific CD4 T cells generated by immunization with Aß and a TLR agonist and polarized in vitro to Th1-, Th2-, or IL-17-producing CD4(+) T cells, were adoptively transferred to APP/PS1 mice at 6 to 7 mo of age. Assessment of animals 5 wk later revealed that Th1 cells, but not Th2 or IL-17-producing CD4(+) T cells, increased microglial activation and Aß deposition, and that these changes were associated with impaired cognitive function. The effects of Th1 cells were attenuated by treatment of the APP/PS1 mice with an anti-IFN-γ Ab. Our study suggests that release of IFN-γ from infiltrating Th1 cells significantly accelerates markers of diseases in an animal model of AD.
Asunto(s)
Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/genética , Encéfalo/inmunología , Interferón gamma/inmunología , Microglía/inmunología , Placa Amiloide/patología , Células TH1/inmunología , Traslado Adoptivo , Enfermedad de Alzheimer/patología , Péptidos beta-Amiloides/inmunología , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Animales , Anticuerpos/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Movimiento Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Expresión Génica , Interferón gamma/antagonistas & inhibidores , Interferón gamma/metabolismo , Interleucina-17/genética , Interleucina-17/inmunología , Ratones , Ratones Transgénicos , Microglía/efectos de los fármacos , Microglía/patología , Placa Amiloide/inmunología , Presenilina-1/genética , Presenilina-1/inmunología , Células TH1/patología , Células TH1/trasplante , Células Th17/inmunología , Células Th17/patología , Células Th17/trasplante , Células Th2/inmunología , Células Th2/patología , Células Th2/trasplanteRESUMEN
Gammagard IVIg is a therapeutic approach to treat Alzheimer's disease currently in phase 3 clinical trials. Despite the reported efficacy of the approach the mechanism of action is poorly understood. We have previously shown that intracranial injection of anti-Aß antibodies into the frontal cortex and hippocampus reveals important information regarding the time course of events once the agent is in the brain. In the current study we compared IVIg, mouse-pooled IgG, and the anti-Aß antibody 6E10 injected intracranially into the frontal cortex and hippocampus of 7-month-old APP/PS1 mice. We established a time course of events ranging from 1 to 21 d postinjection. IVIg and pooled mouse IgG both significantly reduced Aß deposition to the same degree as the 6E10 anti-Aß antibody; however, the clearance was much slower to occur, happening between the 3 and 7 d time points. In contrast, as we have previously shown, Aß reductions were apparent with the 6E10 anti-Aß group at the 1 d time point. Also, neuroinflammatory profiles were significantly altered by the antibody treatments. APP/PS1 transgenic mice at 7 months of age typically exhibit an M2a inflammatory phenotype. All antibody treatments stimulated an M2b response, yet anti-Aß antibody was a more rapid change. Because the neuroinflammatory switch occurs before the detectable reductions in amyloid deposition, we hypothesize that the IVIg and pooled mouse IgG act as immune modulators and this immune modulation is responsible for the reductions in amyloid pathology.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Autoanticuerpos/fisiología , Inmunoglobulinas Intravenosas/administración & dosificación , Mediadores de Inflamación/administración & dosificación , Presenilina-1/antagonistas & inhibidores , Péptidos beta-Amiloides/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Animales , Femenino , Humanos , Inmunoglobulinas Intravenosas/fisiología , Mediadores de Inflamación/fisiología , Inyecciones Intraventriculares , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Presenilina-1/genética , Presenilina-1/inmunología , Factores de Tiempo , Resultado del TratamientoRESUMEN
Abnormalities in T-lymphocyte populations and function are observed in autism. Soluble amyloid precursor protein α (sAPP-α) is elevated in some patients with autism and is known to be produced by immune cells. In light of the well-established role of sAPP-α in proliferation, growth, and survival of neurons, we hypothesized an analogous role in the immune system. Thus, we explored whether sAPP-α could modulate immune development and function, especially aspects of the pinnacle cell of the adaptive arm of the immune system: the T cell. To do this, we generated mice overexpressing human sAPP-α and characterized elements of T-cell development, signal transduction, cytokine production, and innate/adaptive immune functions. Here, we report that transgenic sAPP-α-overexpressing (TgsAPP-α) mice displayed increased proportions of CD8(+) T cells, while effector memory T cells were decreased in the thymus. Overall apoptotic signal transduction was decreased in the thymus, an effect that correlated with dramatic elevations in Notch1 activation; while active-caspase-3/total-caspase-3 and Bax/Bcl-2 ratios were decreased. Greater levels of IFN-γ, IL-2, and IL-4 were observed after ex vivo challenge of TgsAPP-α mouse splenocytes with T-cell mitogen. Finally, after immunization, splenocytes from TgsAPP-α mice displayed decreased levels IFN-γ, IL-2, and IL-4, as well as suppressed ZAP70 activation, after recall antigen stimulation. Given elevated levels of circulating sAPP-α in some patients with autism, sAPP-α could potentially drive aspects of immune dysfunction observed in these patients, including dysregulated T-cell apoptosis, aberrant PI3K/AKT signaling, cytokine alterations, and impaired T-cell recall stimulation.
Asunto(s)
Precursor de Proteína beta-Amiloide/inmunología , Trastorno Autístico/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Precursor de Proteína beta-Amiloide/sangre , Precursor de Proteína beta-Amiloide/genética , Animales , Apoptosis/inmunología , Trastorno Autístico/sangre , Western Blotting , Complejo CD3/inmunología , Complejo CD3/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Citometría de Flujo , Humanos , Inmunidad/inmunología , Memoria Inmunológica/inmunología , Interferón gamma/inmunología , Interferón gamma/metabolismo , Interleucina-2/inmunología , Interleucina-2/metabolismo , Interleucina-4/inmunología , Interleucina-4/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Receptor Notch1/inmunología , Receptor Notch1/metabolismo , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Linfocitos T/metabolismo , Timocitos/citología , Timocitos/inmunología , Timocitos/metabolismoRESUMEN
Immunization against amyloid-ß (Aß) can reduce amyloid accumulation in vivo and is considered a potential therapeutic approach for Alzheimer's disease. However, it has been associated with meningoencephalitis thought to be mediated by inflammatory T-cells. With the aim of producing an immunogenic vaccine without this side effect, we designed CAD106 comprising Aß1-6 coupled to the virus-like particle Qß. Immunization with this vaccine did not activate Aß-specific T-cells. In APP transgenic mice, CAD106 induced efficacious Aß antibody titers of different IgG subclasses mainly recognizing the Aß3-6 epitope. CAD106 reduced brain amyloid accumulation in two APP transgenic mouse lines. Plaque number was a more sensitive readout than plaque area, followed by Aß42 and Aß40 levels. Studies with very strong overall amyloid reduction showed an increase in vascular Aß, which atypically was nonfibrillar. The efficacy of Aß immunotherapy depended on the Aß levels and thus differed between animal models, brain regions, and stage of amyloid deposition. Therefore, animal studies may not quantitatively predict the effect in human Alzheimer's disease. Our studies provided no evidence for increased microhemorrhages or inflammatory reactions in amyloid-containing brain. In rhesus monkeys, CAD106 induced a similar antibody response as in mice. The antibodies stained amyloid deposits on tissue sections of mouse and human brain but did not label cellular structures containing APP. They reacted with Aß monomers and oligomers and blocked Aß toxicity in cell culture. We conclude that CAD106 immunization is suited to interfere with Aß aggregation and its downstream detrimental effects.
Asunto(s)
Enfermedad de Alzheimer/tratamiento farmacológico , Péptidos beta-Amiloides/uso terapéutico , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Inmunoterapia/métodos , Enfermedad de Alzheimer/inmunología , Péptidos beta-Amiloides/efectos adversos , Animales , Células Cultivadas , Ratones , Ratones Transgénicos , Resultado del TratamientoRESUMEN
In vivo detection of Alzheimer's disease (AD) neuropathology in living patients using positron emission tomography (PET) in conjunction with high affinity molecular imaging probes for ß-amyloid (Aß) and tau has the potential to assist with early diagnosis, evaluation of disease progression, and assessment of therapeutic interventions. Animal models of AD are valuable for exploring the in vivo binding of these probes, particularly their selectivity for specific neuropathologies, but prior PET experiments in transgenic mice have yielded conflicting results. In this work, we utilized microPET imaging in a transgenic rat model of brain Aß deposition to assess [F-18]FDDNP binding profiles in relation to age-associated accumulation of neuropathology. Cross-sectional and longitudinal imaging demonstrated that [F-18]FDDNP binding in the hippocampus and frontal cortex progressively increases from 9 to 18months of age and parallels age-associated Aß accumulation. Specificity of in vivo [F-18]FDDNP binding was assessed by naproxen pretreatment, which reversibly blocked [F-18]FDDNP binding to Aß aggregrates. Both [F-18]FDDNP microPET imaging and neuropathological analyses revealed decreased Aß burden after intracranial anti-Aß antibody administration. The combination of this non-invasive imaging method and robust animal model of brain Aß accumulation allows for future longitudinal in vivo assessments of potential therapeutics for AD that target Aß production, aggregation, and/or clearance. These results corroborate previous analyses of [F-18]FDDNP PET imaging in clinical populations.
Asunto(s)
Envejecimiento/patología , Enfermedad de Alzheimer/diagnóstico por imagen , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/inmunología , Anticuerpos Bloqueadores/farmacología , Nitrilos , Tomografía de Emisión de Positrones/métodos , Envejecimiento/inmunología , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Amiloidosis/diagnóstico por imagen , Amiloidosis/genética , Amiloidosis/inmunología , Animales , Antiinflamatorios no Esteroideos/farmacología , Unión Competitiva/inmunología , Modelos Animales de Enfermedad , Radioisótopos de Flúor , Humanos , Naproxeno/farmacología , Ratas , Ratas Sprague-Dawley , Ratas TransgénicasRESUMEN
The neuropathologic hallmarks of Alzheimer's disease (AD) are extracellular plaques and intracellular neurofibrillary tangles. A constituent of senile plaques in AD is beta-amyloid, a hydrophobic peptide of 39-43 amino acids and a fragment of the amyloid precursor protein (APP). APP can be metabolized by at least two pathways, one of which involves generation of soluble APP by an unidentified enzyme named alpha-secretase. This cleavage generates alpha-secretase-cleaved, soluble APP (alpha-sAPP), which in this investigation was measured by a new assay in cerebrospinal fluid (CSF) from members of a Swedish AD family with a pathogenic mutation at APP670/671 (ref. 2). Family members who carry the mutation and are diagnosed with AD had low levels of alpha-sAPP (160 +/- 48 ng ml-1), with no overlap compared with non-carriers (257 +/- 48 ng ml-1). Carriers of the presymptomatic mutation showed intermediate alpha-sAPP levels. Today there exists no antemortem marker in AD with sufficient sensitivity and specificity, but measurement of alpha-sAPP represents a new and promising diagnostic marker.
Asunto(s)
Enfermedad de Alzheimer/diagnóstico , Precursor de Proteína beta-Amiloide/metabolismo , Endopeptidasas/metabolismo , Fragmentos de Péptidos/metabolismo , Adulto , Anciano , Enfermedad de Alzheimer/líquido cefalorraquídeo , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide , Precursor de Proteína beta-Amiloide/inmunología , Ácido Aspártico Endopeptidasas , Biomarcadores/líquido cefalorraquídeo , Western Blotting , Ensayo de Inmunoadsorción Enzimática/métodos , Heterocigoto , Humanos , Persona de Mediana Edad , Mutación , Fragmentos de Péptidos/inmunología , Suecia/epidemiologíaRESUMEN
BACKGROUND/AIMS: Amyloid-ß (Aß) protofibrils are neurotoxic soluble intermediates in the Aß aggregation process eventually forming senile plaques in Alzheimer's disease. This Aß species is a potential biomarker for Alzheimer's disease and also a promising target for immunotherapy. In this study, we investigated the characteristics of conformation-dependent Aß antibodies specific for Aß protofibrils. METHODS: Mice were immunized with Aß protofibrils to generate hybridomas producing Aß-specific monoclonal antibodies. Binding of antibodies to different Aß conformations was investigated with inhibition ELISA. The antibodies' complementarity-determining region (CDR) sequences were determined and compared. RESULTS: A majority of the antibodies were of the IgM class, all selectively binding to aggregated Aß. Two IgG antibodies were generated: one with selective affinity for Aß protofibrils and the other bound Aß in all conformations. A high degree of similarity between the heavy-chain CDRs of the conformation-dependent antibodies was found, and all high-affinity Aß antibodies displayed a high degree of sequence similarity in the light-chain CDRs. CONCLUSION: Sequence similarity in the heavy-chain CDRs is associated with conformation selectivity of the antibodies, while sequence similarity in the light-chain CDRs correlates with the affinity for Aß.
Asunto(s)
Péptidos beta-Amiloides/inmunología , Amiloide/inmunología , Anticuerpos Antiidiotipos/inmunología , Especificidad de Anticuerpos/inmunología , Regiones Determinantes de Complementariedad/inmunología , Cadenas Pesadas de Inmunoglobulina/inmunología , Precursor de Proteína beta-Amiloide/deficiencia , Precursor de Proteína beta-Amiloide/inmunología , Animales , Mapeo Epitopo , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Modelos Animales , Conformación ProteicaRESUMEN
OBJECTIVE: To investigate the therapeutic effect of recombinant adeno-associated virus carrying anti-amyloid ß peptide single-chain antibody gene on Alzheimer's disease (AD) in animal models. METHODS: The recombinant adeno-associated viruses were injected to the leg muscle of mutant amyloid precursor protein transgenic AD mice. The latency of the mice in Morris water maze was tested before and 3, 7, 10 months after drug administration. The animal brains were harvested 10 months after drug administration and sectioned for amyloid plaques staining. RESULTS: The learning and memory abilities of AD model mice were improved significantly 3 months after gene drug administration. Ten months after gene therapy, the numbers of amyloid plaque in hippocampus of model mice decreased. CONCLUSION: The adeno-associated virus carrying anti-amyloid ß peptide single-chain antibody gene has therapeutic effect on AD in model mice.
Asunto(s)
Enfermedad de Alzheimer/terapia , Precursor de Proteína beta-Amiloide/inmunología , Dependovirus/genética , Anticuerpos de Cadena Única/genética , Animales , Modelos Animales de Enfermedad , Femenino , Terapia Genética , Masculino , Ratones , Ratones TransgénicosRESUMEN
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
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
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
In amyloid precursor protein (APP) models of amyloid deposition, the amount of amyloid deposits increase with mouse age. At a first approximation, the extent of amyloid accumulation may either reflect small excesses of production over clearance that accumulate over time or, alternatively, indicate a steady-state equilibrium at that age, reflecting the instantaneous excess of production over clearance, which increases as the organism ages. To discriminate between these options, we reversibly suppressed amyloid deposition in Tg2576 mice with the anti-Abeta antibody 2H6, starting at 8 months, just before the first histological deposits can be discerned. Six months later, we stopped the suppression and monitored the progression of amyloid accumulation in control APP mice and suppressed APP mice over the next 3 months. The accumulation hypothesis would predict that the rate of amyloid from 14 to 17 months would be similar in the suppressed and control mice, while the equilibrium hypothesis would predict that the increase would be faster in the suppressed group, possibly catching up completely with the control mice. The results strongly support the accumulation hypothesis, with no evidence of the suppressed mice catching up with the control mice as predicted by equilibrium models. If anything, there was a slower rate of increase in the suppressed APP mice than the control mice, suggesting that a slow seeding mechanism likely precedes a rapid fibrillogenesis in determining the extent of amyloid deposition.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/patología , Precursor de Proteína beta-Amiloide/metabolismo , Enfermedad de Alzheimer/prevención & control , Precursor de Proteína beta-Amiloide/antagonistas & inhibidores , Precursor de Proteína beta-Amiloide/inmunología , Animales , Anticuerpos/administración & dosificación , Encéfalo/metabolismo , Encéfalo/patología , Proteínas de Drosophila/administración & dosificación , Ratones , Ratones Transgénicos , Placa Amiloide/metabolismo , Placa Amiloide/patología , Factores de TiempoRESUMEN
The epidermal growth factor receptor (EGFR) is usually overexpressed in nasopharyngeal carcinoma (NPC) and is associated with pathogenesis of NPC. However, while EGFR-modulated intracellular proteins have been extensively studied, little is known concerning their extracellular counterparts. To identify EGFR-regulated secreted proteins in NPC, we compared the secretome profiles of TGF-α-stimulated and unstimulated NPC cell line CNE-2. CNE-2 cells were cultured in the absence or presence of TGF-α for 24 h, and secreted proteins were obtained from conditioned serum-free media and enriched by ultrafiltration centrifugation. Using 2-DE and subsequent mass spectrometry, we identified 16 differential secreted proteins, among which the amyloid ß-protein precursor (APP) was up-regulated and cystatin C was down-regulated after TGF-α stimulation. We further showed that the secretory changes of APP and cystatin C in CNE-2 after TGF-α stimulation could be abrogated by pretreatment of EGFR tyrosine kinase inhibitor PD153035 and PI3 kinase inhibitor Wortmannin, validating that APP and cystatin C are EGFR-regulated secreted proteins in NPC cells. Immunohistochemistry showed that the expression level of EGFR was positively correlated with the expression level of APP and negatively correlated with the expression level of cystatin C in NPC tissues, indicating that EGFR also regulates expression of APP and cystatin C in clinical NPC tissues. Furthermore, functional analysis showed that the growth and migration of CNE-2 cells decreased after neutralization of secretory APP in the medium using the anti-APP antibody. Our data provide substantial evidence that APP and cystatin C are target secreted proteins of EGFR in NPC, and upregulation of secretory APP by EGFR may be involved in the pathogenesis of NPC.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Cistatina C/metabolismo , Receptores ErbB/metabolismo , Proteómica/métodos , Precursor de Proteína beta-Amiloide/inmunología , Androstadienos/farmacología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Western Blotting , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Electroforesis en Gel Bidimensional , Inhibidores Enzimáticos/farmacología , Humanos , Inmunohistoquímica , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patología , Quinazolinas/farmacología , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Factor de Crecimiento Transformador alfa/farmacología , WortmaninaRESUMEN
Advances in medical science have led to increased life expectancy and increased median age in the population. Because the symptoms of neurodegenerative diseases generally onset in mid- to late-life, a concomitant increase in the number of persons afflicted with these devastating diseases has occurred. Developing therapies for neurodegenerative diseases is of the highest priority due to the enormous cost of medical care required, as well as for the human suffering involved. Although caused by a variety of genetic and environmental insults, such diseases share commonalities. Many of these diseases are proteinopathies--diseases caused by misfolded, aggregating proteins. Antibodies that can recognize and remove misfolded proteins are ideally suited for proteinopathy therapeutics. The numerous intriguing advances in antibody-based therapies for neurodegenerative diseases are discussed in this review.
Asunto(s)
Inmunoglobulinas Intravenosas/uso terapéutico , Enfermedades Neurodegenerativas/inmunología , Enfermedades Neurodegenerativas/terapia , Precursor de Proteína beta-Amiloide/inmunología , Animales , Modelos Animales de Enfermedad , Humanos , Proteína Huntingtina , Ratones , Proteínas del Tejido Nervioso/inmunología , Proteínas Nucleares/inmunología , Conformación Proteica , alfa-Sinucleína/inmunología , Proteínas tau/inmunologíaRESUMEN
Generation of neurotoxic amyloid beta peptides and their deposition along with neurofibrillary tangle formation represent key pathological hallmarks in Alzheimer's disease (AD). Recent evidence suggests that inflammation may be a third important component which, once initiated in response to neurodegeneration or dysfunction, may actively contribute to disease progression and chronicity. Various neuroinflammatory mediators including complement activators and inhibitors, chemokines, cytokines, radical oxygen species and inflammatory enzyme systems are expressed and released by microglia, astrocytes and neurons in the AD brain. Degeneration of aminergic brain stem nuclei including the locus ceruleus and the nucleus basalis of Meynert may facilitate the occurrence of inflammation in their projection areas given the antiinflammatory and neuroprotective action of their key transmitters norepinephrine and acetylcholine. While inflammation has been thought to arise secondary to degeneration, recent experiments demonstrated that inflammatory mediators may stimulate amyloid precursor protein processing by various means and therefore can establish a vicious cycle. Despite the fact that some aspects of inflammation may even be protective for bystander neurons, antiinflammatory treatment strategies should therefore be considered. Non-steroidal anti-inflammatory drugs have been shown to reduce the risk and delay the onset to develop AD. While, the precise molecular mechanism underlying this effect is still unknown, a number of possible mechanisms including cyclooxygenase 2 or gamma-secretase inhibition and activation of the peroxisome proliferator activated receptor gamma may alone or, more likely, in concert account for the epidemiologically observed protection.
Asunto(s)
Enfermedad de Alzheimer , Encéfalo/patología , Inflamación/etiología , Inflamación/patología , Enfermedad de Alzheimer/complicaciones , Enfermedad de Alzheimer/inmunología , Enfermedad de Alzheimer/terapia , Péptidos beta-Amiloides/inmunología , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/inmunología , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Encéfalo/inmunología , Cromogranina A/inmunología , Cromogranina A/metabolismo , Ciclooxigenasa 2/metabolismo , Citocinas/metabolismo , Humanos , Mediadores de Inflamación/uso terapéutico , Ovillos Neurofibrilares/inmunologíaRESUMEN
Amyloid-beta (A beta) has been identified as a key component in Alzheimer's disease (AD). Significant in vitro and human pathological data suggest that intraneuronal accumulation of A beta peptides plays an early role in the neurodegenerative cascade. We hypothesized that targeting an antibody-based therapeutic to specifically abrogate intracellular A beta accumulation could prevent or slow disease onset. A beta 42-specific intracellular antibodies (intrabodies) with and without an intracellular trafficking signal were engineered from a previously characterized single-chain variable fragment (scFv) antibody. The intrabodies, one with an endoplasmic reticulum (ER) targeting signal and one devoid of a targeting sequence, were assessed in cells harboring a doxycycline (Dox)-regulated mutant human amyloid precursor protein Swedish mutant (hAPP(swe)) transcription unit for their abilities to prevent A beta peptide egress. Adeno-associated virus (AAV) vectors expressing the engineered intrabodies were administered to young adult 3xTg-AD mice, a model that develops amyloid and Tau pathologies, prior to the initial appearance of intraneuronal A beta. Chronic expression of the ER-targeted intrabody (IB) led to partial clearance of A beta 42 deposits and interestingly, in reduced staining for a pathologic phospho-Tau epitope (Thr231). This approach may provide insights into the functional relevance of intraneuronal A beta accumulation in early AD and potentially lead to the development of new therapeutics.
Asunto(s)
Enfermedad de Alzheimer/prevención & control , Precursor de Proteína beta-Amiloide/inmunología , Anticuerpos/inmunología , Dependovirus/genética , Vacunación , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/inmunología , Precursor de Proteína beta-Amiloide/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Retículo Endoplásmico , Vectores Genéticos/administración & dosificación , Humanos , Región Variable de Inmunoglobulina/inmunología , Ratones , Ratones Transgénicos , Proteínas tau/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
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
Amyloid beta-protein (A beta), the principal constituent of senile plaques seen in Alzheimer's disease (AD), is derived by proteolysis from the beta-amyloid precursor protein (beta PP). The mechanism of A beta production in neurons, which are hypothesized to be a rich source of A beta in brain, remains to be defined. In this study, we describe a detailed localization of cell surface beta PP and its subsequent trafficking in primary cultured neurons. Full-length cell surface beta PP was present primarily on perikarya and axons, the latter with a characteristic discontinuous pattern. At growth cones, cell surface beta PP was inconsistently detected. By visualizing the distribution of beta PP monoclonal antibodies added to intact cultures, beta PP was shown to be internalized from distal axons or terminals and retrogradely transported back to perikarya in organelles which colocalized with fluid-phase endocytic markers. Retrograde transport of beta PP was shown in both hippocampal and peripheral sympathetic neurons, the latter using a compartment culture system that isolated cell bodies from distal axons and terminals. In addition, we demonstrated that beta PP from distal axons was transcytotically transported to the surface of perikarya from distal axons in sympathetic neurons. Indirect evidence of this transcytotic pathway was obtained in hippocampal neurons using antisense oligonucleotide to the kinesin heavy chain to inhibit anterograde beta PP transport. Taken together, these results demonstrate novel aspects of beta PP trafficking in neurons, including retrograde axonal transport and transcytosis. Moreover, the axonal predominance of cell surface beta PP is unexpected in view of the recent report of polarized sorting of beta PP to the basolateral domain of MDCK cells.