RESUMO
Quantifying the physical properties of individual exosomes containing amyloid-ß42 (Aß42) is crucial for a better understanding of an underpinning mechanism of Alzheimer's disease expression which is associated with the Aß42 transfer. Because of the lack of proper tools, however, there have been very few studies on how the amount of Aß42 affects the physical properties of exosomes. To answer the question, we investigated the physical properties of exosomes secreted by neuroblastoma by probing individual exosomes using electrostatic force microscopy. Interestingly, we observed that when the higher concentration of Aß42 oligomers was fed to cells, the higher surface charge of the exosomes appeared. This result indicates that the exosomes contain more Aß42 with the increase in Aß42 concentration in cell media, implying that they serve as transport vesicles for Aß42. Our approach could help to better understand how the neuronal exosomes are related to the propagation of neurodegenerative diseases and to seek how to make an early diagnosis of those diseases.
Assuntos
Peptídeos beta-Amiloides/metabolismo , Exossomos/metabolismo , Neurônios/metabolismo , Fragmentos de Peptídeos/metabolismo , Animais , Linhagem Celular , Exossomos/ultraestrutura , Camundongos , Microscopia de Força Atômica , Microscopia Eletroquímica de Varredura , Transporte Proteico , Eletricidade EstáticaRESUMO
Amyloid aggregates have emerged as a significant hallmark of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Although it has been recently reported that microwave heating induces amyloid aggregation compared with conventional heating methods, the mechanism of amyloid aggregate induction has remained unclear. In this study, we investigated the formation of oligomeric amyloid aggregates (OAAs) by microwave irradiation at microscale volumes of solution. Microwave irradiation of protein monomer solution triggered rapid formation of OAAs within 7 min. We characterized the formation of OAAs using atomic force microscopy, thioflavin T fluorescent assay and circular dichroism. In the microwave system, we also investigated the inhibitory effect on the formation of amyloid aggregates by L-ascorbic acid as well as enhanced amyloid aggregation by silver nanomaterials such as nanoparticles and nanowires. We believe that microwave technology has the potential to facilitate the study of amyloid aggregation in the presence of chemical agents or nanomaterials.
Assuntos
Amiloide/química , Micro-Ondas , Agregados Proteicos , Dicroísmo Circular , Lactoglobulinas/química , Nanoestruturas/química , Nanoestruturas/ultraestrutura , Prata/químicaRESUMO
Clearing amyloid-ß (Aß) through immunotherapy is one of the most promising therapeutic approaches to Alzheimer's disease (AD). Although several monoclonal antibodies against Aß have been shown to substantially reduce Aß burden in patients with AD, their effects on improving cognitive function remain marginal. In addition, a significant portion of patients treated with Aß-targeting antibodies experience brain edema and microhemorrhage associated with antibody-mediated Fc receptor activation in the brain. Here, we develop a phagocytosis inducer for Aß consisting of a single-chain variable fragment of an Aß-targeting monoclonal antibody fused with a truncated receptor binding domain of growth arrest-specific 6 (Gas6), a bridging molecule for the clearance of dead cells via TAM (TYRO3, AXL, and MERTK) receptors. This chimeric fusion protein (αAß-Gas6) selectively eliminates Aß plaques through TAM receptor-dependent phagocytosis without inducing NF-kB-mediated inflammatory responses or reactive gliosis. Furthermore, αAß-Gas6 can induce synergistic clearance of Aß by activating both microglial and astrocytic phagocytosis, resulting in better behavioral outcomes with substantially reduced synapse elimination and microhemorrhage in AD and cerebral amyloid angiopathy model mice compared with Aß antibody treatment. Our results suggest that αAß-Gas6 could be a novel immunotherapeutic agent for AD that overcomes the side effects of conventional antibody therapy.
Assuntos
Doença de Alzheimer , Anticorpos de Cadeia Única , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/terapia , Peptídeos beta-Amiloides/metabolismo , Animais , Anti-Inflamatórios/uso terapêutico , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Modelos Animais de Doenças , Camundongos , Camundongos Transgênicos , NF-kappa B , Placa Amiloide/tratamento farmacológico , Receptores Fc/uso terapêutico , Anticorpos de Cadeia Única/uso terapêutico , c-Mer Tirosina QuinaseRESUMO
We report that repeated thermal perturbation by thermal cycling (TC) accelerates the formation rate of amyloid filaments at microliter volumes (10-200 µL) and produces a new conformation of zigzag-shaped filaments. The amyloid filaments have been synthesized under different TC conditions, such as temperature variations (ΔT = 0-86 °C) and the number of cycles (C# = 30-90). In particular, the filament formation was promoted by TC with ΔT ≥ 30 °C. This indicates that the change in binding energy of ß-sheets and the breakage of disulfide bonds induced by TC with large ΔT contributed to the increased filament growth. This molecular interaction was investigated by molecular dynamics simulation. We also found that TC leads to the formation of amyloid filaments with peculiar conformation (zigzag-shaped filaments). Moreover, key structural parameters (tortuosity, segment length, and joint angle) of the amyloid filaments could be fine-tuned by selecting certain ΔT conditions. Taken together, we confirmed that the TC not only promotes the formation of amyloid filaments but also affects the conformational changes of the filaments.