RESUMEN
Amyloid peptide (Aß) is the major protein component of plaques found in Alzheimer's disease, and the aggregation of Aß into oligomeric and fibrillic assemblies has been shown to be an early event of the disease pathway. Visualization of the progressive evolution of nanoscale changes in the morphology of Aß oligomeric assemblies and amyloid fibrils has been accomplished ex situ using atomic force microscopy (AFM) in ambient conditions. In this report, the size and the shape of amyloid ß(1-40) fibrils, as well as the secondary organization into aggregate structures were monitored at different intervals over a period of 5 months. Characterizations with tapping-mode AFM serve to minimize the strong adhesive forces between the probe and the sample to prevent damage or displacement of fragile fibrils. The early stages of Aß growth showed a predominance of spherical seed structures, oligomeric assemblies, and protofibrils; however the size and density of fibrils progressively increased with time. Within a few days of incubation, linear assemblies and fibrils became apparent. Over extended time scales of up to 5 months, the fibrils formed dense ensembles spanning lengths of several microns, which exhibit interesting changes due to self-organization of the fibrils into bundles or tangles. Detailed characterization of the Aß assembly process at the nanoscale will help elucidate the role of Aß in the pathology of Alzheimer's disease.
Asunto(s)
Péptidos beta-Amiloides/química , Amiloide/química , Microscopía de Fuerza Atómica/métodos , Enfermedad de Alzheimer/metabolismo , Amiloide/metabolismo , Amiloide/ultraestructura , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/ultraestructura , Humanos , Nanoestructuras/ultraestructura , Tamaño de la Partícula , Espectrometría de FluorescenciaRESUMEN
Neuronal cytotoxicity observed in Alzheimer's disease (AD) is linked to the aggregation of ß-amyloid peptide (Aß) into toxic forms. Increasing evidence points to oligomeric materials as the neurotoxic species, not Aß fibrils; disruption or inhibition of Aß self-assembly into oligomeric or fibrillar forms remains a viable therapeutic strategy to reduce Aß neurotoxicity. We describe the synthesis and characterization of amyloid aggregation mitigating peptides (AAMPs) whose structure is based on the Aß "hydrophobic core" Aß(17-20), with α,α-disubstituted amino acids (ααAAs) added into this core as potential disrupting agents of fibril self-assembly. The number, positional distribution, and side-chain functionality of ααAAs incorporated into the AAMP sequence were found to influence the resultant aggregate morphology as indicated by ex situ experiments using atomic force microscopy (AFM) and transmission electron microscopy (TEM). For instance, AAMP-5, incorporating a sterically hindered ααAA with a diisobutyl side chain in the core sequence, disrupted Aß(1-40) fibril formation. However, AAMP-6, with a less sterically hindered ααAA with a dipropyl side chain, altered fibril morphology, producing shorter and larger sized fibrils (compared with those of Aß(1-40)). Remarkably, ααAA-AAMPs caused disassembly of existing Aß fibrils to produce either spherical aggregates or protofibrillar structures, suggesting the existence of equilibrium between fibrils and prefibrillar structures.
Asunto(s)
Péptidos beta-Amiloides/metabolismo , Fragmentos de Péptidos/metabolismo , Péptidos/química , Péptidos/metabolismo , Péptidos beta-Amiloides/química , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía de Fuerza Atómica/métodos , Fragmentos de Péptidos/química , Unión Proteica/fisiología , Relación Estructura-ActividadRESUMEN
Considerable research effort has focused on the discovery of mitigators that block the toxicity of the ß-amyloid peptide (Aß) by targeting a specific step involved in Aß fibrillogenesis and subsequent aggregation. Given that aggregation intermediates are hypothesized to be responsible for Aß toxicity, such compounds could likely prevent or mitigate aggregation, or alternatively cause further association of toxic oligomers into larger nontoxic aggregates. Herein we investigate the effect of modifications of the KLVFF hydrophobic core of Aß by replacing N- and C-terminal groups with various polar moieties. Several of these terminal modifications were found to disrupt the formation of amyloid fibrils and in some cases induced the disassembly of preformed fibrils. Significantly, mitigators that incorporate MiniPEG polar groups were found to be effective against Aß(1-40) fibrilligonesis. Previously, we have shown that mitigators incorporating alpha,alpha-disubstituted amino acids (ααAAs) were effective in disrupting fibril formation as well as inducing fibril disassembly. In this work, we further disclose that the number of polar residues (six) and ααAAs (three) in the original mitigator can be reduced without dramatically changing the ability to disrupt Aß(1-40) fibrillization in vitro.