Brains in focus: Uncovering protein aggregate changes across Alzheimer’s disease with single‐molecule imaging

Abstract

AbstractBackgroundOligomers are increasingly implicated in Alzheimer’s disease (AD)1. The characteristics of these protein aggregates remain largely unknown due to their low biological concentration, solubility, small size and structural heterogeneity. This study aims to better classify the diverse protein aggregates found in human brains across AD onset and progression.MethodsPost‐mortem flash‐frozen brain samples (n = 28) spanning the full range of AD stages (Braak 0‐VI) were obtained from London Neurodegenerative Diseases Brain Bank: two brain regions, middle temporal gyrus (MTG) and primary somatosensory cortex (SOM), were obtained from 14 donors. The post‐mortem tissues were homogenised to extract proteins into liquid fractions for single‐molecule studies. Antibodies, including 6E10, HT7, AT8 and SC‐12767, were selected to bind aggregates of amyloid‐β, tau, phosphorylated tau and α‐synuclein respectively. Ultra‐sensitive single‐molecule array (Simoa®) assays measured protein aggregate quantities in the homogenates. Single‐molecule pull‐down (SiMPull) assays, imaged by super‐resolution fluorescence microscopy, measured aggregate size and shape within the homogenates. BCA assays quantified the total protein content of the homogenates for normalisation.ResultsMorphological differences in tau aggregates were observed between disease stages with super‐resolution microscopy. Quantities of phosphorylated and total tau aggregates tend to increase significantly in the MTG and SOM from mid‐stage AD onwards according to Simoa® assays. In comparison to tau, aggregate quantities of amyloid‐β and α‐synuclein displayed less variation between non‐diseased controls and different AD stages.ConclusionsSingle‐molecule techniques can sensitively detect a broad range of protein aggregates from human brains. Novel differences in the morphologies and quantities of aggregates have been identified across AD stages. There is strong potential for uncovering the specific protein aggregates responsible for AD with these methods. This could facilitate earlier disease detection and more effective treatment targeting. 1 Hardy, J. & Selkoe, D. J. The amyloid hypothesis of Alzheimer’s disease: progress and problems on the road to therapeutics. Science 297, 353‐356 (2002).