A High-Throughput Assay for Monitoring and Quantifying Amyloid-β Accumulation and Clearance in Alzheimer's Disease...

Amyloidogenic proteins, such as amyloid-β (Aβ), self-assemble into cross-β fibrils whose accumulation is central to Alzheimer's disease (AD). Measuring Aβ aggregation and clearance in living cells remains challenging using current cell-based assays, which are often low-throughput or not suited for real-time monitoring. This study aimed to (1) develop a robust, quantitative, and scalable fluorescence-based assay using Amytracker to monitor Aβ accumulation and clearance in an Aβ-producing neuronal cell model, and (2) validate its utility for mechanistic studies and therapeutic screening. We established a plate-based fluorescence assay using Amytracker in MC65 neuronal AD model expressing the Amyloid precursor protein C-terminal fragment (APP-C99) that generates Aβ. Accumulation and clearance of Aβ were quantified by measuring Amytracker fluorescence under basal conditions and after inducing Aβ clearance using a Tet-suppressible system. We utilized this assay to evaluate cell death inhibitors ferrostatin-1 and liproxstatin-1 and proteasome activator IU1. Specificity of the assay for amyloidogenic proteins was assessed by treating wild-type neuroblastoma cells with Aβ, human islet amyloid polypeptide (hIAPP), or non-aggregating Aβ controls. Validation included Aβ immunoblotting and cell viability assays. In APP-C99 expressing cells, elevated Amytracker fluorescence correlated with increased Aβ accumulation and reduced cell viability. Supplementation of ferrostatin-1, liproxstatin-1, and IU1, on these cells, markedly reduced Amytracker signal, indicating decreased Aβ burden. Furthermore, the Amytracker assay specifically detected amyloidogenic protein aggregation: wild-type cells exposed to Aβ42 or hIAPP showed high fluorescence, whereas non-aggregating Aβ16 peptide did not. The Amytracker assay provides a simple, non-toxic, and high-throughput platform for quantifying Aβ accumulation and clearance in live cell models. Its sensitivity, specificity, and compatibility with high-throughput screening make it a valuable tool for studying Aβ dynamics, interrogating mechanisms of proteostasis, and identifying therapeutic candidates targeting Aβ.