Author Identifier (ORCID)
Ajish Ariyath: https://orcid.org/0000-0003-1611-2990
Fraulein Denise Arigo: https://orcid.org/0009-0009-5169-0199
Anna Fyfe: https://orcid.org/0009-0009-1654-361X
W. M.A.D.Binosha Fernando: https://orcid.org/0000-0002-8364-7808
Ralph Martins: https://orcid.org/0000-0002-4828-9363
Prashant Bharadwaj: https://orcid.org/0000-0003-4361-9906
Abstract
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. (Figure presented.).
Keywords
amyloid beta, Alzheimer’s disease, protein aggregation, neuronal cells, fluorescence assay, therapeutic screening
Document Type
Journal Article
Date of Publication
5-1-2026
Volume
170
Issue
5
PubMed ID
42141806
Publication Title
Journal of Neurochemistry
Publisher
Wiley
School
Centre of Excellence for Alzheimer's Disease Research and Care / School of Medical and Health Sciences / Sarich and Patricia Neuroscience Research Institute
Funding Information
We acknowledge the support of NH and MRC-ARC dementia research development fellowship (APP1107109) and funding from National Foundation for Medical research innovation (NFMRI) to Prashant Bharadwaj.
Grant Number
ARC Number : APP1107109
Creative Commons License
This work is licensed under a Creative Commons Attribution 4.0 License.
Comments
Ariyath, A., Arigo, F. D., Fyfe, A., Fernando, W. M. a. D. B., Martins, R., & Bharadwaj, P. (2026). A high-throughput assay for monitoring and quantifying amyloid-β accumulation and clearance in Alzheimer’s disease cell models. Journal of Neurochemistry, 170(5), e70473. https://doi.org/10.1111/jnc.70473