Abstract
<jats:title>ABSTRACT</jats:title> <jats:p> Reliable in vitro evaluation of amyloid-β (Aβ) toxicity is essential for the development of anti-amyloid therapeutics, yet experimental workflows often lack standardization. In our previous work, we established a reproducible protocol for the synthesis, characterization and controlled aggregation of highly pure Aβ1-42. Here, we address the biological component of this variability by evaluating the impact of neuronal differentiation and toxicity assays on Aβ-induced neurotoxicity. SH-SY5Y cells were differentiated using retinoic acid and brain-derived neurotrophic factor, generating a neuron-like phenotype validated by immunofluorescence, gene expression profiling and resistance to H <jats:sub>2</jats:sub> O <jats:sub>2</jats:sub> -induced oxidative stress. Using this characterized model, we investigated the effects of non-aggregated and pre-aggregated Aβ1-42 species on cell viability and transcriptional responses. Strikingly, Aβ toxicity was highly dependent on the aggregation state of the peptide, the differentiation status of the target cells and the viability assay employed. Our results suggest that the lack of standardization in peptide quality, aggregation procedures, neuronal maturation and toxicity assessment represents a major source of variability in the amyloid field. Together, these findings provide a methodological framework to improve the reproducibility and translational relevance of in vitro screening strategies for anti-amyloid therapeutics. </jats:p>