Neuronal age drives β-sheet accumulation and, together with ApoE4, enhances synaptic Aβ localization in APP NL-F neurons

Abstract Amyloid-β (Aβ) accumulation and aggregation are defining features of Alzheimer’s disease (AD), but the earliest cellular events driving these processes remain poorly understood. Here, we investigate Aβ dynamics in primary neurons derived from APP NL-F knock-in mice, which express human APP and Aβ at endogenous (non-overexpressed) levels. Using correlative optical photothermal infrared (OPTIR) spectromicroscopy and immunofluorescence, we show that physiological APP NL-F expression is sufficient to drive intraneuronal Aβ accumulation at synapses. Modelling neuronal aging through prolonged culture reveals a marked increase in synaptic Aβ burden accompanied by β-sheet–rich structural transitions Supplementation with astrocyte-derived apolipoprotein E (ApoE) isoforms demonstrates that ApoE4 markedly increases synaptic Aβ accumulation while reducing β-sheet content relative to untreated cultures, suggesting a potential shift toward less fibrillar species. Together, these findings establish APP NL-F neurons as a physiologically relevant system for dissecting early Aβ pathology and show that both Aβ quantity (accumulation) and quality (structural maturation) are regulated by aging and ApoE genotype. This work provides mechanistic insight into the earliest molecular events that may underlie synaptic vulnerability in AD.