In-vitro modelling of Alzheimer's disease using cholinergic neurons derived from human neuroblastoma (SH-SY5Y) retinoic acid-induced differentiation

Background: Alzheimer's disease (AD) is characterised by severe degeneration of cholinergic neurons within the basal forebrain complex (FBC) which is a key regulator of cognitive function. Cholinergic loss represents a central pathological hallmark of AD; however, the underlying molecular mechanisms remain incompletely understood. Although various in-vitro models are available, many are limited by species-specific differences, high cost, and technical complexity. Human neuroblastoma (SH-SY5Y) cells can be differentiated into neuron-like cells and represent a practical alternative for AD research. This study aimed to optimise retinoic acid (RA)-based differentiation conditions to enhance cholinergic characteristics in SH-SY5Y cells and evaluate their susceptibility to AD-related stressors as a simplified, cost-effective model for preliminary high-throughput AD studies.

Methods: A structured literature search (2000-2025) was conducted using PubMed and ScienceDirect. After screening based on predefined criteria, 23 relevant studies were analysed for differentiation inducers, serum concentration, duration, neuronal markers, and cholinergic markers. Here, a simplified RA-only protocol was evaluated using 10µM RA with 1% or 3% heat-inactivated foetal bovine serum (1% or 3% HI-FBS) over 3, 5, and 7 days. Neuronal differentiation was assessed by morphological analysis, neurite length measurement, choline acetyltransferase (ChAT) and acetylcholinesterase (AChE) gene expressions, acetylcholinesterase (AChE) activity. Additionally, model relevance was further evaluated using AD-associated stressors such as streptozotocin (STZ), hydrogen peroxide (H₂O₂), lipopolysaccharide (LPS), and aluminium chloride (AlCl₃).

Results: Although most protocols generated mature neuron-like cells, only ~ 30% reported cholinergic marker expression, with retinoic acid (RA) and brain-derived neurotrophic factor (BDNF) as the most common inducers. This study reports that differentiation with 1% HI-FBS with 10µM RA for 7 days produced pronounced neuronal morphology, significant neurite extension, and extensive branching. These cells demonstrated a cholinergic-like phenotype, with significant upregulation of ChAT and AChE gene expressions, accompanied by increased AChE enzymatic activity. These neuron-like cells also showed dose-dependent responses to STZ, H₂O₂, and AlCl₃, with time-dependent effects observed for H₂O₂ and AlCl₃. Notably, cells were resistant to LPS-induced cytotoxicity.

Conclusion: These findings support the utility of this RA-differentiated SH-SY5Y for neuronal-like cells for cholinergic-like model (1% HI-FBS, 10µM RA) as a practical and cost-effective platform for high-throughput AD drug screening.

Alzheimer’s disease; cell differentiation; cholinergic neurons; cytotoxicity; neuronal differentiation; retinoic acid.