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Background <p>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.</p> Methods <p>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 (<i>ChAT</i>) and acetylcholinesterase (<i>AChE</i>) 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).</p> Results <p>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 <i>ChAT</i> and <i>AChE</i> 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.</p> Conclusion <p>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.</p> Graphical abstract <p></p>

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

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年6月23日

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  • Muhammad-Safuan Zainuddin,
  • Kasthuri Bai Magalingam,
  • Narendra Pamidi,
  • Adzzie Shazleen Azman,
  • Saatheeyavaane Bhuvanendran

摘要

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.

Graphical abstract