Devyatov Alexander, Kozlov Ihor, Das Viswanath
Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University and University Hospital Olomouc, Hněvotínská, Olomouc, 1333/5, 779 00, Czech Republic.
Institute of Molecular and Translational Medicine, Czech Advanced Technologies and Research Institute, Palacký University Olomouc, Křížkovského 511/8, 779 00, Olomouc, Czech Republic.
Mol Neurobiol. 2025 Jun 4. doi: 10.1007/s12035-025-05100-3.
SH-SY5Y cells are widely used as an in vitro neuronal model, yet reliable differentiation protocols tailored for tauopathy research remain limited. Effective differentiation is essential for studying tau aggregation, propagation, and neurodegenerative mechanisms. Here, we present an optimized two-step differentiation protocol for TauP301L-expressing SH-SY5Y cells that enhances neuronal maturation and tauopathy modeling, providing a physiologically relevant system for investigating tau seeding. SH-SY5Y cells expressing TauP301L-EGFP under an inducible system were differentiated using a two-step protocol consisting of retinoic acid (RA) for 72 h, followed by brain-derived neurotrophic factor (BDNF) and RA for 72 h. Differentiated neurons were then exposed to exogenous P301L tau peptide fibrils to assess their susceptibility to tau seeding and aggregation. Differentiation resulted in increased neurite outgrowth, cholinergic marker expression (ChAT upregulation, TH downregulation), and upregulation of the mature 2N4R tau isoform. Western blot analysis showed increased T22 and pSer262 tau immunoreactivity in seeded cells, consistent with tau conformational changes and pathological phosphorylation. These findings may reflect early stages of tau misfolding but do not confirm oligomer formation. Seeding also induced neurite remodeling, varicosity formation, and reduced neurite diameter-features consistent with tau-mediated pathology involving cytoskeletal changes, organelle accumulation, or axonal transport defects. This optimized differentiation protocol provides an experimentally tractable tauopathy model for investigating tau propagation and neuronal dysfunctions in a controlled human cell context. Compared to existing SH-SY5Y differentiation methods, our system provides faster neuronal maturation, controlled TauP301L induction, and enhanced tau isoform expression, making it a valuable platform for studying early tau misfolding events and therapeutic interventions in tauopathies.
SH-SY5Y细胞被广泛用作体外神经元模型，但针对tau蛋白病研究的可靠分化方案仍然有限。有效的分化对于研究tau蛋白聚集、传播和神经退行性机制至关重要。在这里，我们提出了一种优化的两步分化方案，用于表达TauP301L的SH-SY5Y细胞，该方案可增强神经元成熟和tau蛋白病建模，为研究tau蛋白种子提供了一个生理相关系统。在诱导系统下表达TauP301L-EGFP的SH-SY5Y细胞采用两步方案进行分化，第一步用视黄酸（RA）处理72小时，然后用脑源性神经营养因子（BDNF）和RA处理72小时。然后将分化的神经元暴露于外源性P301L tau肽原纤维，以评估它们对tau蛋白种子和聚集的敏感性。分化导致神经突生长增加、胆碱能标志物表达（ChAT上调，TH下调）以及成熟的2N4R tau异构体上调。蛋白质免疫印迹分析显示，接种细胞中T22和pSer262 tau免疫反应性增加，这与tau蛋白构象变化和病理性磷酸化一致。这些发现可能反映了tau蛋白错误折叠的早期阶段，但未证实寡聚体形成。接种还诱导了神经突重塑、静脉曲张形成和神经突直径减小，这些特征与涉及细胞骨架变化、细胞器积累或轴突运输缺陷的tau介导的病理学一致。这种优化的分化方案为在可控的人类细胞环境中研究tau蛋白传播和神经元功能障碍提供了一个易于实验操作的tau蛋白病模型。与现有的SH-SY5Y分化方法相比，我们的系统提供了更快的神经元成熟、可控的TauP301L诱导和增强的tau异构体表达，使其成为研究tau蛋白病早期错误折叠事件和治疗干预的有价值平台。
