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.

• Klíčová slova
• Alzheimer’s disease, Choline Acetyltransferase, Neurodegenerative disease, SH-SY5Y, Tau, Tyrosine Hydroxylase, Vesicular glutamate transporter 1,
• MeSH
• buněčná diferenciace * účinky léků   fyziologie   MeSH
• lidé MeSH
• mozková kůra * patologie   MeSH
• nádorové buněčné linie MeSH
• neurity metabolismus   účinky léků   MeSH
• neurony * patologie   metabolismus   účinky léků   MeSH
• proteiny tau metabolismus   MeSH
• tauopatie * patologie   metabolismus   MeSH
• tretinoin farmakologie   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• proteiny tau MeSH
• tretinoin MeSH

Tau aggregation driven by microtubule-associated protein tau (MAPT) mutations is central to frontotemporal dementia pathology, yet no disease-modifying therapies effectively target mutant tau. Here, we identify purpurin (PUR) and oleocanthal (OLC) as selective inhibitors of mutant tau aggregation using peptide models spanning the R2R3 interface. Biophysical and cellular assays demonstrated that both compounds more effectively inhibit the aggregation of mutant tau peptides compared to wild-type, with PUR preferentially targeting V287I and N279K variants, and OLC showing broader inhibitory activity. Surface plasmon resonance and docking analyses revealed more stable interactions and lower binding free energies with mutant tau, consistent with their enhanced inhibitory effects. Computational studies using monomeric and fibrillar tau structures supported the mutation-specific binding profiles of PUR and OLC. Atomic force microscopy and confocal imaging confirmed reduced fibril formation, while post-transduction treatment assays showed that both compounds significantly suppressed intracellular tau propagation. Additionally, OLC reduced tau phosphorylation and oligomerization in SY5Y-TauP301L-EGFP cells expressing mutant tau. These findings highlight the potential of PUR and OLC as structurally distinct, mutation-targeted inhibitors of tau aggregation and propagation, providing a rationale for their further development as candidate therapeutics for frontotemporal dementia.

• Klíčová slova
• MAPT mutations, frontotemporal dementia, oleocanthal, purpurin, seeding competency, surface plasmon resonance, tau aggregation, tauopathies,
• MeSH
• frontotemporální demence * metabolismus   genetika   farmakoterapie   MeSH
• katecholy * farmakologie   chemie   MeSH
• lidé MeSH
• mutace MeSH
• organofosfáty MeSH
• přemostěné cyklické sloučeniny MeSH
• proteinové agregáty účinky léků   MeSH
• proteiny tau * genetika   chemie   metabolismus   antagonisté a inhibitory   MeSH
• simulace molekulového dockingu MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• CLR01 compound MeSH Prohlížeč
• katecholy * MeSH
• MAPT protein, human MeSH Prohlížeč
• organofosfáty MeSH
• přemostěné cyklické sloučeniny MeSH
• proteinové agregáty MeSH
• proteiny tau * MeSH