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Cells
Volume 14
Issue 19
10.3390/cells14191542
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This is an early access version, the complete PDF, HTML, and XML versions will be available soon.
Article

Spectral Profiling of Early αsyn Aggregation in HEK293 Cells Modified to Stably Express Human WT and A53T-αsyn

by
Priyanka Swaminathan
1,
Karsten Sættem Godø
1,
Eline Bærøe Bjørn
1,
Therése Klingstedt
2,
Debdeep Chatterjee
2,
Per Hammarström
2,3,
Rajeevkumar Raveendran Nair
4 and
Mikael Lindgren
1,*
1
Department of Physics, Faculty of Natural Sciences, Norwegian University of Science and Technology (NTNU), NO-7491 Trondheim, Norway
2
Department of Physics, Chemistry and Biology, Linköping University, SE-581 83 Linköping, Sweden
3
SciLifeLab, Linköping University, SE-581 83 Linköping, Sweden
4
Kavli Institute for Systems Neuroscience, Norwegian University of Science and Technology (NTNU), Olav Kyrres Gate 9, NO-7030 Trondheim, Norway
*
Author to whom correspondence should be addressed.
Cells 2025, 14(19), 1542; https://doi.org/10.3390/cells14191542
Submission received: 29 August 2025 / Revised: 25 September 2025 / Accepted: 30 September 2025 / Published: 2 October 2025
(This article belongs to the Special Issue Applications of Proteomics in Human Diseases and Treatments)
Versions Notes

Abstract

Alpha-synuclein (αsyn) misfolding and aggregation underlie several neurodegenerative disorders, including Parkinson’s disease. Early oligomeric intermediates are particularly toxic yet remain challenging to detect and characterize within cellular systems. Here, we employed the luminescent conjugated oligothiophene h-FTAA to investigate early aggregation events of human wildtype (huWT) and A53T-mutated αsyn (huA53T) both in vitro and in HEK293 cells stably expressing native human-αsyn. Comparative fibrillation assays revealed that h-FTAA detected αsyn aggregation with higher sensitivity and earlier onset than Thioflavin T, with the A53T variant displaying accelerated fibrillation. HEK293 cells stably expressing huWT- or huA53T-αsyn were exposed to respective pre-formed fibrils (PFFs), assessed via immunocytochemistry, h-FTAA staining, spectral emission profiling, and fluorescence lifetime imaging microscopy (FLIM). Notably, huA53T PFFs promoted earlier aggregation patterns and yielded narrower fluorescence lifetime distributions compared with huWT PFFs. Spectral imaging showed h-FTAA emission maxima (~550–580 nm) red-shifted and broadened in cells along with variable lifetimes (0.68–0.87 ns), indicating heterogeneous aggregate conformations influenced by cellular milieu. These findings demonstrate that h-FTAA is useful for distinguishing early αsyn conformers in living systems and, together with stable αsyn-expressing HEK293 cells, offers a platform for probing early αsyn morphotypes. Taken together, this opens for further discovery of biomarkers and drugs that can interfere with αsyn aggregation.
Keywords: alpha-synuclein; Parkinson’s disease; pre-formed fibrils; A53T mutation; fibrillation kinetics assay; ThT; h-FTAA; stable αsyn; HEK293 cells; spectral imaging; fluorescence lifetime imaging alpha-synuclein; Parkinson’s disease; pre-formed fibrils; A53T mutation; fibrillation kinetics assay; ThT; h-FTAA; stable αsyn; HEK293 cells; spectral imaging; fluorescence lifetime imaging

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MDPI and ACS Style

Swaminathan, P.; Godø, K.S.; Bjørn, E.B.; Klingstedt, T.; Chatterjee, D.; Hammarström, P.; Nair, R.R.; Lindgren, M. Spectral Profiling of Early αsyn Aggregation in HEK293 Cells Modified to Stably Express Human WT and A53T-αsyn. Cells 2025, 14, 1542. https://doi.org/10.3390/cells14191542

AMA Style

Swaminathan P, Godø KS, Bjørn EB, Klingstedt T, Chatterjee D, Hammarström P, Nair RR, Lindgren M. Spectral Profiling of Early αsyn Aggregation in HEK293 Cells Modified to Stably Express Human WT and A53T-αsyn. Cells. 2025; 14(19):1542. https://doi.org/10.3390/cells14191542

Chicago/Turabian Style

Swaminathan, Priyanka, Karsten Sættem Godø, Eline Bærøe Bjørn, Therése Klingstedt, Debdeep Chatterjee, Per Hammarström, Rajeevkumar Raveendran Nair, and Mikael Lindgren. 2025. "Spectral Profiling of Early αsyn Aggregation in HEK293 Cells Modified to Stably Express Human WT and A53T-αsyn" Cells 14, no. 19: 1542. https://doi.org/10.3390/cells14191542

APA Style

Swaminathan, P., Godø, K. S., Bjørn, E. B., Klingstedt, T., Chatterjee, D., Hammarström, P., Nair, R. R., & Lindgren, M. (2025). Spectral Profiling of Early αsyn Aggregation in HEK293 Cells Modified to Stably Express Human WT and A53T-αsyn. Cells, 14(19), 1542. https://doi.org/10.3390/cells14191542

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