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

## TL;DR

This study uses a special dye to detect early stages of alpha-synuclein aggregation in cells, which could help in understanding and treating Parkinson's disease.

## Contribution

The study introduces a novel method using h-FTAA for detecting early αsyn aggregation in living cells and identifies differences between wildtype and A53T-mutated αsyn.

## Key findings

- h-FTAA detects αsyn aggregation earlier and more sensitively than Thioflavin T.
- A53T αsyn shows accelerated fibrillation and narrower fluorescence lifetime distributions compared to wildtype αsyn.
- Spectral imaging reveals heterogeneous aggregate conformations influenced by the cellular environment.

## 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.