In Vitro Cell Model Investigation of Alpha-Synuclein Aggregate Morphology Using Spectroscopic Imaging

Area of Science:

• Neuroscience
• Biochemistry
• Molecular Biology

Background:

• Alpha-synuclein (αsyn) protein strain morphology is hypothesized to correlate with clinical subtypes of αsynucleinopathies.
• Direct evidence linking αsyn strain morphology to disease subtypes is lacking due to challenges in conformation-specific characterization.
• Accurate differentiation of αsynucleinopathies is crucial for prognosis and targeted treatment, especially at early disease stages.

Purpose of the Study:

• To develop and validate a novel in vitro cell-based method for characterizing diverse αsyn aggregate morphotypes.
• To investigate the potential of conformation-specific ligands, like h-FTAA, for differentiating αsyn strains.
• To establish a foundation for future in vitro and in vivo toxicity studies of distinct αsyn strains.

Main Methods:

• Developed a HEK293 cell model expressing wildtype-αsyn and A53T-αsyn variants.
• Utilized the amyloid fibril-specific probe h-FTAA for spectroscopic investigation of αsyn aggregates.
• Exposed cells to pre-formed αsyn fibrils and analyzed intracellular aggregate morphotypes via spectral profiles and fluorescence lifetime of h-FTAA binding.

Main Results:

• The h-FTAA probe exhibited a blue-shifted spectrum and longer fluorescence decay time upon binding to αsyn aggregates, indicating hydrophobic binding sites.
• In vitro characterization showed a high binding affinity of h-FTAA to αsyn pre-formed fibrils (K_d < 100 nM).
• The cell-based method successfully characterized intracellular αsyn aggregate morphotypes based on h-FTAA spectral features and fluorescence lifetime.

Conclusions:

• The developed cell culture method, combined with conformation-specific ligands, enables the characterization of αsyn strain morphology.
• This approach provides a pathway for investigating the toxicity of different αsyn strains.
• The methodology holds promise for early, subtype-specific diagnosis of αsynucleinopathies and potentially other neurodegenerative diseases.