Alpha-Synuclein FRET Biosensors Reveal Early Alpha-Synuclein Aggregation in the Endoplasmic Reticulum

Area of Science:

• Neuroscience
• Biochemistry
• Cell Biology

Background:

• Endoplasmic reticulum (ER) dysfunction is implicated in alpha-synuclein (αS) toxicity.
• αS aggregation and conformational changes are key events in neurodegenerative diseases.
• Existing methods like immunofluorescence have limitations in tracking αS dynamics in real-time.

Purpose of the Study:

• To develop and validate novel Förster resonance energy transfer (FRET) biosensors (AFBs) for monitoring αS conformation within cells.
• To investigate the conformational state of αS in native and pro-aggregating conditions, particularly within the ER.
• To assess the utility of AFBs in studying αS behavior in vivo.

Main Methods:

• Development of intermolecular and intramolecular αS FRET biosensors (AFBs) by fusing CFP/YFP to αS.
• Expression of AFBs in SH-SY5Y cells and a stable cell line (i36) with ER-targeted αS.
• Measurement of FRET signals under native and pro-aggregating conditions to infer αS conformation and oligomerization.

Main Results:

• Intramolecular AFBs confirmed a predominantly globular, monomeric αS conformation under native conditions.
• Intermolecular AFBs detected αS oligomer formation under pro-aggregating conditions, especially when ER-localized.
• ER-targeted AFBs demonstrated the ER's potential role in modulating αS conformational changes during aggregation.

Conclusions:

• αS FRET biosensors (AFBs) provide a valuable tool for tracking αS conformational dynamics in living cells.
• The endoplasmic reticulum environment may influence αS conformational transitions and aggregation.
• AFBs offer a sensitive method to study αS behavior in the context of neurodegenerative disease research.