Effect of Stabilizing Alpha Synuclein‐Membrane Interactions on the Protein's Aggregation and Neurotoxicity
Authors: Ysselstein Daniel , Mishra Vartika , McCabe George , Rochet Jean‐Christophe 
Published: November 2023

Oligomerization of the protein alpha‐synuclein (aSyn) is a critical factor in the onset of both familial and sporadic Parkinson's disease (PD). In healthy individuals aSyn exists in a number of conformations, including a membrane‐bound state that displays resistance to aggregation. How this aggregation‐resistant state is altered is a significant problem in PD research, as a better understanding of the events that initiate aSyn aggregation is critical for designing neuroprotective strategies. We hypothesize that disruption of interactions between aSyn and phospholipid membranes leads to a shift to a conformation that is more susceptible to the formation of neurotoxic aSyn oligomers due to exposure of the protein's central hydrophobic region. Therefore, stabilization of membrane‐bound aSyn should prevent membrane‐induced aggregation and alleviate aSyn neurotoxicity.

We generated aSyn variants predicted to disrupt aSyn‐membrane interactions, including the familial mutants A30P and G51D. We characterized the variants in terms of their aggregation on synthetic phospholipid vesicles and their ability to permeate the membrane by measuring release of encapsulated fluorescent dye. Dopaminergic neurotoxicity was examined using adenoviral‐mediated expression in a primary midbrain culture model. To examine the protective effects of stabilizing aSyn‐membrane interactions, we first investigated the impact of endosulfine alpha (ENSA), a protein found to interact with membrane‐bound aSyn. Additionally, we performed a phage display screen to identify small peptides that interact with membrane‐bound aSyn. These potential “stabilizers” of membrane‐bound aSyn were examined for their effects on membrane‐induced aSyn aggregation, membrane permeabilization, and dopaminergic neurotoxicity.

We found that aSyn variants that displayed enhanced accumulation of SDS‐resistant aggregates at the membrane, including A30P and G51D, also displayed increased neurotoxicity. Upon co‐incubating A30P and G51D aSyn with ENSA in the presence of vesicles, we observed a reduction in membrane‐induced aSyn aggregation. This reduction was partially abrogated when the experiment was repeated with the S109E mutant of ENSA, indicating that residue S109 (previously shown to be important for the interaction of ENSA with membrane‐bound aSyn) is involved in the protective effect of ENSA. Co‐expression of ENSA with A30P and G51D in primary midbrain cultures alleviated dopaminergic cell death, suggesting that “stabilizers” of membrane‐bound aSyn may protect against aSyn neurotoxicity. Lastly, we identified several peptides that bind membrane‐associated aSyn and inhibit membrane‐induced aggregation and membrane permeabilization. These peptide stabilizers could be a powerful tool to examine how membrane‐induced aggregation plays a role in aSyn neurotoxicity.

Support or Funding Information

National Institutes of Health (NINDS), Branfman Foundation, Floss Endowment Graduate Research Award