Exploring the Release of Toxic Oligomers from α-Synuclein Fibrils with Antibodies and STED Microscopy.

Alessandra Bigi,Roberta Cascella,Serene W Chen,Cristina Cecchi,Emilio Ermini

doi:10.3390/life11050431

Abstract

α-Synuclein (αS) is an intrinsically disordered and highly dynamic protein involved in dopamine release at presynaptic terminals. The abnormal aggregation of αS as mature fibrils into intraneuronal inclusion bodies is directly linked to Parkinson’s disease. Increasing experimental evidence suggests that soluble oligomers formed early during the aggregation process are the most cytotoxic forms of αS. This study investigated the uptake by neuronal cells of pathologically relevant αS oligomers and fibrils exploiting a range of conformation-sensitive antibodies, and the super-resolution stimulated emission depletion (STED) microscopy. We found that prefibrillar oligomers promptly penetrate neuronal membranes, thus resulting in cell dysfunction. By contrast, fibril docking to the phospholipid bilayer is accompanied by αS conformational changes with a progressive release of A11-reactive oligomers, which can enter into the neurons and trigger cell impairment. Our data provide important evidence on the role of αS fibrils as a source of harmful oligomers, which resemble the intermediate conformers formed de novo during aggregation, underling the dynamic and reversible nature of protein aggregates responsible for α-synucleinopathies.

Highlights

The abnormal aggregation of αS, a 14 kDa intrinsically disordered protein implicated in neurotransmitter release at presynaptic terminals, is associated with the onset and progression of Parkinson’s disease (PD) and other debilitating neurodegenerative conditions collectively referred to as α-synucleinopathies [1]
The different fluorescence intensities relative to activated caspase-3 were plotted versus the time elapsed after the addition of αS aggregates to the culture medium, and the resulting kinetic plots were fitted by means of a single exponential (1) or a sigmoidal (2) functions of the forms: F (t) = F + A exp(−kt)
By using the conformation-sensitive and oligomer-specific A11 antiindeed, the exposure of neuronal cells to such assemblies induces the aggregation of the body, we observed the presence of oligomeric assemblies inserted into the lipid bilayer endogenous αS and the formation of inclusions that are morphologically and biochemically and internalized into the cytosol of dopaminergic neurons treated with short fibrils (SF), as well as with similar to those found in the brain of PD patients [13,14,15,16,17]

Summary

Introduction

The abnormal aggregation of αS, a 14 kDa intrinsically disordered protein implicated in neurotransmitter release at presynaptic terminals, is associated with the onset and progression of Parkinson’s disease (PD) and other debilitating neurodegenerative conditions collectively referred to as α-synucleinopathies [1]. There is increasing evidence that metastable oligomeric assemblies, formed during the early phases of αS aggregation, play a crucial role in neuronal injury [4,5,6,7,8,9,10]. Despite their prominent role in the pathogenesis of α-synucleinopathies, the isolation and structural characterization of αS oligomers is extremely difficult because of their instability and transient nature. We have previously described the mechanisms of membrane perturbation and neuronal damage triggered by a range of well-characterized and stable αS aggregates, referred to as type-B*

Methods

Results

Conclusion