Two conformationally distinct α-synuclein oligomers share common epitopes and the ability to impair long-term potentiation

Abstract

Parkinson’s Disease (PD) is a neurodegenerative disease for which there currently is no cure. Aggregation of the pre-synaptic protein α-synuclein (aSN) into oligomers (αSOs) is believed to play a key role in PD pathology, but little is known about αSO formation in vivo and how they induce neurodegeneration. Both the naturally occurring polyunsaturated fatty acid docosahexaenoic acid (DHA) and the lipid peroxidation product 4-hydroxynonenal (HNE), strongly upregulated during ROS conditions, stimulate the formation of αSOs, highlighting a potential role in PD. Yet, insight into αSOs structure and biological effects is still limited as most oligomer preparations studied to date are heterogeneous in composition. Here we have aggregated aSN in the presence of HNE and DHA and purified the αSOs using size exclusion chromatography. Both compounds stimulate formation of spherical αSOs containing anti-parallel β-sheet structure which have the same shape as unmodified αSOs though ca. 2-fold larger. Furthermore, the yield and stabilities of these oligomers are significantly higher than for unmodified aSN. Both modified and unmodified αSOs permeabilize synthetic vesicles, show high co-localisation with glutamatergic synapses and decrease Long Term Potentiation (LTP), in line with the reported synaptotoxic effects of αSOs. We conclude that DHA- and HNE-αSOs are convenient models for pathogenic disease-associated αSOs in PD.

The interactome of stabilized α‐synuclein oligomers and neuronal proteins

“…D,E). We have previously shown that only a small part of the DHA‐αSOs dissociates back into monomers after αSO purification using SEC , but the absence of staining upon incubation with 2.5 μ m of monomer shows that these dissociated monomers will not interfere with the identification of αSO binding partners. The lack of binding of monomeric αSN is a vindication of our strategy to work with stabilized αSOs in a cellular context to minimize potential dissociation of the oligomeric state.…”

“…The experimental set‐up was validated by coupling DHA‐αSOs to the 5G4 antibody, previously shown to bind aggregated αSN and DHA‐αSOs . DHA‐αSOs were labelled with 0.1 or 0.5 m m of sulfo‐LC‐SDA crosslinker and were then crosslinked to the 5G4 antibody using either 15 or 30 min of UV irradiation.…”

“…Note that the relatively high αSO concentration used (1 μ m monomer equivalents) prevented the calculation of colocalization of αSO puncta with vGluT1 puncta as previously shown , since puncta size becomes too large to be automatically identified as individual puncta. However, based on the lack of differences in αSO binding pattern and intensity between labelled and nonlabelled αSOs, it is reasonable to assume that labelled DHA‐αSOs continue to colocalize with glutamatergic synapses, as previously shown for nonlabelled DHA‐αSOs .…”

“…Furthermore, αSOs impair long‐term potentiation (LTP), a molecular process underlying synaptic plasticity, learning and memory, most likely via N ‐methyl‐ d ‐aspartate (NMDA) receptor activation, which are located at excitatory synapses . We have recently shown that docosahexaenoic acid (DHA)‐modified αSOs bind to mature hippocampal neurons in a punctate pattern and colocalize with the glutamatergic synapses . These interactions are likely important for the αSO toxicity, and the identification of involved proteins could be vital for the unravelling the mechanism of αSO toxicity.…”

α‐synuclein oligomers and fibrils: a spectrum of species, a spectrum of toxicities

“…Oligomers formed and stabilized by lipid peroxidation products show much the same neuronal binding and toxicity properties as their unmodified counterparts, whereas more radical changes in preparation procedures ( e.g. , use of iron salts, ethanol, and/or different shaking regimes) can have substantial impact on their cellular activity profiles (Van Diggelen et al ),(Danzer et al ; Qin et al ; Ehrnhoefer et al ; Cremades et al ).…”