While it is generally accepted that a-synuclein oligomers (aSOs) play an important role in neurodegeneration in Parkinson's disease, the basis for their cytotoxicity remains unclear. We have previously shown that docosahexaenoic acid (DHA) stabilizes aSOs against dissociation without compromising their ability to colocalize with glutamatergic synapses of primary hippocampal neurons, suggesting that they bind to synaptic proteins. Here, we develop a proteomic screen for putative aSO binding partners in rat primary neurons using DHA-stabilized human aSOs as a bait protein. The protocol involved co-immunoprecipitation in combination with a photoactivatable heterobifunctional sulfo-LC-SDA crosslinker which did not compromise neuronal binding and preserved the interaction between the aSOsbinding partners. We identify in total 29 proteins associated with DHA-aSO of which eleven are membrane proteins, including synaptobrevin-2B (VAMP-2B), the sodium-potassium pump (Na + /K + ATPase), the V-type ATPase, the voltage-dependent anion channel and calcium-/calmodulin-dependent protein kinase type II subunit gamma; only these five hits were also found in previous studies which used unmodified aSOs as bait. We also identified Rab-3A as a target with likely disease relevance. Three out of four selected hits were subsequently validated with dot-blot binding assays. In addition, likely binding sites on these ligands were identified by computational analysis, highlighting a diversity of possible interactions between aSOs and target proteins. These results constitute an important step in the search for disease-modifying treatments targeting toxic aSOs. AbbreviationsCo-IP, co-immunoprecipitation; LC-MS/MS, liquid chromatography coupled to mass spectrometric analysis and tandem mass spectrometry; NHS, N-hydroxysuccinimide; NMDA, N-methyl-D-aspartate; PD, Parkinson's disease; sulfo-LC-SDA, sulfo-linker carbon-succinimidyl-diazirine; aSOs, a-synuclein oligomers.Proteomic identification of aSN oligomer ligands F. van Diggelen et al.

Functional amyloid (FA) proteins have evolved to assemble into fibrils with a characteristic cross-β structure, which stabilizes biofilms and contributes to bacterial virulence. Some of the most studied bacterial FAs are the curli protein CsgA, expressed in a wide range of bacteria, and FapC, produced mainly by members of the Pseudomonas genus. Though unrelated, both CsgA and FapC contain imperfect repeats believed to drive the formation of amyloid fibrils. While much is known about CsgA biogenesis and fibrillation, the mechanism of FapC fibrillation remains less explored. Here, we show that removing the three imperfect repeats of FapC (FapC ΔR1R2R3) slows down the fibrillation but does not prevent it. The increased lag phase seen for FapC ΔR1R2R3 allows for disulfide bond formation, which further delays fibrillation. Remarkably, these disulfide-bonded species of FapC ΔR1R2R3 also significantly delay the fibrillation of human α-synuclein, a key protein in Parkinson’s disease pathology. This attenuation of α-synuclein fibrillation was not seen for the reduced form of FapC ΔR1R2R3. The results presented here shed light on the FapC fibrillation mechanism and emphasize how unrelated fibrillation systems may share such common fibril formation mechanisms, allowing inhibitors of one fibrillating protein to affect a completely different protein.

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.

Ionic surfactants such as sodium dodecyl sulfate (SDS) unfold proteins in a much more diverse yet effective way than chemical denaturants such as guanidium chloride (GdmCl). But how these unfolding...

Parkinson’s disease is mainly caused by aggregation of α-synuclein (α-syn) in the brain. Exchange of α-syn between the brain and peripheral tissues could have important pathophysiological and therapeutic implications, but the trafficking mechanism of α-syn across the blood brain-barrier (BBB) remains unclear. In this study, we therefore investigated uptake and transport mechanisms of α-syn monomers and oligomers across an in vitro BBB model system. Both α-syn monomers and oligomers were internalized by primary brain endothelial cells, with increased restriction of oligomeric over monomeric transport. To enlighten the trafficking route of monomeric α-syn in brain endothelial cells, we investigated co-localization of α-syn and intracellular markers of vesicular transport. Here, we observed the highest colocalization with clathrin, Rab7 and VPS35, suggesting a clathrin-dependent internalization, preferentially followed by a late endosome retromer-connected trafficking pathway. Furthermore, STED microscopy revealed monomeric α-syn trafficking via Rab7-decorated carriers. Knockdown of Caveolin1, VPS35, and Rab7 using siRNA did not affect monomeric α-syn uptake into endothelial cells. However, it significantly reduced transcytosis of monomeric α-syn in the luminal-abluminal direction, suggesting a polarized regulation of monomeric α-syn vesicular transport. Our findings suggest a direct role for Rab7 in polarized trafficking of monomeric α-syn across BBB endothelium, and the potential of Rab7 directed trafficking to constitute a target pathway for new therapeutic strategies against Parkinson’s disease and related synucleinopathies.