Bro̷chner Bo Volf, Zhang Xialin, Nielsen Janni, Kjems Jo̷rgen, Otzen Daniel E, Malle Mette Galsgaard
Interdisciplinary Nanoscience Center (iNANO), Aarhus University, Gustav Wieds Vej 14, 8000 Aarhus C, Denmark.
Department of Molecular Biology and Genetics, Aarhus University, Universitetsbyen 81, 8000 Aarhus C, Denmark.
ACS Nano. 2025 Sep 16;19(36):32108-32122. doi: 10.1021/acsnano.5c04005. Epub 2025 Aug 12.
Neurodegenerative disorders, such as Parkinson's disease (PD) pose significant health challenges. A major hallmark of PD is the aggregation of α-synuclein into toxic oligomers (αSO) and fibrils. While many efforts focus on slowing disease progression, the molecular origins and mechanisms of αSO toxicity remain poorly understood, particularly regarding its proposed link to membrane disruption. To address this, we have developed a single-vesicle analysis platform for direct, and real-time measurements of αSO and membrane interaction. This platform allows us to demonstrate real-time translocation of dyes through αSO pores with single-particle resolution and use single-channel electrical recordings to analyze pore formation in planar lipid bilayers. Across methods, our data provide evidence for a three-stage model of αSO and membrane interactions, comprising initial membrane recruitment followed by partial pore insertion and subsequent full pore formation. Notably, while αSO recruitment was found to favor curved membranes, pore formation occurred more efficiently in less curved membranes, hence, recruitment is decoupled from a membrane charge-promoted reorientation and pore integration. Single αSO pore formations undergo multiple translocation steps making pore formation highly dynamic, cycling back and forth between partial insertion and full pore formation. The dynamic nature of pore formation can be modulated by lipid charge, lipid headgroup class, and ligand binding. Our findings suggest that increased dynamic pore formation could imply increased membrane toxicity. Evidence for the three-stage model is important for developing future targeting strategies to block αSO-mediated PD-related cellular dysfunction. We envision that the single-vesicle assay will enable screening of ligands modulating the pore formation.
神经退行性疾病，如帕金森病（PD），对健康构成了重大挑战。PD的一个主要标志是α-突触核蛋白聚集成有毒的寡聚体（αSO）和原纤维。虽然许多努力都集中在减缓疾病进展上，但αSO毒性的分子起源和机制仍知之甚少，特别是关于其与膜破坏的假定联系。为了解决这个问题，我们开发了一个单囊泡分析平台，用于直接实时测量αSO与膜的相互作用。该平台使我们能够以单颗粒分辨率证明染料通过αSO孔的实时转运，并使用单通道电记录来分析平面脂质双层中的孔形成。通过各种方法，我们的数据为αSO与膜相互作用的三阶段模型提供了证据，包括初始膜募集，随后是部分孔插入和随后的完全孔形成。值得注意的是，虽然发现αSO募集有利于弯曲的膜，但孔形成在弯曲程度较小的膜中更有效地发生，因此，募集与膜电荷促进的重新定向和孔整合解耦。单个αSO孔的形成经历多个转运步骤，使得孔形成高度动态，在部分插入和完全孔形成之间来回循环。孔形成的动态性质可以通过脂质电荷、脂质头基类别和配体结合来调节。我们的研究结果表明，增加的动态孔形成可能意味着增加的膜毒性。三阶段模型的证据对于开发未来的靶向策略以阻断αSO介导的与PD相关的细胞功能障碍很重要。我们设想单囊泡测定将能够筛选调节孔形成的配体。
