Previous studies have shown that aggregated alpha-synuclein (α-s) protein, a key pathological marker of Parkinson’s disease (PD), can propagate between cells, thus participating in disease progression. This prion-like propagation has been widely studied using in vivo and in vitro models, including rodent and human cell cultures. In this study, our focus was on temporal assessment of functional changes during α-s aggregation and propagation in human induced pluripotent stem cell (hiPSC)-derived neuronal cultures and in engineered networks. Here, we report an engineered circular tripartite human neuronal network model in a microfluidic chip integrated with microelectrode arrays (MEAs) as a platform to study functional markers during α-s aggregation and propagation. We observed progressive aggregation of α-s in conventional neuronal cultures and in the exposed (proximal) compartments of circular tripartite networks following exposure to preformed α-s fibrils (PFF). Furthermore, aggregated forms propagated to distal compartments of the circular tripartite networks through axonal transport. We observed impacts of α-s aggregation on both the structure and function of neuronal cells, such as in presynaptic proteins, mitochondrial motility, calcium oscillations and neuronal activity. The model enabled an assessment of the early, middle, and late phases of α-s aggregation and its propagation during a 13-day follow-up period. While our temporal analysis suggested a complex interplay of structural and functional changes during the in vitro propagation of α-s aggregates, further investigation is required to elucidate the underlying mechanisms. Taken together, this study demonstrates the technical potential of our introduced model for conducting in-depth analyses for revealing such mechanisms.

先前的研究表明，聚集的α-突触核蛋白（α-s）蛋白是帕金森病（PD）的关键病理标志物，可以在细胞之间传播，从而参与疾病进展。这种类似朊病毒的传播已使用体内和体外模型（包括啮齿动物和人类细胞培养物）进行了广泛研究。在这项研究中，我们的重点是对人类诱导多能干细胞 (hiPSC) 衍生的神经元培养物和工程网络中 α-s 聚集和传播过程中的功能变化进行时间评估。在这里，我们报告了一种在与微电极阵列（MEA）集成的微流控芯片中设计的圆形三方人类神经元网络模型，作为研究α-s聚集和传播过程中功能标记的平台。我们观察到，在传统的神经元培养物中以及暴露于预先形成的 α-s 原纤维 (PFF) 后的圆形三方网络的暴露（近端）隔室中，α-s 逐渐聚集。此外，聚集形式通过轴突运输传播到圆形三方网络的远端隔室。我们观察了 α-s 聚集对神经元细胞结构和功能的影响，例如突触前蛋白、线粒体运动、钙振荡和神经元活动。该模型能够在 13 天的随访期内评估 α-s 聚集的早期、中期和晚期阶段及其传播。虽然我们的时间分析表明 α-s 聚集体的体外传播过程中结构和功能变化存在复杂的相互作用，但仍需要进一步研究来阐明潜在的机制。 总而言之，这项研究证明了我们引入的模型在进行深入分析以揭示此类机制方面的技术潜力。