Modeling interregional propagation of α-synuclein in human striatal–midbrain assembloids

Abstract Parkinson’s disease is characterized by the progressive degeneration of dopaminergic (DA) neurons in the substantia nigra that project to the striatum. Postmortem studies have found that α-synuclein pathology spreads across brain regions in a prion-like manner. Human three-dimensional neural tissues provide experimentally tractable platforms for clarifying the molecular pathology of synucleinopathy that overcome the limitations of postmortem brain studies. In this study, we established a human striatal–midbrain assembloid model enabling spatially and temporally controlled α-synuclein induction after formation of the nigrostriatal pathway within hStrMAs. Human induced pluripotent stem cells (hiPSCs) were differentiated into striatal spheroids and midbrain spheroids, which were fused to reconstruct the nigrostriatal pathway in vitro. Histological, electrophysiological, and single-cell RNA sequencing analyses confirmed the generation of region-specific neuronal populations, including striatal GABAergic neurons and midbrain DA neurons, and demonstrated that distinct forebrain and ventral midbrain identities were preserved within assembloids. To model synucleinopathy, we generated a doxycycline-inducible α-synuclein hiPSC line, permitting selective induction of α-synuclein expression in the striatal region after assembloid formation. Spatially restricted α-synuclein induction resulted in the progressive accumulation of phosphorylated and fibrillar α-synuclein in midbrain DA neurons, indicating interregional propagation through established neural circuits. Transcriptomic analysis of the midbrain region revealed significant downregulation of DA identity and synaptic function genes, suggesting functional impairment of DA neurons following α-synuclein propagation. These findings demonstrate that the human neural circuit architecture contributes to the interregional propagation of α-synuclein pathology. Our controllable human assembloid platform provides a powerful experimental framework for dissecting the mechanisms of pathological protein spread and developing therapeutic strategies targeting molecular pathology in neurodegenerative diseases. Significance statement One pathological feature of Parkinson’s disease is the progressive spread of α-synuclein pathology across brain regions. However, the molecular mechanisms underlying this process remain poorly understood. In this study, we established a human striatal–midbrain assembloid model in which α-synuclein expression can be spatially restricted and temporally induced after the nigrostriatal pathway is formed, thus enabling the experimental dissection of interregional α-synuclein propagation across brain regions. We demonstrated that α-synuclein pathology initiated in the striatal region propagated to midbrain dopaminergic neurons, leading to disease-relevant molecular and transcriptional alterations. These findings provide direct evidence that human neural circuits contribute to the spread of synucleinopathy and offer a controllable human model for studying disease progression and therapeutic strategies. Competing Interest Statement The authors have declared no competing interest.