Multiscale brain-wide mapping of α-synuclein-driven dopaminergic degeneration and white matter impairment in α-synucleinopathy mice

Abstract The spatiotemporal relationships among α-synuclein inclusion formation, dopaminergic degeneration, and white matter microstructural changes remain poorly defined. To address this, we unilaterally injected preformed fibrils (PFFs) or monomeric α-synuclein into the substantia nigra pars compacta (SNc) of wild-type mice. At 12 and 20 weeks post-injection (wpi), we performed ex vivo magnetic resonance imaging (MRI) at 9.4T to assess microstructural, volumetric and paramagnetic changes, along with light-sheet microscopy (LSM) to map α-synuclein aggregates, and dopaminergic neuron at single-cell resolution across the whole brain. We developed a Python-based, automated registration pipeline that achieves <40 µm alignment error between ex vivo MRI and LSM and computes clearing-induced distortion, enabling scalable deformation analysis and multimodal multiscale data integration. Unilateral SNc injection of α-syn PFFs induced dense phospho-α-syn pathology in the ipsilateral SNc at 12 wpi, which decreased by 20 wpi in parallel with the loss of tyrosine hydroxylase-positive dopaminergic neuron. Pathogenic α-synuclein spreads along with dopaminergic denervation in the nigrostriatal pathway to the ipsilateral striatum and contralateral SNc. Our ex vivo MRI-LSM platform provides a scalable, open-source framework for understanding circuit-level and whole-brain propagation of pathology in an α-synucleinopathy model and beyond. Highlights Open-source, automated pipeline aligns ex vivo 9.4T MRI with light-sheet microscopy (LSM) at <40 µm error, quantifies clearing-induced tissue distortion, and enables multiscale, whole-brain multimodal analysis. Whole-brain, single-cell-resolution mapping of α-synuclein pathology and dopaminergic neuron loss in an α-synucleinopathy mouse model using an optimized iDISCO+ protocol that achieves uniform antibody penetration even in dense regions like the striatum. Unilateral SNc injection of α-syn PFFs drives spread of p-α-syn pathology along the nigrostriatal pathway to the ipsilateral striatum, BST, CEA, and contralateral SNc, along with dopaminergic degeneration. Ipsilateral SNc shows high pS129 pathology at 12 weeks post-injection but significant reduction by 20 wpi, concurrent with loss of tyrosine hydroxylase-positive neurons. Multiparametric MRI reveals microstructural and iron-related alterations in striatum, SNc, and VTA, with cross-modal correlations linking pS129 burden, TH loss, and magnetic susceptibility changes. Competing Interest Statement CH and RMN are employees and shareholders of Neurimmune AG, Schlieren, Switzerland. The other authors have no competing interests. Footnotes we update the analysis pipeline, MRI data and include additional samples.