Enhanced ex vivo 3D whole-brain mapping and automated analysis of Parkinson's disease pathologies in mice

Parkinsons disease (PD) is characterized by the accumulation of -synuclein (-syn) aggregates, which are thought to drive neurodegeneration in vulnerable neuronal populations, particularly within the dopaminergic nigrostriatal pathway. Here, we aimed to investigate the spatiotemporal relationship between -syn aggregation and dopaminergic cell degeneration by developing an optimized analysis pipeline for efficient whole-brain mapping. We injected -syn preformed fibrils (PFFs) into the substantia nigra pars compacta (SNc) of wild-type mice, using monomeric -syn-injected mice as the control group. Aggregate propagation and dopaminergic degeneration were analyzed using antibodies against phosphorylated -syn (pS129) and tyrosine hydroxylase, followed by brain clearing combined with high-resolution (<4 {micro}m) light-sheet microscopy (LSM), coupled with automated pipeline data analysis. Whole-brain LSM mapping revealed dense somatic and neuritic phosphorylated -syn aggregates within dopaminergic neurons of the SNc 12 weeks post-unilateral PFF injection, accompanied by significant loss of tyrosine hydroxylase-positive neurons and prion-like propagation of -syn aggregates to anatomically connected brain regions, including the striatum. The distribution pattern of -syn pathology visualized by LSM was validated by whole-brain immunohistochemical analysis of PFF-injected mouse brains. The 3D LSM approach introduced here uniquely captures the spatial organization and propagation of -syn pathology and dopaminergic degeneration across interconnected brain networks.