Distinct alpha-synuclein strains derived from Parkinson's disease patient tissues trigger differential inclusion pathology in a novel biosensor cell model
Background: -Synuclein (Syn) can misfold and aggregate to form fibrillar {beta}-sheet-rich aggregates ("strains") that are phosphorylated (p-Syn) and deposited into intracellular inclusions in the brain, the pathological hallmark of synucleinopathies including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Previously, we reported that seed amplification assays such as real-time quaking-induced conversion (RT-QuIC) amplifies and detects Syn strains from the patient skin. However, whether skin-derived Syn strains induce disease-specific pathological features in a biological system is unknown. Methods: We generated a U251 human glioblastoma cell line expressing fluorescently tagged Syn carrying the PD-linked A53T mutation and fluorescence resonance energy transfer (FRET)-based U251 biosensor cells. Using fluorescence microscopy coupled with in situ detergent extraction, FRET-Flow cytometry and high-content confocal imaging, we examined the pathological burden and morphology of p-Syn inclusions seeded by RT-QuIC-amplified patient skin and brain Syn strains in Syn-expressing U251 cells, FRET-based Syn biosensor cells and Syn biosensor cell-derived neurons. Results: U251 cells allow robust and rapid in situ detection of detergent-insoluble intracellular Syn inclusions triggered by exogenous Syn seeds. In U251 FRET-based biosensor cells, PD skin-amplified strains induce a greater pathological burden and distinct p-Syn inclusion morphology from PD brain-amplified and DLB skin-amplified strains. Inclusion morphology of DLB and MSA skin- and brain-amplified strains are comparable. Furthermore, skin-amplified Syn strains induce neuronal inclusions and cause degeneration of induced neurons reprogrammed from the U251 biosensor cells. Finally, biosensor cell-propagated PD skin Syn strains induce higher seeding activity measured by RT-QuIC than PD brain and DLB skin Syn strains, linking intracellular pathological burden to in vitro seeding activity. Conclusions: We report the detection of distinct PD strains derived from patient skin and brain tissues that trigger unique pathological phenotypes in U251 Syn biosensor cells and cause degeneration of reprogrammed neurons from the same cell lineage. Moreover, DLB and MSA skin Syn strains mimic pathological features of their brain Syn strains in these biosensor cells. Therefore, the U251 Syn biosensor cell model is a robust tool to potentially discriminate PD and DLB synucleinopathies and to study Syn tissue- and strain-specific etiology and pathogenesis.