Selective imaging of internalized proteopathic α-synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies

Direct cell-to-cell transmission of proteopathic alpha-synuclein (alpha-syn) aggregates is thought to underlie the progression of neuro-degenerative synucleinopathies. However, the specific intracellular processes governing this transmission remain unclear because currently available model systems are limited. For example, in cell culture models of alpha-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied alpha-syn seed species. Herein, we employed fluorescently labeled alpha-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized alpha-syn seeds in cultured primary neurons and to quantitatively characterize the concentration dependence, time course, and inhibition of pff uptake. To study the long-term fates of exogenous alpha-syn pffs in neurons, we developed a pff species labeled at amino acid residue 114 with the environmentally insensitive fluorophore BODIPY or the pH-sensitive dye pHrodo red. We found that pffs are rapidly trafficked along the endolysosomal pathway, where most of the material remains for days. We also found that brief pharmacological perturbation of lysosomes shortly after the pff treatment causes aberrations in intracellular processing of pff seeds concomitant with an increased rate of inclusion formation via recruitment of endogenous alpha-syn to a relatively small number of exogenous seeds. Our results validate a quantitative assay for pff uptake in primary neurons, implicate lysosomal processing as the major fate of internalized proteopathic seeds, and suggest lysosomal integrity as a significant rate-determining step in the transmission of alpha-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.