Abstract
Direct cell-to-cell transmission of proteopathic α-synuclein (α-syn) aggregates is thought to underlie the progression of neurodegenerative 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 α-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied α-syn seed species. Herein, we employed fluorescently labeled α-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized α-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 α-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 α-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 α-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.
Highlights
Direct cell-to-cell transmission of proteopathic ␣-synuclein (␣-syn) aggregates is thought to underlie the progression of neurodegenerative synucleinopathies
It was determined that independent thresholds for quantifying intracellular objects within each uptake experiment should be determined by relationship to the autofluorescence from PBStreated neurons, such that no significant signal contribution from unquenched fibrils or cellular autofluorescence would be identified in each image frame
Intercellular transmission of ␣-syn pathology is widely considered to be a mediator of the spatiotemporal spread of synucleinopathies in the brain, a lack of model systems capable of unambiguous visualization of internalized proteopathic seeds has obscured the fate of these seeds in the initial hours and days after uptake
Summary
Trypan blue is widely used in cell culture applications as a vital stain due to its polarity and as a fluorescence quencher capable of accepting energy from the excited state of fluorophores through dipole– dipole interactions (16 –18). Trypan blue provides a fluorescent label of the plasma membrane when interacting with extracellular membrane-bound proteins, affording visual confirmation of the boundaries of each cell when serially acquired GFP and trypan blue frames are overlaid (Fig. 1E, right) These results represent the first time that intracellular mSyn–GFP pff puncta have been quantitatively imaged in both neuronal somata and processes with confocal resolution and no signal contribution from extracellular pffs. It was found that uptake is contingent on a number of factors, including endocytic activity, membrane surface binding, and fibril size These inhibition results validate trypan blue quenching of extracellular fluorescent pffs as an appropriate method to determine pff uptake under a number of conditions. Uptake appears saturable, suggesting a facilitated rather than nonspecific mechanism of uptake
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