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

Abstract

Edited by Paul E. FraserDirect cell-to-cell transmission of proteopathic ␣-synuclein (␣-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 ␣-syn-seeded aggregation, it is difficult to discern intracellular from extracellular exogenously applied ␣-syn seed species. Herein, we employed fluorescently labeled ␣-syn preformed fibrils (pffs) in conjunction with the membrane-impermeable fluorescence quencher trypan blue to selectively image internalized ␣-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 ␣-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 ␣-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 ␣-syn pathology. Further, lysosomal processing of transmitted seeds may represent a new therapeutic target to combat the spread of synucleinopathies.Mounting evidence implicates direct cell-to-cell transmission of misfolded amyloidogenic protein species as a central component underlying the spatiotemporal progression of pathophysiology in numerous proteinopathies (1, 2). In synucleinopathies, including Parkinson's disease, Parkinson's disease with dementia, dementia with Lewy bodies, and multiple system atrophy, the amyloidogenic protein ␣-synuclein (␣-syn) 3 aggregates into Lewy bodies, Lewy neurites, and glial cytoplasmic inclusions (3, 4). These intracellular proteinaceous inclusions have been widely recapitulated in a number of in vivo and cell-based model systems via introduction of pathological, insoluble ␣-syn species from either brain extracts of diseased postmortem tissue or preformed fibrils of recombinant origin (5-10). Despite strong evidence gleaned from these models implicating a causal relationship between exposure of neurons to insoluble ␣-syn aggregates and the subsequent development of inclusions bearing pathological hallmarks of synucleinopathies, the specific processes underlying cell-to-cell transmission of proteopathic seeds and resulting intracellular events leading to the development of pathological ...

Cellular milieu imparts distinct pathological α-synuclein strains in α-synucleinopathies

“…PK digestion has been performed in the same way as above described for GCI-α-Syn and LB-α-Syn. Treatment of primary neurons with chloroquine was performed as previous described 19 .…”

“…1c–d), we asked whether the high potency of GCI-α-Syn is due to its resistance to degradation. Previously we showed that exogenously added misfolded α-Syn accumulate in lysosomes and that treating the cells with chloroquine (lysosomal inhibitor) could increase the amount of α-Syn pathology induced 19 . To test this hypothesis, primary neurons seeded with GCI-α-Syn, LB-α-Syn or PFFs were treated with chloroquine.…”

Mechanisms of Cell-to-Cell Transmission of Pathological Tau

“…Experiments using labeled α-synuclein PFFs have demonstrated that these synthetic PFFs do transit through acidified compartments in late endosomes and lysosomes. 47,73 Furthermore, enhanced cellular seeding of tau and α-synuclein is evident on inhibition of lysosomal processing of tau seeds using the small molecule inhibitor chloroquine. 47,73 However, it remains unclear whether this process is mediated by disruption of lysosomal integrity, which allows these proteopathic seeds to escape degradation in lysosomes and encounter cellular substrates for templated fibrillization or whether there are other mechanisms to account for this, including mechanisms linked to decreased clearance of these seeds.…”

Best Practices for Generating and Using Alpha-Synuclein Pre-Formed Fibrils to Model Parkinson’s Disease in Rodents

“…For instance, some groups have reported issues generating aSyn PFFs using lyophilized protein or tagged aSyn protein [31]. However, these issues have been overcome by others [32, 43]. To increase the probability of success when first adopting this model, it is recommended that the investigator either generates and validates their own protein or purchases validated aSyn monomeric protein specifically-formulated to generate aSyn PFFs.…”