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 ...

Covalent side-chain cross-linking has been shown to be a viable strategy to control peptide folding. We report here that an oxime side-chain linkage can elicit α-helical folds from peptides in aqueous solution. The bio-orthogonal bridge is formed rapidly under neutral buffered conditions, and the resulting cyclic oximes are capable of dynamic covalent exchange.

Characterization of the amyloidogenic Parkinson’s Disease protein α-synuclein (αS) has proven difficult due to its structural plasticity. Here, we present a number of complementary methods to site-specifically introduce fluorescent probes to examine αS fibril formation and cellular uptake. By using various combinations of conventional Cys modification, amber codon suppression, transferase mediated N-terminal modification, and native chemical ligation, several variants of singly- and doubly-labeled αS were produced. We validated the nonperturbative nature of the label by a combination of in vitro aggregation kinetics measurements and imaging of the resulting fibrils. The labeled αS can then be used to monitor conformational changes during fibril formation or cellular uptake of αS fibrils in models of disease propagation.