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Selective imaging of internalized proteopathic α‐synuclein seeds in primary neurons reveals mechanistic insight into transmission of synucleinopathies
Author(s) -
Karpowicz Richard J.,
Haney Conor M.,
Mihaila Tiberiu S.,
Sandler Raizel M.,
Petersson E. James,
Lee Virginia M.Y.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.118.3
Subject(s) - synucleinopathies , intracellular , microbiology and biotechnology , extracellular , biochemistry , fibril , biophysics , chemistry , biology , alpha synuclein , pathology , medicine , disease , parkinson's disease
Direct cell‐to‐cell transmission of proteopathic alpha‐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. Support or Funding Information This work was supported by funding from the National Institutes of Health (NS053488‐08 to VMYL and NIH NS081033 to EJP), the Ofer Nemirovsky Family Fund, as well as the University of Pennsylvania. Instruments supported by the National Science Foundation include: CD spectrometer (NSF DMR05‐20020) and MALDI MS (NSF MRI‐0820996). CMH is supported by an Age Related Neurodegenerative Disease Training Grant fellowship (NIH T32AG000255). This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .