Sensitive fluorescence polarization technique for rapid screening of alpha-synuclein oligomerization/fibrillization inhibitors.
Parkinson's disease (PD) is characterized by the accumulation of fibrillar alpha-synuclein (alpha-Syn) inclusions known as Lewy bodies (LBs) and Lewy neurites. Mutations in the alpha-Syn gene or extra copies thereof cause familial PD or dementia with LBs (DLB) in rare kindreds, but abnormal accumulations of wildtype alpha-Syn also are implicated in the pathogenesis of sporadic PD, the most common movement disorder. Insights into mechanisms underlying alpha-Syn mediated neurodegeneration link alpha-Syn oligomerization and fibrillization to the onset and progression of PD. Thus, inhibiting alpha-Syn oligomer or fibril formation is a compelling target for discovering disease modifying therapies for PD, DLB, and related synucleinopathies. Although amyloid dyes recognize alpha-Syn fibrils, efficient detection of soluble oligomers remains a challenge. Here, we report a novel fluorescence polarization (FP) technique for examining alpha-Syn assembly by monitoring changes in its relative molecular mass during progression of normal alpha-Syn from highly soluble monomers to higher order multimers and thence insoluble amyloid fibrils. We report that FP is more sensitive than conventional amyloid dye methods for the quantification of mature fibrils, and that FP is capable of detecting oligomeric alpha-Syn, allowing for rapid automated screening of potential inhibitors of alpha-Syn oligomerization and fibrillization. Furthermore, FP can be combined with an amyloid dye in a single assay that simultaneously provides two independent biophysical readouts for monitoring alpha-Syn fibrillization. Thus, this FP method holds potential to accelerate discovery of disease modifying therapies for LB PD, DLB, and related neurodegenerative synucleinopathies.
Luk
Kelvin C
KC
Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19143, USA.
Hyde
Edward G
EG
Trojanowski
John Q
JQ
Lee
Virginia M-Y
VM
D000975:Antioxidants / Q000494:pharmacology; D000978:Antiparkinson Agents / Q000494:pharmacology; D019281:Dimerization; D004298:Dopamine / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004353:Drug Evaluation, Preclinical / Q000379:methods; D004837:Epinephrine / Q000494:pharmacology; D044950:Flavanones / Q000494:pharmacology; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000494:pharmacology; D007980:Levodopa / Q000494:pharmacology; D009454:Neurofibrils / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D009638:Norepinephrine / Q000494:pharmacology; D011108:Polymers; D051844:alpha-Synuclein / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*
D000975:Antioxidants; D000978:Antiparkinson Agents; D044950:Flavanones; D005456:Fluorescent Dyes; D011108:Polymers; D051844:alpha-Synuclein; D007980:Levodopa; C006680:baicalein; D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine