title: Addition of exogenous α-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous α-synuclein to Lewy body and Lewy neurite-like aggregates.
abstract: This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of the Lewy bodies and Lewy neurites found in Parkinson's disease brains and other synucleinopathies. This model allows investigation of aggregate formation, their impact on neuron function, and development of therapeutics. Addition of preformed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds the recruitment of endogenous α-syn into aggregates characterized by detergent insolubility and hyperphosphorylation. Aggregate formation follows a lag phase of 2-3 d, followed by formation in axons by days 4-7, spread to somatodendritic compartments by days 7-10 and neuron death ~14 d after PFF addition. Here we provide methods and highlight the crucial steps for PFF formation, PFF addition to cultured hippocampal neurons and confirmation of aggregate formation. Neurons derived from various brain regions from nontransgenic and genetically engineered mice and rats can be used, allowing interrogation of the effect of specific genes on aggregate formation.
authors.affiliation: Department of Neurology, Center for Neurodegeneration and Experimental Therapeutics, Civitan International Research Center, University of Alabama at Birmingham, Birmingham, Alabama, USA.
authors.forename: Laura A
authors.initials: LA
authors.lastname: Volpicelli-Daley
authors.affiliation: Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Kelvin C
authors.initials: KC
authors.lastname: Luk
authors.affiliation: Department of Pathology and Laboratory Medicine, Institute on Aging and Center for Neurodegenerative Disease Research, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA.
authors.forename: Virginia M-Y
authors.initials: VM
authors.lastname: Lee
mesh_terms: D000818:Animals; D018929:Cell Culture Techniques / Q000379:methods; D005455:Fluorescent Antibody Technique; D015151:Immunoblotting; D016631:Lewy Bodies / Q000378:metabolism*; D051379:Mice; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism*; D051381:Rats; D051844:alpha-Synuclein / Q000378:metabolism*
chemical_list: D046912:Multiprotein Complexes; D051844:alpha-Synuclein