Addition of exogenous alpha-synuclein preformed fibrils to primary neuronal cultures to seed recruitment of endogenous alpha-synuclein to Lewy body and Lewy neurite-like aggregates

作者：Volpicelli-Daley, Laura A.^*; Luk, Kelvin C.; Lee, Virginia M-Y

来源：Nature Protocols, 2014, 9(9): 2135-2146.

DOI：10.1038/nprot.2014.143

• PARKINSONS-DISEASE
• DEMENTIA
• TRANSFECTION
• INCLUSIONS
• BODIES
• NEURODEGENERATION
• TRANSMISSION
• HIPPOCAMPUS
• PROPAGATION
• EFFICIENCY

This protocol describes a primary neuronal model of formation of alpha-synuclein (alpha-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 alpha-syn to neurons seeds the recruitment of endogenous alpha-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 similar to 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.

• 出版日期2014-9