Addition of exogenous α-Synuclein Pre-formed fibrils to Primary Neuronal Cultures to seed recruitment of endogenous α-Synuclein to Lewy body and Lewy Neurite-like aggregates

摘要

This protocol describes a primary neuronal model of formation of α-synuclein (α-syn) aggregates that recapitulate features of 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 pre-formed fibrils (PFFs) synthesized from recombinant α-syn to neurons seeds recruitment of endogenous α-syn into aggregates characterized by detergent-insolubility, and hyperphosphorylation. Aggregate formation follows a lag phase of 2–3 days, followed by formation in axons by days 4–7, spread to somatodendritic compartments by days 7–10, and neuron death around 14 days post-PFF. Here, we provide methods and highlight critical steps for PFF formation, addition to cultured hippocampal neurons, and confirmation of aggregate formation. Neurons derived from various brain regions from non-transgenic and genetically-engineered mice and rats can be used, allowing interrogation of the impact of specific genes on aggregate formation.