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PFF-induced modeling of phosphorylated α-synuclein accumulation in primary dopamine neurons provides a reliable tool to study the underlying mechanisms mediating formation and elimination of α-synuclein inclusions, with the opportunity for high-throughput drug screening and cellular phenotype analysis.
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These data are quantified (e.g., counting the number of phospho-α-synuclein-containing dopamine neurons per well) with the use of free software that provides a platform for unbiased high-content phenotype analysis. Multispectral fluorescence images are obtained via automated microscopy of 96 well plates.
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At the end of the experiment, the cells are fixed with paraformaldehyde for immunocytochemistry and fluorescence microscopy imaging. Culturing the neurons in 96 well plates increases the robustness and power of the experimental setups. In vitro cell culture conditions are also suitable for the application and evaluation of treatments preventing α-synuclein accumulation, such as small molecule drugs and neurotrophic factors, as well as lentivirus vectors for genetic manipulation (e.g., with CRISPR/Cas9). Accumulation of endogenous phosphorylated α-synuclein in the soma of dopamine neurons is detected by immunostaining already at 7 days after the PFF addition. In this protocol, the hallmark histopathology of Parkinson’s disease, Lewy bodies (LB), is mimicked by the addition of α-synuclein pre-formed fibrils (PFFs) directly to neuronal culture media.
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Primary midbrain cultures provide a reliable source of bona fide embryonic dopamine neurons. Combined with immunostaining and unbiased automated image analysis, this model allows for the analysis of the effects of drugs and genetic manipulations on α-synuclein aggregation in neuronal cultures. The goal of this protocol is to establish a robust and reproducible model of α-synuclein accumulation in primary dopamine neurons.