Organotypic slice culture model demonstrates inter-neuronal spreading of alpha-synuclein aggregates

Here we describe the use of an organotypic hippocampal slice model for studying alpha-synuclein aggregation and inter-neuronal spreading initiated by microinjection of pre-formed alpha-synuclein fibrils (PFFs). PFF injection at dentate gyrus (DG) templates the formation of endogenous alpha-synuclein aggregates in axons and cell bodies of this region that spread to CA3 and CA1 regions. Aggregates are insoluble and phosphorylated at serine-129, recapitulating Lewy pathology features found in Parkinson's disease and other synucleinopathies. The model was found to favor anterograde spreading of the aggregates. Furthermore, it allowed development of slices expressing only serine-129 phosphorylation-deficient human alpha-synuclein (S129G) using an adeno-associated viral (AAV) vector in alpha-synuclein knockout slices. The processes of aggregation and spreading of alpha-synuclein were thereby shown to be independent of phosphorylation at serine-129. We provide methods and highlight crucial steps for PFF microinjection and characterization of aggregate formation and spreading. Slices derived from genetically engineered mice or manipulated using viral vectors allow testing of hypotheses on mechanisms involved in the formation of alpha-synuclein aggregates and their prion-like spreading.