Organotypic slice culture model demonstrates interneuronal spreading of alpha-synuclein aggregates

Here we describe the use of an organotypic hippocampal slice model for studying α-synuclein aggregation and inter-neuronal spreading initiated by injection of preformed α-synuclein filaments (PFFs). PFF injection at dentate gyrus templates the endogenous α-synuclein to form aggregates in axons and cell bodies that spread to CA3 and CA1 regions. Aggregates were insoluble and phosphorylated at serine 129, recapitulating Lewy pathology features found in Parkinson’s disease and other synucleinopathies. The spreading of the aggregates were favoring the anterograde direction in the slice model. The model allowed development of slices expressing only serine-129 phosphorylation-deficient human α-synuclein (S129G) using adeno-associated viral (AAV) vector in α-synuclein knockout slices. Processes of aggregation and spreading of α-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 by using viral vectors allow testing of hypotheses on mechanisms involved in formation of α-synuclein aggregates and their prion-like spreading.