Human amyloid-β enriched extracts: evaluation of in vitro and in vivo internalization and molecular characterization

BackgroundIntracerebral inoculation of extracts from post-mortem human Alzheimer's disease brains into mice produces a prion-like spreading effect of amyloid-. The differences observed between these extracts and the synthetic peptide, in terms of amyloid- internalization and seed and cell-to-cell transmission of cytosolic protein aggregates, suggest that brain extracts contain key contributors that enhance the prion-like effect of amyloid-. Nevertheless, these potential partners are still unknown due to the complexity of whole brain extracts.MethodsHerein, we established a method based on sequential detergent solubilization of post-mortem samples of human brains affected by Alzheimer's disease that strongly enrich amyloid- aggregates by eliminating 92% of the remaining proteins. Internalization of A(1-42) from the enriched AD extracts was evaluated in vitro, and internalization of fluorescent-labeled AD extracts was also investigated in vivo. Furthermore, we carried out a molecular characterization of the A-enriched fraction using label-free proteomics, studying the distribution of representative components in the amygdala and the olfactory cortex of additional human AD brain samples by immunohistochemistry.ResultsA(1-42) from the enriched AD extracts are internalized into endothelial cells in vitro after 48h. Furthermore, accumulation of fluorescent-labeled A-enriched extracts into mouse microglia was observed in vivo after 4months of intracerebral inoculation. Label-free proteomics (FDR<0.01) characterization of the amyloid--enriched fraction from different post-mortem samples allowed for the identification of more than 130 proteins, several of which were significantly overrepresented (i.e., ANXA5 and HIST1H2BK; p<0.05) and underrepresented (i.e., COL6A or FN1; p