Evaluation of the amyloid beta-GFP fusion protein as a model of amyloid beta peptides-mediated aggregation: a study of DNAJB6 chaperone

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid beta (A beta) peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid beta peptides may also play a role in disease pathogenesis. This would suggest that intracellular Heat Shock Proteins (HSP) that maintain cellular protein homeostasis might be candidates for disease amelioration. We recently found that DNAJB6, a member of DNAJ family of heat shock proteins, effectively prevented the aggregation of short aggregation prone peptides containing large poly glutamines (associated with GAG repeat diseases) both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of A beta 42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular A beta peptides using transfection of human embryonic kidney 293 (HEK293) cells with A beta-green fluorescent protein (GFP) fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses A beta-GFP aggregation, but not seeded aggregation initiated by extracellular A beta peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the A beta-GFP fusion protein and its J-domain was crucial for its anti-aggregation effect. In addition, other DNAJ proteins as well as HSPA1a overexpression also suppressed A beta-GFP aggregation efficiently. Our findings suggest that A beta aggregation differs from poly glutamine (Poly Q) peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of A beta-GFP fusion construct for studying A beta peptide aggregation in cells.