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

Abstract

Alzheimer's disease (AD) is a progressive neurodegenerative disease characterized by the accumulation and aggregation of extracellular amyloid β (Aβ) peptides and intracellular aggregation of hyper-phosphorylated tau protein. Recent evidence indicates that accumulation and aggregation of intracellular amyloid β 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 CAG repeat diseases) both in vitro and in cells. Moreover, recent in vitro data showed that DNAJB6 can delay the aggregation of Aβ42 peptides. In this study, we investigated the ability of DNAJB6 to prevent the aggregation of extracellular and intracellular Aβ peptides using transfection of human embryonic kidney 293 (HEK293) cells with Aβ-green fluorescent protein (GFP) fusion construct and performing western blotting and immunofluorescence techniques. We found that DNAJB6 indeed suppresses Aβ-GFP aggregation, but not seeded aggregation initiated by extracellular Aβ peptides. Unexpectedly and unlike what we found for peptide-mediated aggregation, DNAJB6 required interaction with HSP70 to prevent the aggregation of the Aβ-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 Aβ-GFP aggregation efficiently. Our findings suggest that Aβ aggregation differs from poly glutamine (Poly Q) peptide induced aggregation in terms of chaperone handling and sheds doubt on the usage of Aβ-GFP fusion construct for studying Aβ peptide aggregation in cells.

Keywords: Alzheimer’s disease; Aβ-GFP; DNAJB6; amyloid beta aggregation; chaperones; heat shock proteins.

Figure 1

DNAJB6 prevents the aggregation of Aβ42-GFP. (A) Scheme of generation of Aβ42-GFP intracellularly. Cells were transfected with Ub-Aβ42-GFP. Upon expression, Ub-Aβ42-GFP is cleaved by endogenous ubiqutin C-terminal hydrolases after the RGG amino acid sequence to give rise to Ub and Aβ42-GFP at 1:1 ratio. (B) HEK293 cells were transfected with 0.5 μg Ub-Aβ42-GFP with FRTTO-DNAJB6b-V5 at 1:1, 1:2 ratio or FRTTO as a control (1:0 ratio). After 48 h, cells were lysed and separated into Triton X100 soluble (S) and pellet (P) fractions (left panel) or total lysate (right panel). A β42-GFP and DNAJB6-V5 were detected by Western blotting with 6E10 and anti V5 antibodies respectively. β-actin was used as a loading control. The quantification of pellet/Soluble ratio relative to FRTTO were depicted in the chart above the blot. Values represent mean ± SE of three independent experiments. (C) HEK293 cells were transfected with Ub-Aβ42-GFP and either DNAJB6-V5 at 1:3 ratio or FRTTO as a control. Cells were fixed and immunostained with α-V5 (red) antibody to detect DNAJB6-V5. α-GFP (green) was used to detect Aβ-GFP and DAPI (blue) was used for DNA staining. The panel shows confocal images of mock cells (upper panel), cells transfected with Ub-Aβ-GFP with FRTTO (middle panel), cells transfected with Ub-Aβ-GFP and DNAJB6 (lower panel). Scale bar = 5 μm. (D) HEK293 cell line, stably expressing DNAJB6 was transfected with 0.2, 0.3 or 0.5 μg of Ub-Aβ42-GFP. Tetracycline was added to switch on the expression of DNAJB6. Cell lysates were fractionated into Soluble (S) and Pellet (P) and analyzed using western blotting as in panel (B).

Figure 2

DNAJB6 depends on HPD motif to prevent Aβ42-GFP aggregation. (A) HEK293 cells were transfected with Ub-Aβ42-GFP with either DNAJA1, DNAJB1, DNAJB6 (Wt), DNAJB6 (H31Q) mutants at 1:3 ratio for 48 h. Cells were lysed, fractionated into T, S, P and Western blotting was performed (B) HEK293 cells were transfected with Ub-Aβ42-GFP with either FRTTO (control) or HSPA1a at 1:3 ratio for 48 h. Quantification of the pellet/soluble ratio of Aβ-GFP relative to FRTTO was depicted in the charts above the blots. Values represent mean ± SE of two independent experiments. *P < 0.05, ***P < 0.001, ns = non significant.

Figure 3

Canonical members of HSPB family prevent the aggregation of Aβ-GFP. Cells were transfected with HSPB1, HSPB5, HSPB7 or FRTTO at 1:3 ratio for 48 h. Quantification of the pellet/soluble ratio of Aβ-GFP relative to FRTTO was depicted in the chart above the blot. Values represent mean ± SE of two independent experiments. ^**P < 0.01, ns = non significant.

Figure 4

Aβ₄₂-GFP aggregates do not co-localize with extracellular Aβ42-ATTO peptides. (A) One micromolar of Aβ42-ATTO 550 fibrils were added into the culture medium of HEK293 cells for 24 h Cells were fixed and stained with DAPI for nuclear staining. Confocal images were acquired by Leica SP8 confocal microscope. Extracellular Aβ peptides can enter the cells and form intracelluar aggreagtes. (B) HEK293 cells were transfected with DNAJB6-V5 in 1:3 ratio. After 24 h transfection, 1 μM of Aβ42 fibrils were added into the culture medium of cells for another 24 h and cells were fixed and stained with anti V5. Confocal images show that DNAJB6 cannot prevent the extracellular Aβ mediated aggregation. (C) Cells were transfected with 1μg Ub-Aβ-GFP. After 24 h transfection, 1 μM of Aβ42 fibrils were added into the culture medium of cells for another 24 h Cells were fixed. Confocal images show that extracellular Aβ peptides do not co-localize with Aβ-GFP. (D) Cells were transfected with Ub-Aβ-GFP and DNAJB6 in 1:3 ratio for 24 h then 1 μM of Aβ42-ATTO 550 fibrils were added into the culture medium of HEK293 cells for another 24 h Cells were fixed and immunostained as mentioned above except that DNAJB6 is not stained for. Images show that DNAJB6 can still decrease the aggregation of intracellular Aβ42-GFP in presence of extracellular Aβ peptides. ATTO 550 was used to detect extracellular Aβ peptides, GFP for Aβ42-GFP, anti V5 for DNAJB6 and DAPI for nuclei. DIC: differential interference contrast. Scale bar = 5 μm. (E) Western blot of cells transfected and fractionated as mentioned above.