Control of Alzheimer's amyloid beta toxicity by the high molecular weight immunophilin FKBP52 and copper homeostasis in Drosophila

Abstract

FK506 binding proteins (FKBPs), also called immunophilins, are prolyl-isomerases (PPIases) that participate in a wide variety of cellular functions including hormone signaling and protein folding. Recent studies indicate that proteins that contain PPIase activity can also alter the processing of Alzheimer's Amyloid Precursor Protein (APP). Originally identified in hematopoietic cells, FKBP52 is much more abundantly expressed in neurons, including the hippocampus, frontal cortex, and basal ganglia. Given the fact that the high molecular weight immunophilin FKBP52 is highly expressed in CNS regions susceptible to Alzheimer's, we investigated its role in Abeta toxicity. Towards this goal, we generated Abeta transgenic Drosophila that harbor gain of function or loss of function mutations of FKBP52. FKBP52 overexpression reduced the toxicity of Abeta and increased lifespan in Abeta flies, whereas loss of function of FKBP52 exacerbated these Abeta phenotypes. Interestingly, the Abeta pathology was enhanced by mutations in the copper transporters Atox1, which interacts with FKBP52, and Ctr1A and was suppressed in FKBP52 mutant flies raised on a copper chelator diet. Using mammalian cultures, we show that FKBP52 (-/-) cells have increased intracellular copper and higher levels of Abeta. This effect is reversed by reconstitution of FKBP52. Finally, we also found that FKBP52 formed stable complexes with APP through its FK506 interacting domain. Taken together, these studies identify a novel role for FKBP52 in modulating toxicity of Abeta peptides.

Figure 1. Effects of dFKBP59 mutations on Aβ42 toxicity in Drosophila.

(A–D) Eye phenotypes. (A) wild-type eyes of FKBP59^c01413/FKBP59^c01413 flies. (B) rough-eye phenotype of Aβ flies. (C) enhanced rough-eye phenotype of Aβ flies carrying the loss-of-function mutation dFKBP59^c01413. (D) suppressed rough-eye phenotype of Aβ flies carrying the gain-of-function mutation dFKBP59^EY03538. (E–F) Lifespan analysis of Aβ flies carrying dFKBP59 mutations. (E) CNS-directed expression of Aβ42 (blue) causes a shorter lifespan compared to flies expressing only the elavGal4 driver (green). (F) A gain-of-function mutation in dFKBP59 rescues the Aβ phenotype. Comparison of Aβ-only (blue) and Aβ/dFKBP59^EY03538 (red). (G) A loss-of-function mutation in dFKBP59 rescues the Aβ phenotype. Comparison of Aβ-only (blue) and Aβ/dFKBP59^c01413 (red). (H) Over-expression of FKBP59 (FKBP59gof) in 15–17 day old Aβ42- expressing flies reduces the levels of total Aβ42.

Figure 2. Effects of copper on Aβ42 phenotypes.

(A–D) Eye phenotypes of 15–17 day old flies. (A) wild type flies on 1 mM Cu (B) Aβ flies on normal food. (C) Aβ flies on food supplemented with 1 mM Cu. (D) Aβ flies on food supplemented with 1 mM BCS. (E–F) Quantification of the effects of Cu (E) and BCS (F) feeding on the rough eye phenotype. Phenotypes were evaluated as mild (light gray), moderate (dark gray) or severe (black) and the percent distribution of these phenotypes is shown. The graphs show a shift in the distribution of phenotypes when flies are raised on supplemented food. (G) Dose-dependent increase in levels of copper in flies raised on Cu-supplemented food.

Figure 3. Mutations in Ctr1, Atox1 and dFKBP59 alter levels of Cu in Drosophila heads and mouse cells.

(A–B) Copper measurements in Drosophila heads. (A) Control flies (oreR), flies expressing Aβ42 (Abeta) or flies over-expressing Ctr1A (UAS-Ctr1). (B) Wild type flies (oreR) and flies carrying loss-of-function mutations in the Atox1 and FKBP59 genes. Stars denote statistical significance. (C–D) Copper measurements in MEF cells. (C) intracellular copper in cells treated with BCS. (D) Intracellular copper in FKBP52 knock-out MEF cells.

Figure 4. Interaction of dFKBP59 and copper and effects on Aβ levels.

(A–C) Lifespan analysis of Aβ flies carrying mutations in dFKBP59 and raised on normal food or food supplemented with 1 mM BCS. (A) Aβ/BCS flies (green) compared to Aβ flies (blue). (B) Aβ/dFKBP59^lof flies (red) compared to Aβ/dFKBP59^lof/BCS flies (green). (C) Aβ/dFKBP59^gof flies (red) compared to Aβ/dFKBP59^gof/BCS flies (green). (D) APP and FKBP52 expression in APP695-HEK cells transiently transfected with FKBP52. (E) Aβ levels in conditioned medium collected after 24 h from cells in (D). (F) APP and FKBP52 expression in APP695-FKBP52 knockout MEF cells transiently transfected with FKBP52. (G) Aβ levels in conditioned medium collected after 48 h from cells in (F).

Figure 5. FKBP52 interacts with APP in endogenous and overexpression systems.

(A) HEK cells were transiently co-transfected with Myc-APP-FLAG and FKBP52-V5, immuniprecipitated with anti-Myc or anti-FLAG antibody and detected by western blot using anti-V5 antibody or anti-Myc antibody. (B) Western blot analysis for endogenous expression levels of both APP and FKBP52 in whole-cell lysates from human neuroblastoma cell (SH-SY5Y) and Human Kidney Epithelial cell (HEK). (C) HEK cells were treated with or without 0.5 µM FK506 for 5 hr and immunoprecipitated with anti-APP antibody followed by western blot using anti FKBP52 antibody. Expression level of APP or FKBP52 was detected by western blot using anti-APP or anti-FKBP52 antibody. (D) HEK cells were transiently transfected with full-length FKBP52-V5 or FKBP52 domain I-II-V5 and immunoprecipitated with anti-APP antibody followed by western blot using an anti V5 antibody.