Figure 1.
Effect of amyloid protein on the cell viability.
(A) Cortical neurons, BSMC, hepatocytes and dermal fibroblasts were treated with 10 µM fAβ (25–35) viability. (B) Cortical neurons (circles) and BSMC (triangles) were treated with fAβ (25–35) or vehicle (ionized water) at the indicated concentrations. MTT reducing activity was determined 48 h later. MTT reducing activity was determined 48 h later. Data are expressed as means ± S.E.M. (n = 4). *P<0.05, **P<0.01, compared with control (vehicle) by ANOVA followed by Dunnett's test.
Figure 2.
Effect of 15d-PGJ2 on the cell viability.
(A) Cortical neurons, BSMC, hepatocytes and dermal fibroblasts were treated with 10 µM 15d-PGJ2 or vehicle (0.1% ethanol). (B) Cortical neurons (circles) and BSMC (triangles) were treated with 15d-PGJ2 at the indicated concentrations. MTT-reducing activities of cortical neurons and other cells were determined 24 h or 48 h later, respectively. Data are expressed as means ± S.E.M. (n = 4). *P<0.05, **P<0.01, compared with control by ANOVA followed by Dunnett's test.
Figure 3.
15d-PGJ2 induced morphorogical degeneration in cortical neurons and BSMC.
Cortical neurons (A and B) and BSMC (C and D) were treated with vehicle (A and C) or 10 µM 15d-PGJ2 (B and D). Vehicle was 0.1% ethanol. Cortical neurons and BSMC were examined by phase-contrast microscopy 24 h and 48 h later, respectively.
Figure 4.
15d-PGJ2 downregulated cortical neurons and BSMC.
(A) Cortical neurons and BSMC were treated with vehicle (control) or 10 µM 15d-PGJ2. Vehicle was 0.1% ethanol. Cell densities (open columns) and MTT-reducing activities (closed columns) in cortical neurons and BSMC were determined 24 h or 48 h later, respectively. (B) Cortical neurons and BSMC were treated with vehicle (control), PGD2, PGJ2, Δ12-PGJ2 or 15d-PGJ2 at 10 µM. Vehicle was 0.1% ethanol. MTT-reducing activities in cortical neurons (open columns) and BSMC (closed columns) were determined 8 h or 48 h later, respectively. Data are expressed as means ± S.E.M. (n = 4). **P<0.01, compared with control by ANOVA followed by Dunnett's test.
Figure 5.
Binding assay of [3H]15d-PGJ2 to subcellular fractions.
(A) Proteins fractionated to plasma membranes, nuclear, cytosol or microsome from cortical neurons were incubated with 10 nM [3H]15d-PGJ2 at 25°C for 1 h in the absence or presence of 10 µM unlabeled 15d-PGJ2. Total binding, nonspecific binding and specific binding were hatched columns, open columns and closed columns, respectively. Data are expressed as means (n = 2). (B) Effects of 15d-PGJ2 and its precursors on the binding of 10 nM [3H]15d-PG J2 to cortical neurons (open columns) and BSMC (closed columns). Plasma membranes (10 µg/protein) were incubated with [3H]15d-PGJ2 at 4°C for 24 h in the presence of unlabeled PGD2, PGJ2, Δ12-PGJ2 or 15d-PGJ2 at the indicated concentrations. The control value of [3H]15d-PGJ2 binding in cortical neurons and BSMC were 2523 cpm and 1309 cpm, respectively. Data are expressed as means ± S.E.M. (n = 4). *P<0.05, **P<0.01, compared with control by ANOVA followed by Dunnett's test.
Table 1.
Comparison of the specific binding sites for [3H]15d-PGJ2 in plasma membranes to authentic receptors, DP1 and DP2.
Figure 6.
Biotinylated 15d-PGJ2 induced neuronal cell death.
(A) Cortical neurons were treated with 15d-PGJ2 (open circles) or biotinylated 15d-PGJ2 (closed circles) at the indicated concentrations in the serum-free medium. MTT-reducing activities were determined 18 h later. Data are expressed as means ± S.E.M. (n = 4). **P<0.01, compared with control by ANOVA followed by Dunnett's test. (B) Cortical neurons were treated with vehicle (control), 3 µM 15d-PGJ2 or 3 µM biotinylated 15d-PGJ2 in the serum-free medium. Vehicle was 0.1% ethanol. Cortical neurons were photographed by phase-contrast microscopy 18 h later.
Figure 7.
Identification of biotinylated 15d-PGJ2-modified proteins in the plasma membrane of cortical neurons.
Western blotting: Membrane proteins (400 µg) were incubated with 1 µM biotinylated 15d-PGJ2 at 4°C for 24 h in the presence of vehicle (A), Magnified photograph including spot 1-21 (B), 10 µM unlabeled 15d-PGJ2 (C) and 100 µM unlabeled 15d-PGJ2 (D). SYPRO Ruby: Membrane proteins (400 µg) were incubated with 1 µM biotinylated 15d-PGJ2 at 4°C for 24 h in the presence of vehicle (E). The proteins were separated by isoelectrofocusing (pH 3-10) and then by SDS-PAGE. The white circles denote spots excised for subsequent identification by MALDI-TOF analysis, as described under Expreimental Procedures.
Figure 8.
MALDI-TOF mass spectrum of the tryptic digest of spot 8.
Spot 8 from Figure 7D was digested in gel with trypsin, and the resulting peptides were analyzed by MALDI-TOF MS as detailed in the experimental section. (A) Typical mass spectrum from a representative experiment. (B) Probability based Mowse Score. (C) Positions of matched peptides in the sequence of GFAP.
Figure 9.
Peptide matches of spot 8 with GFAP.
List of the monoisotopic masses of some of the peptides identified showing their position in the sequence of GFAP.
Table 2.
Membrane proteins targeted for 15d-PGJ2.
Figure 10.
Interactions of 15d-PGJ2 with targets.
Membrane proteins (400 µg) were incubated with 1 µM biotinylated 15d-PGJ2 at 4°C for 24 h. Membrane lysates were incubated with Streptavidin Agarose. The presence of targeted proteins was detected by immunoblot analysis, and the incorporation of biotinylated 15d-PGJ2 into immunoprecipitates was detected with ECL.
Table 3.
Regions homologous to the binding site of 15d-PGJ2 in targeted proteins.
Figure 11.
Hypothetical roles of targets for 15d-PGJ2 on amyloidoses.
Membrane target proteins for 15d-PGJ2 were glycolytic enzymes (Enolase 2, PKM1 and GAPDH), molecular chaperones (Hsp8a and TCP1α), and cytoskeletal proteins (Actin β, CapZα2, Tubulin β and Internexin α). These proteins were factors associated with the two remarks of AD, the amyloid plaque and the neurofibrillary tangle. Beyond classical roles as glycolytic enzymes and molecular chaperones, GAPDH, Enolase2 and Hsp8a appear to form the complex of PMOs and contribute to the generation of reacting oxygen species by 15d-PGJ2.