γ-Secretase processing and effects of γ-secretase inhibitors and modulators on long Aβ peptides in cells

Abstract

Understanding how different species of Aβ are generated by γ-secretase cleavage has broad therapeutic implications, because shifts in γ-secretase processing that increase the relative production of Aβx-42/43 can initiate a pathological cascade, resulting in Alzheimer disease. We have explored the sequential stepwise γ-secretase cleavage model in cells. Eighteen BRI2-Aβ fusion protein expression constructs designed to generate peptides from Aβ1-38 to Aβ1-55 and C99 (CTFβ) were transfected into cells, and Aβ production was assessed. Secreted and cell-associated Aβ were detected using ELISA and immunoprecipitation MALDI-TOF mass spectrometry. Aβ peptides from 1-38 to 1-55 were readily detected in the cells and as soluble full-length Aβ proteins in the media. Aβ peptides longer than Aβ1-48 were efficiently cleaved by γ-secretase and produced varying ratios of Aβ1-40:Aβ1-42. γ-Secretase cleavage of Aβ1-51 resulted in much higher levels of Aβ1-42 than any other long Aβ peptides, but the processing of Aβ1-51 was heterogeneous with significant amounts of shorter Aβs, including Aβ1-40, produced. Two PSEN1 variants altered Aβ1-42 production from Aβ1-51 but not Aβ1-49. Unexpectedly, long Aβ peptide substrates such as Aβ1-49 showed reduced sensitivity to inhibition by γ-secretase inhibitors. In contrast, long Aβ substrates showed little differential sensitivity to multiple γ-secretase modulators. Although these studies further support the sequential γ-secretase cleavage model, they confirm that in cells the initial γ-secretase cleavage does not precisely define subsequent product lines. These studies also raise interesting issues about the solubility and detection of long Aβ, as well as the use of truncated substrates for assessing relative potency of γ-secretase inhibitors.

Keywords: Alzheimer Disease; Amyloid; Amyloid Precursor Protein; Intramembrane Proteolysis; Protease.

FIGURE 1.

BRI2-Aβ1-x overexpression and production profile in transiently transfected HEK 293T cells. A, IP/MS spectra from 1 ml of conditioned media from transiently transfected HEK 293T cells expressing the various BRI2-Aβ1-x vectors. The m/z value of peaks representing Aβ1-x peptides are labeled (also see Table 1). B, selected subset of IP/MS spectra from Triton X-100 cell lysates from the transiently transfected cells expressing the various BRI2-Aβ1-x vectors. C, overexpression of the BRI2-Aβ1-x constructs as detected by Western blotting of the cell lysates using an anti-BRI2 antibody ITM2b and the anti-Aβ_1–16 antibody 6E10.

FIGURE 2.

Processing of BRI2-Aβ1-x in transiently transfected HEK 293T cells is detectable by Aβ ELISA. A, Aβx-40 and Aβx-42 ELISA of conditioned media from transiently transfected HEK 293T cells expressing the various BRI2-Aβ1-x vectors. B, percentage of inhibition of Aβx-42 determined by ELISA production for each construct in the presence of 1 μm LY411,575 compared with DMSO control. The numbers below each bar indicate the length of the corresponding BRI2-Aβ1-x constructs.

FIGURE 3.

Constructs longer than Aβ1–48 are efficiently processed by γ-secretase in stable CHO cells. A, Aβx-40 and Aβx-42 ELISA of conditioned media from stable cell line transfected with various BRI2-Aβ1-x constructs. B, the ratio of Aβx-42 to Aβx-40 production of each BRI2-Aβ1-x construct is shown. C, percentage of inhibition of Aβx-40 and Aβx-42 determined by ELISA for each construct in the presence of 1 μm LY411,575 compared with DMSO control. D, IP/MS spectra of select BRI2-Aβ1-x constructs. 10 ml of conditioned media was used for the BRI2-Aβ1–47 and BRI2-Aβ1–48 constructs, with 1 ml used for the remaining constructs. Labels in the spectra indicate the corresponding Aβ peptide, e.g., 37 for Aβ1–37. Intact Aβ1–47, 48, and 49 are labeled with asterisks. Peaks without labeling are nonspecific. E, IP/MS spectra of fresh Ham's F-12 medium with 1 μm synthetic Aβ1–49 (top panel) and media from CHO cells that was incubated with 1 μm Aβ1–49 overnight (bottom panel).

FIGURE 4.

PSEN1 mutants increase Aβ42:Aβ40 from CHO cells expressing BRI2-C99 and BRI2-Aβ1–51 but not from cells expressing BRI2-Aβ1–49. PSEN1 WT, variant M139V, or Δexon9 was transiently co-transfected with BRI2-C99 (A), BRI2-Aβ1–49 (B), or BRI2-Aβ1–51 (C), and then Aβx-40 and Aβx-42 were measured by ELISA. The data represent the results from four to seven independent experiments, with two or three replicates in each experiment per condition. Statistical analysis was performed by one-way analysis of variance. *, p < 0.05; **, p < 0.01; ***, p < 0.001.

FIGURE 5.

Effects of GSI on γ-secretase cleavage of long BRI2-Aβ1-x constructs. A and B, Aβx-40 production in the presence of 1 μm γ-secretase inhibitors L685,458 (A) or 0.1 μm MRK560 (B) using transiently transfected HEK cells. The ratio of Aβx-40 in the presence of inhibitors to Aβx-40 in the absence of inhibitors (DMSO) were used to evaluate the extent of inhibition. The data are expressed and graphed as means ± S.E. with statistical analysis performed by one-way analysis of variance followed by Dunnett's post hoc testing for group differences. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001. C and D, dose response of L685,458 on Aβx-40 production in APP wild type and BRI2-Aβ1–49 from transiently transfected HEK-293 cells (C) or stable CHO cells (D), respectively. E, effect of MRK560 on Aβx-40 production in an in vitro γ-secretase assay using recombinant C99 and synthetic Aβ1–49.

FIGURE 6.

γ-Secretase cleavage of BRI2-Aβ1-x constructs can be modulated by GSMs. A, Aβx-42 levels from CHO stable line cells expressing the indicated BRI2-Aβ1-x vectors as measured by ELISA after treatment with 1 μm GSM1, 1 μm compound 2, 50 μm fenofibrate, or 1 μm (Z-LL)₂ ketone were compared with the DMSO group. Statistical analysis was performed by one-way analysis of variance. *, p < 0.05; **, p < 0.01. B and C, dose response of GSM1 (B) and compound 2 (C) on BRI2-C99 and BRI2-Aβ1-x constructs in CHO stable line cells is shown.

FIGURE 7.

Mass spectra of Aβ from BRI2-Aβ1–49, 1–51, and 1–55 constructs in response to select GSMs. A, IP/MS spectra of conditioned media (1 ml) from CHO stable line cells expressing the indicated BRI2-Aβ1-x 49, 1–51, and 1–55 vectors treated with 1 μm GSM1, compound 2, μm (Z-LL)₂ ketone, or 50 μm fenofibrate, respectively. *, peak at m/z 4150 is nonspecific. B, Aβ1-x production in the presence of GSMs. The sum of Aβ1–36 through Aβ1–42 peak intensities is assigned as 100%. Peaks with intensity of <5% or signal:noise ratio of <5 are not shown.