Signature amyloid β profiles are produced by different γ-secretase complexes

Abstract

γ-Secretase complexes are involved in the generation of amyloid-β (Aβ) in the brain. Therefore, γ-secretase has been proposed as a potential therapeutic target in Alzheimer disease (AD). Targeting γ-secretase activity in AD requires the pharmacological dissociation of the processing of physiological relevant substrates and the generation of "toxic" Aβ. Previous reports suggest the differential targeting of γ-secretase complexes, based on their subunit composition, as a valid strategy. However, little is known about the biochemical properties of the different complexes, and key questions regarding their Aβ product profiles should be first addressed. Here, we expressed, purified, and analyzed, under the same conditions, the endopeptidase and carboxypeptidase-like activities of the four γ-secretase complexes present in humans. We find that the nature of the catalytic subunit in the complex affects both activities. Interestingly, PSEN2 complexes discriminate between the Aβ40 and Aβ38 production lines, indicating that Aβ generation in one or the other pathway can be dissociated. In contrast, the APH1 subunit mainly affects the carboxypeptidase-like activity, with APH1B complexes favoring the generation of longer Aβ peptides. In addition, we determined that expression of a single human γ-secretase complex in cell lines retains the intrinsic attributes of the protease while present in the membrane, providing validation for the in vitro studies. In conclusion, our data show that each γ-secretase complex produces a characteristic Aβ signature. The qualitative and quantitative differences between different γ-secretase complexes could be used to advance drug development in AD and other disorders.

Keywords: Alzheimer Disease; Amyloid Beta; Amyloid Precursor Protein; Enzyme Mechanisms; Gamma-Secretase; Neurobiology; Secretases.

FIGURE 1.

Purification of γ-secretase complexes. A, schematic representation of the subunit composition and structural heterogeneity of the γ-secretase complex. A color code, indicated on top, is applied to the PSEN and APH1 subunits to show local homology between PSEN1 and PSEN2 or APH1A and APH1B subunits, respectively (based on ClustalW using Blosum matrix). PSEN1 and PSEN2 show 65% homology, whereas APH1A and APH1B show 56% homology at the amino acid level. Stars on presenilins TMD6 and TMD7 denote catalytic aspartate residues Asp-257 and Asp-385, respectively (46). Stars on the ectodomain of NCT indicate complex glycosylation. B, schematic representation of the purification methodology. Baculovirus expressed γ-secretase complex is purified using the GFP tag linked to the cytoplasmic domain of NCT. The complex is eluted with Prescission protease. C, Coomassie staining of purified γ-secretase complexes. Next to the γ-secretase subunits, the Prescission protease remains present in all purified samples. An unidentified band is also visible in all purifications (asterisk). D, Western blot of purified γ-secretase complexes. PEN-2 subunit immunoreactivity was used to estimate and normalize γ-secretase complex levels used in further in vitro activity assays.

FIGURE 2.

Schematic overview of the current model for γ-secretase activity. APP-C99 is first cleaved by an endopeptidase activity to release the intracellular domain and a long Aβ peptide (Aβ₄₈ or Aβ₄₉). These longer Aβ peptides are then further processed by a consecutive carboxypeptidase-like activity at every third amino acid, except for the processing of Aβ₄₂ into Aβ₃₈, where four amino acids are released. Methods used for the detection and quantification of AICD and Aβ products are indicated.

FIGURE 3.

PSEN2 containing γ-secretase complexes display lower endopeptidase activity (ϵ-cleavage), relative to PSEN1 complexes. A, total AICD generated by the different γ-secretase complexes in the presence or absence of 10 μm inhibitor X (Inh X; L-685,458, Merck). B, total AICD levels plotted as % of AICD generated by the PSEN1·APH1A γ-secretase complex (mean ± 95% CI, n = 12). C, detection of AICD50–99 and total AICD using a neo epitope (Neo) antibody and the FLAG-M2 antibody, respectively. Relative staining is not different. ns, not significant.

FIGURE 4.

γ-Secretase complexes generate distinctive Aβ product profiles. A, Aβ products were measured using ELISA techniques and normalized to initial AICD generation. Results are plotted as % of the Aβ products generated by the PSEN1·APH1A γ-secretase complex (mean ± 95% CI, n = 12). Aβ₃₈ peptides were undetectable in reactions with the PSEN2·APH1B complex (ND). B, the sum of Aβ₃₈, Aβ₄₀, Aβ₄₂, and Aβ₄₃ was plotted as % of the PSEN1·APH1A complex (mean ± 95% CI, n = 12). Lower total Aβ levels suggest the accumulation of longer Aβ (>43) peptides. C, Aβ profiles analyzed in urea-based gels corroborate the accumulation of longer Aβ peptides by these complexes. Aβ₄₂ and Aβ₄₃ are running as one band, the asterisk denotes an aspecific band (between Aβ₄₀ and Aβ₃₈).

FIGURE 5.

The APH1 subunit affects the fourth cleavage in vitro. Aβ product/substrate ratios for the fourth turnover were plotted as % of the ratio generated by the PSEN1/APH1A complex (mean ± 95% CI, n = 12). Aβ₃₈/Aβ₄₂ ratios could not be calculated for the PSEN2/APH1B complex because the levels of Aβ₃₈ were undetectable (ND). APH1B containing γ-secretase complexes lower the efficiency of the fourth cycle of γ-secretase activity, when compared with the corresponding APH1A containing complexes.

FIGURE 6.

Cell-based assays confirm the effects of APH1 and PSEN subunit composition on γ-secretase activities. A, expression of γ-secretase subunits in MEFs deficient for the two endogenous PSEN and the three endogenous APH1 subunits and rescued with either human PSEN1 or PSEN2 and with either human APH1A or APH1B. B, expression of endogeneous APP and APP-C83 and the introduced human APP-C99-GFP and its products in the presence or absence of 10 μm semagacestat (Janssen Pharmaceutica). Pictures come from the same blot developed at one exposure, so protein levels can be mutually compared. C, plotted as % of the ratio generated by the MEF cell line expressing PSEN1 and APH1A (mean ± 95% CI, n = 5).