Generation of stable cell lines overexpressing all human γ-secretase components with FAD-linked PS1 variants.

<p>MEF PS1/2^−/− were stably co-transduced with lentiviral vectors carrying genes encoding hNCT-V5, Flag-hPEN2, hAPH1aL-HA and clones were isolated by limiting dilution to generate a cell line, designated as γ- PS1/2, that overexpresses high amount of the three subunits. γ- PS1/2 MEFs were further transduced with hPS1 variants harbouring FAD-linked mutations or mutations in the catalytic aspartate residue(s), or PS1-WT, and cloned. Each clone, derived form the γ- PS1/2, was conveniently named according to the mutation present in PS1 preceded by the symbol γ and followed by the number of the clone (γ-MEF) in order to distinguish them from wild-type MEF (WT MEF) and MEF PS1/2^−/−. Two clones per γ-secretase variant were selected for characterization. (A–B) Whole cell protein extracts of the different cell lines were prepared in 1% NP40-HEPES buffer, separated by SDS-PAGE on 4–12% Bis-Tris or 12% Tris-Glycine gels and analysed by immunostaining to detect the γ-secretase core components NCT (NCT164), PS1 (NTF, MAB1563; CTF; MAB5232), APH1aL-HA (3F10), and Flag-PEN2 (M2) (A), and endogenous APP (A8717) (B). β-Actin was used as a loading control. Each lane represents one selected clone. <i>CTF</i>: C-terminal fragment, <i>FL</i>: full-length, <i>im.</i>: immature NCT; <i>m.</i>: mature NCT, <i>N</i>: N-glycosylated, <i>NTF</i>: N-terminal fragment, <i>O</i>: O-glycosylated.</p

Enzymatic activity of partially purified γ-secretase complexes with FAD-linked PS1 mutants.

<p>(A) γ-Secretase activity assays performed with γ-MEF and γ - PS1/2 microsomal extracts prepared in 1% CHAPSO-HEPES buffer. Equal protein levels from the different extracts were diluted to 0.25% CHAPSO-HEPES buffer and incubated for 4 h at 37°C with lipids and 1 µM of recombinant human APP-based substrate (C100-Flag). Samples were analyzed by SDS-PAGE and immunostained with anti-Flag (M2) or anti-PS1 (MAB1563). The relative amounts of AICD-Flag generated in the reactions, reflecting γ-secretase activity, were estimated by densitometry. PS1 immunostaining was used to assess the amount of input material. (B) Equal amounts of microsomal proteins were immunoprecipitated overnight at 4°C with either anti-Flag M2 or anti-HA affinity resins, and submitted to a C100-His assay according to the same protocol as in (A). Protein samples were separated by SDS-PAGE and analysed by immunostaining for γ-secretase subunits ((NCT164 (NCT), MAB1563 (PS1-NTF), or UD1 (PEN2)). AICD-His cleavage products were immunostained with an anti-APP-CTF antibody (A8717). *Indicates a non-specific band corresponding to the IgG light chains. (C) Aβ1–40 and Aβ1–42 were quantified by sandwich ELISA and represented in pg/mL (left Y-axis) or in percentage (right Y-axis) of the mean of Aβ1–40 levels generated by the two wild-type clones. Aβ1–42/Aβ1–40 ratios are indicated on the top of the bars. The results were confirmed in three independent experiments and a representative dataset is shown.</p

Aβ production in cell lines overexpressing human γ-secretase components with FAD-linked PS1 variants.

<p>WT MEF, γ-MEF and γ - PS1/2 were transduced with an APP-based substrate corresponding to the 99 C-terminal residues of human APP fused to the APP signal peptide in N-terminus (SPA4CT <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035133#pone.0035133-Lichtenthaler1" target="_blank">[36]</a>) and a Flag Tag in C-terminus. Cell proteins were extracted in 1% NP40-HEPES buffer, separated by SDS-PAGE on 12% Tris-Glycine gels and analysed by immunostaining with an antibody targeting the C-terminal part of APP (A8717) (A, C). Aβ1–40 and Aβ1–42 levels were also measured in the corresponding cell culture media (B, D). Data corresponds to three independent experiments (Mean ± SEM).</p

Enzymatic activity of highly purified γ-secretase complexes with FAD-linked or aspartate PS1 mutants.

<p>Equal amounts of the different purified γ-secretase preparations characterized in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035133#pone-0035133-g004" target="_blank">Figure 4</a> were tested for activity on C100-Flag, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035133#pone-0035133-g003" target="_blank">Figure 3</a>. The resulting cleavage products were separated by SDS-PAGE and detected by immunostaining with an anti-Flag antibody (M2) for C100-Flag or AICD-Flag (A), and by sandwich ELISA for Aβ1–40 or Aβ1–42 (B). Note that the levels of Aβ produced from FAD-linked γ-secretase complexes were all in the non-linear range of the ELISA standards, close to the detection limit. Whenever possible, Aβ1–42/Aβ1–40 ratios were quantified and indicated on the top of the bars. Two independent purifications were performed on each clone and similar results were obtained. A representative dataset is shown.</p

High-grade purification of human γ-secretase complexes with FAD-linked PS1 mutants.

<p>(A) Schematic representation of the γ-secretase purification process. Briefly, Presenilin double-knockout MEFs were used to first generate cell lines that stably overexpress human γ-secretase complexes containing different PS1 variants. Next, these cell lines were used for a multi-step purification procedure as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035133#s4" target="_blank">material and methods</a>. (B) Blue-Native PAGE analysis of purified γ-secretase complexes made of different PS1 variants. Equal volumes of the different purified γ-secretase preparations were separated by native-PAGE on a 4–16% Bis-Tris gel, and stained with silver nitrate (top panel), or immunostained for NCT (NCT164, middle panel) or PS1-NTF (ab10281, bottom panel) as indicated. γ-Secretase complexes appeared on the gel as high molecular weight complexes (HMWCs) of ∼350 kDa. Note that the levels of HMWCs were similar for all clones. (C) Equal volumes of purified γ-secretase complexes with FAD-linked PS1 mutants were separated under denaturing conditions (SDS-PAGE) and immunostained with anti-NCT (NCT164), anti-PS1-NTF (MAB1563), anti-PS1-CTF (MAB5232), anti-HA (3F10), or anti-Flag (M2) antibodies. Two independent purifications were performed on each clone with similar results. A representative dataset is shown.</p

Generation of Monoclonal Antibody Fragments Binding the Native γ‑Secretase Complex for Use in Structural Studies

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimer’s disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation

Discovery of a Novel Pharmacological and Structural Class of Gamma Secretase Modulators Derived from the Extract of Actaea racemosa

A screen of a library of synthetic drugs and natural product extracts identified a botanical extract that modulates the processing of amyloid precursor protein (APP) in cultured cells to produce a lowered ratio of amyloid-beta peptide (1–42) (Aβ42) relative to Aβ40. This profile is of interest as a potential treatment for Alzheimer’s disease. The extract, from the black cohosh plant (Actaea racemosa), was subjected to bioassay guided fractionation to isolate active components. Using a combination of normal-phase and reverse-phase chromatography, a novel triterpene monoglycoside, <b>1</b>, was isolated. This compound was found to have an IC₅₀ of 100 nM for selectively reducing the production of amyloidogenic Aβ42 while having a much smaller effect on the production of Aβ40 (IC₅₀ 6.3 μM) in cultured cells overexpressing APP. Using IP-MS methods, this compound was found to modulate the pool of total Aβ produced by reducing the proportion of Aβ42 while increasing the relative amounts of shorter and less amyloidogenic Aβ37 and Aβ39. Concentrations of <b>1</b> sufficient to lower levels of Aβ42 substantially (up to 10 μM) did not significantly affect the processing of Notch or other aspects of APP processing. When <b>1</b> (10 μg) was administered to CD-1 normal mice intracerebroventricularly, the level of Aβ42 in brain was reduced. Assays for off-target pharmacology and the absence of overt signs of toxicity in mice dosed with compound <b>1</b> suggest a comparatively selective pharmacology for this triterpenoid. Compound <b>1</b> represents a new lead for the development of potential treatments for Alzheimer’s disease via modulation of gamma-secretase