Alzheimer's Disease-Linked Mutations in Presenilin-1 Result in a Drastic Loss of Activity in Purified γ-Secretase Complexes

BACKGROUND: Mutations linked to early onset, familial forms of Alzheimer's disease (FAD) are found most frequently in PSEN1, the gene encoding presenilin-1 (PS1). Together with nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2), the catalytic subunit PS1 constitutes the core of the γ-secretase complex and contributes to the proteolysis of the amyloid precursor protein (APP) into amyloid-beta (Aβ) peptides. Although there is a growing consensus that FAD-linked PS1 mutations affect Aβ production by enhancing the Aβ1-42/Aβ1-40 ratio, it remains unclear whether and how they affect the generation of APP intracellular domain (AICD). Moreover, controversy exists as to how PS1 mutations exert their effects in different experimental systems, by either increasing Aβ1-42 production, decreasing Aβ1-40 production, or both. Because it could be explained by the heterogeneity in the composition of γ-secretase, we purified to homogeneity complexes made of human NCT, APH1aL, PEN2, and the pathogenic PS1 mutants L166P, ΔE9, or P436Q. METHODOLOGY/PRINCIPAL FINDINGS: We took advantage of a mouse embryonic fibroblast cell line lacking PS1 and PS2 to generate different stable cell lines overexpressing human γ-secretase complexes with different FAD-linked PS1 mutations. A multi-step affinity purification procedure was used to isolate semi-purified or highly purified γ-secretase complexes. The functional characterization of these complexes revealed that all PS1 FAD-linked mutations caused a loss of γ-secretase activity phenotype, in terms of Aβ1-40, Aβ1-42 and APP intracellular domain productions in vitro. CONCLUSION/SIGNIFICANCE: Our data support the view that PS1 mutations lead to a strong γ-secretase loss-of-function phenotype and an increased Aβ1-42/Aβ1-40 ratio, two mechanisms that are potentially involved in the pathogenesis of Alzheimer's disease

The Gamma-Secretase-Mediated Proteolytic Processing of APP C-Terminal Fragments as a Therapeutic Target for Alzheimer's Disease

Alzheimer's disease (AD) is a devastating neurodegenerative disorder which severely impairs cognitive functions by triggering neuronal cell death and synaptic loss, and finally leads the patients to death. Two main histopathological hallmarks can be found in the brain tissue of AD patients: the amyloid plaques composed of aggregated Amyloid-β peptides (Aβ) and the neurofibrillary tangles (NFTs) constituted of hyperphosphorylated tau protein. To explain the underlying molecular mechanisms of AD pathogenesis, the amyloid cascade hypothesis states that early accumulation of Aβ peptides (especially the Aβ42 specie identified as the most toxic one) triggers their assembly into oligomers and their subsequent extracellular aggregation into dense fibrillar deposits. The oligomeric Aβ assemblies further initiate a succession of neurotoxic events including disruptions at the synaptic level, inflammation, neuritic injury, altered calcium homeostasis, oxidative stress, and altered phosphorylation activity leading to the hyperphosphorylation of tau and its aggregation into NFTs. Finally, this cascade of neuronal deleterious effects induces cognitive dysfuntions leading to dementia. The Aβ peptides originate from the amyloidogenic processing of the amyloid precursor protein (APP), a type I membrane protein that undergoes a first cleavage by β-secretase to liberate the soluble APP domain (sAPPβ) in the extracellular space, and the membrane bound APP-C99 fragment. Then, APP-C99 is processed by the intramembrane aspartyl-protease gamma-secretase (γ-secretase) composed of four subunits (presenilin (PS), nicastrin (NCT), anterior pharynx-defective protein 1 (APH1), and presenilin enhancer 2 (PEN2)) to release the toxic Aβ peptides in the lumen, and the APP intracellular domain (AICD) in the cytosol. Alternatively, APP can undergo the non-amyloidogenic processing, in which it is first shedded by α-secretase to generate the sAPPα domain and the APP-C83 fragment. The latter is further cleaved by γ-secretase into the non toxic p3 peptide and the AICD. AD is the most frequent form of dementia in elderly humans. Despite this evidence, no treatment is currently available to prevent or cure this disease. Thus, multiple potential drugs are tested in clinical trials or are still being developed in preclinical studies. In this work, a new therapeutic approach to lower Aβ production by specifically targeting the γ-secretase-mediated processing of APP-C99 is presented. Monoclonal antibodies against APP-C99 were generated and characterized following mice immunization with the active recombinant substrate APP-C99. When tested in cells, these antibodies decreased the γ-secretase-dependent processing of APP-C99, thus lowering Aβ production. Furthermore, the intracerebroventricular injection of anti-APP-C99 antibodies in a mouse model of AD decreased total soluble Aβ levels. These results validated this new approach as a potential immunotherapeutic strategy to prevent and/or delay the neurotoxic effects caused by Aβ in AD pathogenesis. Next, the γ-secretase-mediated processing of the APP-carboxy-terminal fragments (APP-CTFs) regulating the toxic versus non-toxic pathways was further investigated with the help of in vitro activity assays. This comparative study of APP-C99 and APP-C83 processing by γ-secretase revealed that both substrates were identically processed by the enzyme, with the same AICDs being produced. In addition, the effects of familial AD (FAD) mutations in the PS subunit of highly pure and homogeneous γ-secretase complexes were also investigated, and showed a drastic loss-of-function phenotype linked to these mutants. Overall, in vitro studies of the APP-CTFs processing by γ-secretase underlined particular functional aspects of this processing, which could be related to the cellular pathways involved in AD pathogenesis such as the competition between the amyloidogenic and non-amyloidogenic pathways and the FAD-linked impaired metabolism of APP

Therapeutic antibodies targeting app-c99

The present invention relates to an isolated and/or purified antibody, antibody fragment or derivative thereof able to block the gamma-secretase-dependent processing of the amyloid precursor protein (APP) and to pharmaceutical compositions containing said antibody/ies

Selective neutralization of APP-C99 with monoclonal antibodies reduces the production of Alzheimer's Aβ peptides

The toxic amyloid-β (Aβ) peptides involved in Alzheimer's disease (AD) are produced after processing of the amyloid precursor protein-C-terminal fragment APP-C99 by γ-secretase. Thus, major therapeutic efforts have been focused on inhibiting the activity of this enzyme. However, preclinical and clinical trials testing γ-secretase inhibitors revealed adverse side effects most likely attributed to impaired processing of the Notch-1 receptor, a γ-secretase substrate critically involved in cell fate decisions. Here we report an innovative approach to selectively target the γ-secretase-mediated processing of APP-C99 with monoclonal antibodies neutralizing this substrate. Generated by immunizing mice with natively folded APP-C99, these antibodies bound N- or C-terminal accessible epitopes of this substrate, and decorated extracellular amyloid deposits in AD brain tissues. In cell-based assays, the same antibodies impaired APP-C99 processing by γ-secretase, and reduced Aβ production. Furthermore, they significantly decreased brain Aβ levels in the APPPS1 mouse model of AD after intracerebroventricular injection. Together, our findings support APP-C99 substrate-targeting antibodies as new immunotherapeutic and Notch-sparing agents to lower the levels of Aβ peptides implicated in AD

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

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

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

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