Secreted Amyloid β-Proteins in a Cell Culture Model Include N-Terminally Extended Peptides That Impair Synaptic Plasticity

Evidence for a central role of amyloid β-protein (Aβ) in the genesis of Alzheimer’s disease (AD) has led to advanced human trials of Aβ-lowering agents. The “amyloid hypothesis” of AD postulates deleterious effects of small, soluble forms of Aβ on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aβ could be therapeutically advantageous, it is important to understand the full range of soluble Aβ derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aβ species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aβ40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aβ-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aβ. These N-terminally extended Aβ-containing monomeric fragments are distinct from soluble Aβ oligomers formed from Aβ1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing β-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aβ-containing species can arise from APP processing events N-terminal to the classical β-secretase cleavage site

IgG Conformer's Binding to Amyloidogenic Aggregates

Amyloid-reactive IgGs isolated from pooled blood of normal individuals (pAbs) have demonstrated clinical utility for amyloid diseases by in vivo targeting and clearing amyloidogenic proteins and peptides. We now report the following three novel findings on pAb conformer's binding to amyloidogenic aggregates: 1) pAb aggregates have greater activity than monomers (HMW species > dimers > monomers), 2) pAbs interactions with amyloidogenic aggregates at least partially involves unconventional (non-CDR) interactions of F(ab) regions, and 3) pAb's activity can be easily modulated by trace aggregates generated during sample processing. Specifically, we show that HMW aggregates and dimeric pAbs present in commercial preparations of pAbs, intravenous immunoglobulin (IVIg), had up to ~200- and ~7-fold stronger binding to aggregates of Aβ and transthyretin (TTR) than the monomeric antibody. Notably, HMW aggregates were primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated IVIg IgGs. Human pAb conformer's binding to amyloidogenic aggregates was retained in normal human sera, and mimicked by murine pAbs isolated from normal pooled plasmas. An unconventional (non-CDR) component to pAb's activity was indicated from control human mAbs, generated against non-amyloid targets, binding to aggregated Aβ and TTR. Similar to pAbs, HMW and dimeric mAb conformers bound stronger than their monomeric forms to amyloidogenic aggregates. However, mAbs had lower maximum binding signals, indicating that pAbs were required to saturate a diverse collection of binding sites. Taken together, our findings strongly support further investigations on the physiological function and clinical utility of the inherent anti-amyloid activities of monomeric but not aggregated IgGs

Unfractionated, Aβ- and Cibacron blue-isolated human IgGs binding to plate-immobilized PFs.

<p>¹Mon stands for IgG monomers.</p><p>^2,3SEC-isolated IgG monomers (mon), dimers, and HMW aggregates as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g002" target="_blank">2</a> & <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g003" target="_blank">3</a>.</p><p>Each value for EC₅₀ and maximum signal amplitude was determined from the average of two to three sigmoidal fitted antibody binding curves, as shown in Figs <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g003" target="_blank">3</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0137344#pone.0137344.g008" target="_blank">8</a>.</p

Aβ-isolated but not heat-induced Avastin aggregates have enhanced avidity for PFs.

<p>(<b>A</b>) Left panel: SEC chromatograms for 0.3 mg/mL of Aβ-isolated Avastin IgGs, untreated Avastin, and for the antibody diluted into elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound Avastin IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: Antibody binding curves against PFs for unfractionated IVIg and Avastin, and for Aβ-isolated Avastin IgGs. (<b>B</b>) Left panel: SEC chromatograms for ~5 mg/mL of unfractionated Avastin in PBS, pH 7.4, and for IgG conformers contained in supernatant of 71°C heated Avastin monomers (A_400nm 0.5 sup) in PBS, pH 7.4. Right panel: Antibody binding curves against PFs for soluble (A_400nm 0.5 sup) and insoluble (A_400nm 0.5 pellet) IgG conformers of heat-treated Avastin monomers, and for untreated Avastin and IVIg.</p

A human mAb generated against a non-amyloid target binds aggregated Aβ.

<p>(<b>A</b>) Left panel: SEC chromatograms for ~15 mg/mL of mAb Avastin (anti-VEGF) and IVIg. SEC was carried out using a Superdex 200 Increase 10/300 GL column (GE Healthcare) equilibrated in PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for unfractionated (Unfrac) IVIg, and for Avastin used unfractionated or as SEC-isolated monomers and dimers. (<b>B</b>) The top Western blots show immunoprecipitation (IP) of synthetic Aβ conformers (monomers (Mon), dimers, and PFs) by 100 μg/mL of Avastin and IVIg, and by 200 μg/mL of a pan-Aβ reactive polyclonal antibody, AW8. The blots were probed for Aβ using an Aβ N-terminal reactive mAb, 6E10 (Signet Laboratories). The lower Western blots show 20 μg/mL mAb Avastin's ability to IP 5 μg/mL of Aβ dimers and PFs in the presence of a 5-molar excess (with respect to Avastin) of a N-terminal 165-amino acid fragment of its immunogen VEGF (VEGF-165). Control IP experiments (Ctls) were carried out using 5 μg/mL mAb 6E10 and a mixture of Aβ dimers and PFs, or with 20 μg/mL Avastin and 1 μg/mL VEGF-165. The blots were probed for Aβ and VEGF-165 using mAb 6E10 and Avastin, respectively. In IPs carried out in the absence of VEGF-165, cross-reactivity of the secondary antibody, goat anti-human IgG (heavy and light, Jackson Immunoresearch Laboratories Inc), with Avastin’s Ig light chain caused a faint band that migrated near VEGF-165. (<b>C</b>) Avastin IgG conformers binding curves against plate-immobilized PFs in the presence or absence of a 1:10 dilution of IgG-depleted normal human sera. (<b>D</b>) Bar charts for solution-phase PF's, Aβ monomers, and non-amyloid native and aggregated molecule's inhibition of Avastin monomers an dimers binding to plate-immobilized PFs. Competition studies were carried out using 0.1 mg/mL competitors and concentrations of Avastin conformers that were equivalent to their EC₅₀ values for PFs: 500 nM IgG Mon, and 200 nM IgG dimer. Each competition curve was carried out in duplicate, and bars represent the standard error.</p

IgG aggregates are primarily responsible for the enhanced anti-amyloid activities of Aβ- and Cibacron blue-isolated pAb IgGs.

<p>(<b>A</b>) Left panel: SEC chromatograms for ~0.5 mg/mL of Aβ-isolated IVIg IgGs, and for IVIg, untreated, or diluted into column elution buffer (0.1 M glycine, pH 2.7) that was used to elute Aβ-bound IVIg IgGs. SEC was carried out using a Superdex 200 increase 10/300 GL column (GE Healthcare) that was equilibrated with PBS, pH 7.4. Right panel: IgG binding curves against plate-immobilized PFs for untreated IVIg, and for Aβ column-isolated IVIg IgGs that were used unfractionated (Unfrac) or as SEC-isolated monomers (SEC Mon) or aggregates (SEC Aggs). SEC Aggs consisted of a pool of IgG conformers (dimers and HMW species) that eluted before the monomeric antibody. (<b>B</b>) Left panel: SEC chromatograms for ~0.5 mg/mL dye-isolated IVIg IgGs, and for unfractionated IVIg that was untreated or diluted into column elution buffer (PBS containing 1.5 M NaCl, pH 7.4) that was used to elute dye-bound IVIg IgGs. Right panel: IgG binding curves against plate-immobilized PFs for unfractionated and SEC-isolated conformers of dye-isolated IVIg IgGs, and for untreated IVIg.</p

Dimeric pAbs bind more tightly to PFs than their monomeric form.

<p>(<b>A</b>) Left panel: SEC chromatograms for ~1 mg/mL of protein A-purified pAbs and IVIg in PBS, pH 7.4. SEC was carried out using a Hiprep16/60 Sephacryl S300 HR column (GE Healthcare) that was equilibrated in PBS, pH 7.4. Arrows indicate the elution of protein molecular weight standards. Right panel: Antibody binding curves against PFs for unfractionated (Unfrac) and SEC-isolated monomeric pAbs. (<b>B</b>) Left panel: Antibody binding curves against PFs for unfractionated and SEC-isolated monomeric and dimeric IVIg IgGs. Right panel: SEC chromatographs for ~0.2 mg/ml IVIg dimers and monomers in PBS, pH 7.4, after incubation under ELISA-like conditions (4 h at 37°C). (<b>C</b>) Dynamic light scattering for 0.8 mg/mL and 0.06 mg/mL of SEC-isolated IVIg monomers and (<b>D</b>) dimers, respectively. Dynamic light scattering was determined at room temperature immediately after SEC-isolation of each IgG conformer in PBS, pH 7.4. <i>R</i>_<i>h</i> and pd are abbreviations for hydrodynamic radius and polydispersity, respectively.</p

IgG F(ab)ˈs but not Fc mediate IgG binding to PFs.

<p>Antibody binding curves are shown against plate-immobilized PFs for intact and fragmented IgGs from preparations of unfractionated IVIg (<b>A</b>) and for Cibacron blue-isolated IVIg IgGs (<b>B</b>). Antibody binding studies were carried out in triplicate and bars represent the standard error.</p

IVIg and Protein A-purified human and murine pAbs have similar anti-amyloid activities.

<p>(<b>A</b>) IgG binding curves against plate-immobilized PFs for IVIg and protein A-purified human (Hu) and murine (Mu) pAbs from pooled normal plasmas. Antibody binding curves are also shown for pAbs present in or dosed back into plasma. (<b>B</b>) Hybrid capture/competition ELISA curves for pAb's and IVIg's dose-dependent inhibition of PFs binding by plate-immobilized IVIg F(ab') fragments. The assay was carried out using 8 μg/ml solution-phase PFs. (<b>C</b>) Antibody binding curves for Hu and Mu pAb's nM cross-reactivity with plate-immobilized Aβ and TTR fibrils. (<b>D</b>) Left panel: Competition curves for solution-phase PF's and Aβ monomer's (Mon) inhibition of pAbs and IVIg binding to plate-immobilized PFs. Right panel: SAgg's and native TTR's (Nat) inhibition of pAbs and IVIg binding to plate-immobilized TTR fibrils. Competition studies were carried out using IgG concentrations, ~500 nM, which were equivalent to their EC₅₀ values for binding to PFs or TTR fibrils. Each binding or competition curve was carried out in duplicate, and bars represent the standard errors.</p

PAb aggregates have diverse avidities for Aβ.

<p>(<b>A</b>) Left panel: SEC chromatograms for untreated IVIg and for supernatants of ~4 mg/mL of SEC-isolated monomeric IVIg in PBS, pH 7.4, which was heated at 71°C until light scattering at A_400nm was 0.6 or 3.9, respectively. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, Aβ-isolated IVIg IgGs, and for IgG supernatants (sup) and PBS resuspendend pellets (pellet) of heat-treated IVIg. <b>(B</b>) Left panel: SEC chromatograms for ~4 mg/mL of untreated IVIg and for IVIg that was buffer exchanged at room temperature from gentle elution buffer (Pierce), pH 6.6, into PBS, pH 7.4. Right panel: Antibody binding curves against plate-immobilized PFs for untreated IVIg, buffered exchanged IVIg that was used unfractionated or as SEC-isolated monomers, and for Aβ-isolated IVIg IgGs.</p