Systematic Evaluation of Candidate Ligands Regulating Ectodomain Shedding of Amyloid Precursor Protein

Despite intense interest in the proteolysis of the β-Amyloid Precursor Protein (APP) in Alzheimer’s disease, how the normal processing of this type I receptor-like glycoprotein is physiologically regulated remains ill-defined. In recent years, several candidate protein ligands for APP, including F-spondin, Reelin, β1 Integrin, Contactins, Lingo-1, and Pancortin, have been reported. However, a cognate ligand for APP that regulates its processing by α- or β-secretase has yet to be widely confirmed in multiple laboratories. Here, we developed new assays in an effort to confirm a role for one or more of these candidate ligands in regulating APP ectodomain shedding in a biologically relevant context. A comprehensive quantification of APPsα and APPsβ, the immediate products of secretase processing, in both non-neuronal cell lines and primary neuronal cultures expressing endogenous APP yielded no evidence that any of these published candidate ligands stimulate ectodomain shedding. Rather, Reelin, Lingo-1, and Pancortin-1 emerged as the most consistent ligands for significantly inhibiting ectodomain shedding. These findings led us to conduct further detailed analyses of the interactions of Reelin and Lingo-1 with APP

Structural change induced in αS vs. NAA-αS by negatively charged vesicles.

<p><b>A:</b> Example of CD spectroscopy of PS vesicles titrated into NAA-αS solutions at 25°C. Spectra are shown at ascending lipid/protein ratios. Note the isobestic point (at 204 nm) indicative of a pure two state coil-helix transition. The arrow indicates the decrease in ellipticity at 222 nm as a consequence of helical folding. B–D: CD spectroscopy measurements of αS (5 µM) titration with PS (POPC/POPS 4∶1, 40 mM) (<b>B</b>), GM3 (POPC/GM3 4:1, 40 mM) (<b>C</b>) and GM1 (POPC/GM1 4:1, 40 mM) (<b>D</b>). Decrease in MRE at 222 nm upon titration of αS with PS, GM3 and GM1 containing vesicles correlates with increased α-helical structure in either NAA-αS or non-acetylated αS.</p

Thermodynamic and structural parameters for NAA-αS and αS upon SUV titration.

<p>Experiments were performed at 25°C. Depicted values are means values from 3 independent measurements with standard deviations shown for the ITC measurements. Helicity values are means from 3 independent measurements.</p

N-alpha acetylation has a functional impact on αS aggregation into amyloid fibrils.

<p>Aggregation of NAA-αS and αS (0.6 mg/ml) in the absence or presence of GM1 containing vesicles (GM1/protein 10∶1 mol/mol) measured by an increase in thioflavin T fluorescence. Standard deviations are calculated from N = 4 experiments. The presence of N-acetylation or GM1 vesicles binding each led to increased resistance to aggregation, with the effects being cumulative. A 2-way ANOVA test was used to test for significance at selected time points. **** = p<0.0001.</p

ITC-measured heat release curves of NAA-αS and αS upon titration with negatively charged lipid vesicles.

<p><b>A:</b> Example of primary heat signal obtained from titration calorimetry of PS vesicles titrated into solutions of NAA-αS at 25°C measured in tandem with the CD spectra in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103727#pone-0103727-g002" target="_blank">Fig. 2</a>. <b>B:</b> Integrated heat signals of titrations of PS vesicles (POPC/POPS 4∶1, 40 mM) into protein solutions of NAA-αS and αS (5 µM). The sigmoidal titration curve suggests simple protein-lipid binding with saturable binding sites. <b>C:</b> Titrations of GM3 vesicles (POPC/GM3 4:1, 40 mM) into solutions of NAA-αS and αS (5 µM) at 25°C. <b>D:</b> Titration of GM1 vesicles (POPC/GM1 4:1, 40 mM) vesicles into the two protein solutions (5 µM). Note the doubled heat release for NAA-αS compared to non-acetylated αS. All measurements were conducted at 25°C.</p

Effects of Membrane Lipids on the Activity and Processivity of Purified γ-Secretase

The 19-transmembrane multisubunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer’s disease (AD) by intramembrane proteolysis of the β-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human γ-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerization, and membrane lipid polar headgroups on γ-secretase function. We analyzed γ-secretase activity and processivity [i.e., sequential cleavages in the APP transmembrane domain that convert longer Aβ species (e.g., Aβ₄₆) into shorter ones (e.g., Aβ₄₀)] by quantifying the APP intracellular domain (AICD) and various Aβ peptides, including via a bicine/urea gel system that detects multiple Aβ lengths. These assays revealed several trends. (1) Switching from a <i>cis</i> to a <i>trans</i> isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased γ-activity, did not affect Aβ₄₂:Aβ₄₀ ratios, but decreased the ratio of long (≥42) versus short (≤41) Aβ peptides. (2) Increasing the FA carbon chain length (14, 16, 18, and 20) increased γ-activity, reduced longer Aβ species, and reduced the Aβ₄₂:Aβ₄₀ ratio. (3) Shifting the position of the double bond in 18:1­(Δ9-<i>cis</i>) PC to the Δ6 position substantially reduced activity. (4) Gangliosides increased γ-activity but decreased processivity, thus elevating the Aβ₄₂:Aβ₄₀ ratio. (5) Phosphatidylserine decreased γ-activity but increased processivity. (6) Phosphatidylinositol strongly inhibited γ-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence γ-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as presenilin or APP mutations do