Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase

Abstract

A hallmark of Alzheimer's disease (AD) is the aggregation of β-amyloid peptides (Aβ) into amyloid plaques in patient brain. Cleavage of amyloid precursor protein (APP) by the intramembrane protease γ-secretase produces Aβ of varying lengths, of which longer peptides such as Aβ42 are thought to be more harmful. Increased ratios of longer Aβs over shorter ones, exemplified by the ratio of Aβ42 over Aβ40, may lead to formation of amyloid plaques and consequent development of AD. In this study, we analyzed 138 reported mutations in human presenilin-1 (PS1) by individually reconstituting the mutant PS1 proteins into anterior-pharynx-defective protein 1 (APH-1)aL-containing γ-secretases and examining their abilities to produce Aβ42 and Aβ40 in vitro. About 90% of these mutations lead to reduced production of Aβ42 and Aβ40. Notably, 10% of these mutations result in decreased Aβ42/Aβ40 ratios. There is no statistically significant correlation between the Aβ42/Aβ40 ratio produced by a γ-secretase variant containing a specific PS1 mutation and the mean age at onset of patients from whom the mutation was isolated.

Keywords: Alzheimer’s disease; Aβ peptides; amyloid hypothesis; cleavage activity; γ-secretase.

Fig. 1.

Preliminary analysis of 10 AD-derived PS1 mutations. (A) A schematic diagram of the major workflow in this study. The WT human γ-secretase and 138 variants, each containing an AD-derived mutation in PS1, were individually overexpressed and purified. The peak fractions were incubated with APP-C99 and examined for the production of Aβ42 and Aβ40 using the AlphaLISA assay. (B) Effect of 10 AD-derived PS1 mutations on the cleavage activities of the corresponding γ-secretases. Shown here is the combined production of Aβ42 and Aβ40 peptides by the 10 γ-secretase variants. The activity of WT γ-secretase was normalized as 1. Each experiment was repeated three times, and the SD is shown. (C) Effect of 10 AD-derived PS1 mutations on the generation of Aβ42 and Aβ40. (D) All but two variants show increased molar ratios of Aβ42 over Aβ40 compared with WT γ-secretase. Only the variant A285V displays a slightly lower Aβ42/Aβ40 ratio, and S365A is nearly identical to WT γ-secretase. (E) The combined production of Aβ42 and Aβ40 by each of the 10 γ-secretase variants shows no obvious correlation with the mean AAO. (F) The Aβ42/Aβ40 ratio exhibits no obvious correlation with the mean AAO.

Fig. S1.

AD-derived mutations in PS1. (A) A topology diagram of PS1. The residues, each mutated to only one type of amino acid, are colored red. The residues, each mutated to two or more types of amino acid, are colored yellow. (B) A flowchart on the selection of 138 PS1 mutations for in-depth analysis. These 138 mutations include 129 missense mutations, seven truncations, and two insertions. All 121 AD-targeted amino acids are covered by these 138 mutations.

Fig. S2.

WT γ-secretase and variants used in this study. WT γ-secretase and variants are visualized on SDS/PAGE gels by Coomassie staining. For most variants, five polypeptide bands are clearly visible, representing Nicastrin (NCT), the N-terminal fragment (NTF) of PS1, APH-1aL, the C-terminal fragment (CTF) of PS1, and PEN-2. Each variant is identified by a specific PS1 mutation labeled at the top of the lane. Each lane is identified by two numbers, with the first representing the gel and the second specifying location within the gel. For example, WT γ-secretase is in lane 1-9 (gel 1, lane number 9).

Fig. S3.

γ-secretase and variants used in this study. (A) γ-secretase variants are visualized on SDS/PAGE gels by Coomassie staining. (B) Thirteen engineered γ-secretase variants used in this study are visualized on SDS/PAGE gels by Coomassie staining.

Fig. S4.

Eight γ-secretase variants abolish autoproteolysis. (A) These eight γ-secretase variants are visualized on a SDS/PAGE gel by Coomassie staining. In contrast to WT γ-secretase, these variants nearly abrogated endoproteolysis, as evidenced by the intact PS1 and missing NTF/CTF bands. (B) These eight variants exhibit severely compromised cleavage activities compared with WT γ-secretase. (C) Seven of the eight residues affected by these mutations map to the vicinity of the active site in PS1. Shown here are two perpendicular views of PS1, with the mutated residues colored red. The two catalytic aspartate residues are shown in spheres.

Fig. 2.

Effect of 138 AD-derived PS1 mutations on the abilities of the corresponding γ-secretase variants to generate Aβ42 and Aβ40. The amounts of Aβ42 and Aβ40 produced by WT γ-secretase are normalized. The relative amounts of Aβ42 and Aβ40 produced by these 138 γ-secretase variants are shown here. The vast majority of these variants exhibit reduced production of both Aβ42 and Aβ40. In particular, production of Aβ42 as well as Aβ40 was abrogated to background levels for 11 γ-secretase variants, including S178P, S212Y, Q223R, S230I, I238M, K239N, T245P, L271V, T274R, T291P, and E318G. Only 10 γ-secretase variants exhibit increased cleavage activities toward both Aβ42 and Aβ40 compared with WT γ-secretase.

Fig. 3.

Effect of 138 AD-derived PS1 mutations on the abilities of the corresponding γ-secretase variants to modulate the Aβ42/Aβ40 ratios. (A) Effect of 138 AD-derived PS1 mutations on the combined production of Aβ42 and Aβ40 by the corresponding γ-secretase variants. The vast majority of the mutations negatively affect the combined production of Aβ42 and Aβ40. Only 13 variants exhibit increased total cleavage activity compared with WT γ-secretase. (B) Effect of 138 AD-derived PS1 mutations on the abilities of the corresponding γ-secretase variants to modulate the Aβ42/Aβ40 ratios. The vast majority of these mutations lead to increased Aβ42/Aβ40 ratios. Only 13 variants exhibit decreased Aβ42/Aβ40 ratios compared with WT γ-secretase.

Fig. 4.

The Aβ42/Aβ40 ratios generated in liposome- and cell-based cleavage assays are similar to those obtained in detergent micelle-based assays. (A) A schematic diagram of the liposome-based cleavage assay. The WT and mutant γ-secretases were individually reconstituted into the liposomes together with APP-C99 and examined for their abilities to generate Aβ42 and Aβ40 using the AlphaLISA assay. (B) The Aβ42/Aβ40 ratios generated in the liposome-based cleavage assays are similar to those obtained in detergent micelle-based assays. Results from 11 representative γ-secretase variants are shown here. (C) A schematic diagram of the cell-based cleavage assay. The CRISPR/Cas9 technology was used to generate PS1^−/− N2a cells. The pCAG vectors, each encoding WT or a distinct γ-secretase variant, were individually transfected into N2a cells for assessment of production of Aβ42 and Aβ40. (D) The Aβ42/Aβ40 ratios generated in the cell-based cleavage assays are similar to those obtained in detergent micelle-based assays.

Fig. S5.

Negatively stained electron microscopy images of the (Left) empty liposomes and (Right) γ-secretase-loaded proteoliposomes. These liposomes are mostly intact.

Fig. S6.

PSEN1 deletion in N2a cells. (A) DNA sequencing results around the targeted deletion region of the coding sequences of PSEN1. Sequencing was performed on genomic DNA derived from WT N2a cells. The dinucleotide CG, present in WT PSEN1, has been designed for deletion. (B) Sequencing of genomic DNA derived from a mixture of PSEN1^+/+, PSEN1^+/−, and PSEN1^−/− N2a cells. (C) Sequencing of genomic DNA derived from the PSEN1^−/− N2a cells. (D) The production of Aβ40 is drastically reduced in PSEN1^−/− N2a cells.

Fig. 5.

There is no statistically significant correlation between the Aβ42/Aβ40 ratios produced by γ-secretase variants and the mean AAOs. (A) A plot of the Aβ42/Aβ40 ratio versus the mean AAO. Altogether, 90 data points are shown here. For 42 γ-secretase variants, the Aβ42/Aβ40 ratio cannot be computed due to background levels of Aβ42 and/or Aβ40. For six PS1 mutations, there are no reported AAOs. (B) A plot of the total cleavage activity of γ-secretase variant versus the mean AAO. Altogether, 127 data points are shown here. For 11 PS1 mutations, there are no reported AAOs. (C) Higher ratios of Aβ42/Aβ40 correlate with lower AAOs from a group of nine PS1 mutations. The correlation coefficient is 0.99, with a P value of less than 0.001. (D) Lower ratios of Aβ42/Aβ40 correlate with lower AAOs from a group of nine PS1 mutations. The correlation coefficient is 0.92, with a P value of less than 0.001.

Fig. S7.

Analysis of potential correlation between the Aβ42/Aβ40 ratios and the mean AAOs. (A) A forced fit of all data points into a straight line yields a statistically insignificant correlation coefficient of 0.038 and a P value of 0.06. (B) A plot of Aβ42/Aβ40 ratio versus mean AAO for the TOP10 variants. The TOP10 variants exhibit increased production of both Aβ42 and Aβ40. (C) A plot of Aβ42/Aβ40 ratio versus mean AAO for the RATIO31 variants. The RATIO31 variants exhibit the 31 highest ratios of Aβ42/Aβ40.

Fig. S8.

Comparison with published studies on potential correlations between AAO and the Aβ42/Aβ40 ratio. (A) Five PS1 mutations appear to exhibit a positive correlation between the Aβ42/Aβ40 ratios and AAOs. This is mainly contributed by two mutations, A246E and L250S. These five PS1 mutations were investigated in a published study (43), where a weak correlation between AAO and the Aβ42/Aβ40 ratio was observed. (B) Six PS1 mutations appear to exhibit a negative correlation between the Aβ42/Aβ40 ratios and AAOs. These five PS1 mutations were investigated in a published study (41), where a perfectly negative correlation between AAO and the Aβ42/Aβ40 ratio was noted.

Fig. 6.

γ-secretase variants with engineered PS1 mutations exhibit similar biochemical properties as the variants with AD-derived mutations. (A) Location of the 13 amino acids affected by engineered mutations. Except TM4, each of the other eight TMs contributes at least one residue for mutation. Shown here is a ribbon representation of PS1 structure. The residues targeted for mutation are colored magenta, and the two catalytic Asp residues are shown in spheres. (B) All 13 engineered mutations result in compromised protease activities of the corresponding γ-secretase variants for the generation of both Aβ42 and Aβ40. (C) All 13 engineered mutations lead to decreased levels of protease activity as judged by the combined production of Aβ42 and Aβ40. (D) Seven mutations cause increased ratios of Aβ42/Aβ40 compared with WT. Only three mutations (I140V, I253V, and I414V) have no significant effect on the Aβ42/Aβ40 ratios.

Fig. S9.

Comparison of Aβ42 and Aβ40 production by 10 γ-secretase variants containing either APH-1aL or APH-1b. (A) A plot of the total amount of Aβ42 and Aβ40 generated by γ-secretase variants containing either APH-1aL or APH-1b. (B) A plot of the total amount of Aβ42 and Aβ40 versus mean AAO for the APH-1b–containing γ-secretase variants. (C) The ratios of Aβ42 over Aβ40 produced by these γ-secretase variants. (D) A plot of the Aβ42/Aβ40 ratio versus mean AAO for the APH-1b–containing γ-secretase variants.