Identification of Alzheimer’s disease–associated rare coding variants in the ECE2 gene
Xinxin Liao, Fang Cai, Zhanfang Sun, Yun Zhang, Juelu Wang, Bin Jiao, Jifeng Guo, Jinchen Li, Xixi Liu, Lina Guo, Yafang Zhou, Junling Wang, Xinxiang Yan, Hong Jiang, Kun Xia, Jiada Li, Beisha Tang, Lu Shen, Weihong Song
Xinxin Liao, Fang Cai, Zhanfang Sun, Yun Zhang, Juelu Wang, Bin Jiao, Jifeng Guo, Jinchen Li, Xixi Liu, Lina Guo, Yafang Zhou, Junling Wang, Xinxiang Yan, Hong Jiang, Kun Xia, Jiada Li, Beisha Tang, Lu Shen, Weihong Song
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Research Article Neuroscience

Identification of Alzheimer’s disease–associated rare coding variants in the ECE2 gene

Abstract

Accumulation of amyloid β protein (Aβ) due to increased generation and/or impaired degradation plays an important role in Alzheimer’s disease (AD) pathogenesis. In this report, we describe the identification of rare coding mutations in the endothelin-converting enzyme 2 (ECE2) gene in 1 late-onset AD family, and additional case-control cohort analysis indicates ECE2 variants associated with the risk of developing AD. The 2 mutations (R186C and F751S) located in the peptidase domain in the ECE2 protein were found to severely impair the enzymatic activity of ECE2 in Aβ degradation. We further evaluated the effect of the R186C mutation in mutant APP–knockin mice. Overexpression of wild-type ECE2 in the hippocampus reduced amyloid load and plaque formation, and improved learning and memory deficits in the AD model mice. However, the effect was abolished by the R186C mutation in ECE2. Taken together, the results demonstrated that ECE2 peptidase mutations contribute to AD pathogenesis by impairing Aβ degradation, and overexpression of ECE2 alleviates AD phenotypes. This study indicates that ECE2 is a risk gene for AD development and pharmacological activation of ECE2 could be a promising strategy for AD treatment.

Authors

Xinxin Liao, Fang Cai, Zhanfang Sun, Yun Zhang, Juelu Wang, Bin Jiao, Jifeng Guo, Jinchen Li, Xixi Liu, Lina Guo, Yafang Zhou, Junling Wang, Xinxiang Yan, Hong Jiang, Kun Xia, Jiada Li, Beisha Tang, Lu Shen, Weihong Song

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Figure 3

Effect of ECE2 mutants on Aβ levels and in vitro enzymatic activity.

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Effect of ECE2 mutants on Aβ levels and in vitro enzymatic activity. 20E...
20E2 cells were transfected with vector, ECE2WT, ECE2R186C, or ECE2F751S constructs for 72 hours. Levels of Aβ40 and Aβ42 from conditioned medium and cell lysates were measured using sandwich ELISA. (A) Aβ40 level in the conditioned media of 20E2 cells (n = 5). (B) Aβ42 level in the conditioned media of 20E2 cells (n = 5). (C) Aβ40 level in cell lysates of 20E2 cells (n = 4). For in vitro enzymatic activity test, HEK293 cells were transiently transfected with vector, ECE2WT, ECE2R186C, or ECE2F751S constructs for 48 hours, and the proteins were purified under native conditions. (D) McaBk2 peptide (10 μM) in 0.2 M sodium acetate buffer (pH 5.5) was incubated with purified vector, ECE2WT, ECE2R186C, or ECE2F751S proteins at 37°C for 1 hour. Relative fluorescent units (RFU) were recorded at Ex/Em = 320 nm/405 nm (n = 3). (E) Identification of purified proteins by Coomassie staining (upper) and Western blotting (bottom) (n = 3). (F) 20E2 cells were transfected with vector, ECE2WT, ECE2R186C, or ECE2F751S plasmids, and cell lysates were blotted for APP, C-terminal fragments (C83 and C99), and ECE2. Full-length APP and APP C-terminal fragments were detected with C20 antibody. ECE2 variants were detected by 9E10 antibody. C83 (G) and C99 (H) quantified and expressed as the ratio of C83 or C99 level in vector-expressing cells (n = 3). All results are expressed as mean ± SEM. Statistical significance was determined by ANOVA followed by Bonferroni’s multiple-comparisons test.