Authors:

Rayan Lahlou-Nabil

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About Paper:

Alzheimer's disease affects approximately 55 million individuals worldwide and is traditionally explained by the amyloid hypothesis, which states that amyloid-B (AB) accumulation initiates neurodegeneration [1]. AB is generated through B-secretase cleavage of amyloid precursor protein (APP) [2]. However, failed B-site APP-cleaving enzyme (BACE) inhibitor clinical trials suggest that additional APP processing pathways may contribute to disease progression. Recently, Dr. Michael Willem identified a new pathway, the n-secretase pathway, through analysis of human cerebrospinal fluid (CSF), producing AETA fragments that can be further cleaved into An-a and An-f [3]. Experimental evidence indicates that AETA fragments disrupt NMDA receptor signaling and impair memory-related processes, suggesting an alternative mechanism of toxicity. We hypothesize that characterizing AETA production and clearance kinetics in humans will clarify the role of n-secretase in Alzheimer's disease. To test this, Western blotting, immunoprecipitation (IP), and liquid chromatography/Mass Spectrometry were used to quantify AETA-a and AETA-B production and clearance in cell culture and human CSF using Stable Isotope Labeling Kinetics (SILK). Method development used *°C-leucine—labeled H4APPwt cell media to optimize antibody selection, IP conditions, protease digestion, and mass spectrometry parameters. The validated workflow was then applied to unlabeled and labeled CSF samples from healthy older adults and individuals with Alzheimer's disease to generate kinetic curves. BACE inhibitor LY2886721 (3 uM) was successful in suppressing B-secretase-mediated APP cleavage compared to BACE inhibitor C3, with sAPP® levels reduced to 3.02% and 0.58% at 1 uM and 3 uM, respectively. On the other hand, there were increases in sAPPa and AETA-a, which indicate an increase in a-secretase activity. These results are guiding optimization of the AETA kinetic analysis. Overall, this project advances understanding of APP metabolism beyond the classical amyloid pathway and may inform BACE inhibitor dosing strategies and development of early-stage Alzheimer's biomarkers.

Source:

Northwestern University

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Co-authors:

Rayan Lahlou-Nabil