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[P1–168]: QUANTIFICATION OF PHOSPHOLIPIDS AND EVALUATION OF GAMMA‐SECRETASE ACTIVITY IN CYNOMOLGUS MONKEY BRAINS
Author(s) -
Kawatsuki Akihiro,
Morita Shinya,
Nishimura Masaki
Publication year - 2017
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2017.06.235
Subject(s) - phosphatidylethanolamine , phosphatidylserine , biochemistry , phosphatidylcholine , phosphatidic acid , liposome , cholesterol , chemistry , enzyme , membrane , biology , microbiology and biotechnology , phospholipid
Background: Relative increase in aggregation-prone Ab species generation causes not only Presenilin mutation-caused familial Alzheimer’s disease (AD) but also sporadic AD. Ab species are produced by g-secretase, which is an intramembrane protease, and lipid compositions of the membrane reportedly alter and modify g-secretase activity of cultured cells. However, relationship between lipid composition and g-secretase activity in brain remains to be elucidated. In this study, we measured contents of phospholipids and cholesterol and evaluated g-secretase activity using membrane fraction of monkey brains. Methods: Frozen tissues of temporal cortices from 42 cynomolgus monkeys (Macaca fascicularis, 4-36 years of age) were used. All experimental procedures were performed according to the Guide for the Care and Use of Laboratory Animals. Frozen tissues were homogenized, and membrane pellets were obtained by centrifugation of post-nuclear supernatants at 100,000 g. To measure phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidic acid (PA), sphingomyeline (SM), and cholesterol, enzymatic fluorometric methods detecting H2O2 production was utilized (Morita, et al. 2015 Sci. Rep. 5,11737). For a cell-free assay of g-secretase activity, Ab42 and Ab40 generated in a mixture of 1% CHAPSO-solubilized membrane and a recombinant humanAPP C-terminal fragment of 99 amino acids were measured by ELISA after 6 hr incubation at 37 C. Results:We optimized enzymatic measurement of phospholipids and cholesterol for membrane fractions from monkey brains. Our measurements of PC, PE, PS, PA, SM, and cholesterol in 1% CHAPSO soluble membrane fractions were reasonably sensitive and reproducible. Age-related increase in relative Ab42 generation was observed, as previously reported. The preliminary data suggested weak downregulation of the PE/PC ratio during ageing, though so far we have not found any correlated change in lipid compositions andg-secretase activity. We still continue the detailed analysis of the phospholipids and g-secretase activity in monkey brains of various age. Conclusions: Compared to conventional methods such as thin-layer chromatography (TLC) and mass spectrometry (MS), our enzymatic method has advantage in sensitivity, reproducibility, and applicability to high-throughputmeasurement of phospholipids. Thismethod is beneficial to investigate the involvement of phospholipid alterations in AD pathogenesis.