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[P3–162]: GENOMEWIDE SCREENING OF MOLECULES INVOLVED IN UPTAKE OF ALZHEIMER DISEASE‐ASSOCIATED PROTEINS BY CRIPSR/CAS9 SYSTEM
Author(s) -
Tomita Taisuke,
Ebinuma Ihori,
Tsuruya Naoki,
Hori Yukiko
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.2017.06.1374
Subject(s) - biology , crispr , endocytic cycle , cas9 , population , genome editing , extracellular , microbiology and biotechnology , endocytosis , gene , genetics , cell , medicine , environmental health
Background: Fifty percent of amyloid-b (Ab) in the human central nervous system is cleared via direct transport across the blood-brain barrier (BBB) and reabsorption from CSF into the blood. This suggests thatmodulatingAb clearance from the blood could be a possible new therapeutic strategy for AD. Resident immune cells in the brain can participate in Ab clearance via phagocytosis and proteolytic degradation, but the role of peripheral immune cells in Ab clearance from blood remains unknown. Therefore the aim of this study was to determine phagocytic capacity of Ab and the expression of two important Ab degrading enzymes, neprilysin and insulin-degrading enzyme (IDE), in white blood cells.Methods:For this proof of principle, total white blood cells were isolated fromwhole blood of anonymous donors visiting the VU University Medical center in Amsterdam, The Netherlands. A method to measure Ab40 and Ab42 phagocytosis was developed. Total white blood cells were exposed to fluorescent Ab for 30 minutes and subsequently washed. Next, extracellular Ab was quenched with 0.2% trypan blue and Ab phagocytosis was detected with flow cytometry. Neprilysin and IDE activity was measured in lysates of monocytes, lymphocytes and granulocytes with in-house activity assays. Results:Here we report the measurement of specific Ab clearance capacities in peripheral immune cells. Flow cytometry showed that both granulocytes and monocytes possess Ab40 and Ab42 phagocytic activity. Interestingly, neprilysin activity was highest in granulocytes followed by monocytes and lymphocytes (2.8, 0.04 and 0.01 mMneprilysin activity per 10.000 cells respectively). IDE activity was also highest in granulocytes followed by lymphocytes, but absent in monocytes (0.1 ng IDE and 0.02 ng IDE per 10.000 cells respectively). This shows that monocytes, lymphocytes and granulocytes all participate in peripheral Ab clearance. Conclusions:Next to monocytes and lymphocytes, we identified granulocytes as peripheral immune cells that possess high capacity to degrade Ab and to phagocytose both Ab40 and Ab42. As Granulocytes outnumber monocytes and lymphocytes in blood, they could be key players in Ab clearance.