Milenkovic, Vladimir M. and Slim, Dounia and Bader, Stefanie and Koch, Victoria and Heinl, Elena-Sofia and Alvarez-Carbonell, David and Nothdurfter, Caroline and Rupprecht, Rainer and Wetzel, Christian H. (2019) CRISPR-Cas9 Mediated TSPO Gene Knockout alters Respiration and Cellular Metabolism in Human Primary Microglia Cells. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 20 (13): 3359. ISSN 1422-0067,
Full text not available from this repository. (Request a copy)Abstract
The 18 kDa translocator protein (TSPO) is an evolutionary conserved cholesterol binding protein localized in the outer mitochondrial membrane. It has been implicated in the regulation of various cellular processes including oxidative stress, proliferation, apoptosis, and steroid hormone biosynthesis. Since the expression of TSPO in activated microglia is upregulated in various neuroinflammatory and neurodegenerative disorders, we set out to examine the role of TSPO in an immortalized human microglia C20 cell line. To this end, we performed a dual approach and used (i) lentiviral shRNA silencing to reduce TSPO expression, and (ii) the CRISPR/Cas9 technology to generate complete TSPO knockout microglia cell lines. Functional characterization of control and TSPO knockdown as well as knockout cells, revealed only low de novo steroidogenesis in C20 cells, which was not dependent on the level of TSPO expression or influenced by the treatment with TSPO-specific ligands. In contrast to TSPO knockdown C20 cells, which did not show altered mitochondrial function, the TSPO deficient knockout cells displayed a significantly decreased mitochondrial membrane potential and cytosolic Ca2+ levels, as well as reduced respiratory function. Performing the rescue experiment by lentiviral overexpression of TSPO in knockout cells, increased oxygen consumption and restored respiratory function. Our study provides further evidence for a significant role of TSPO in cellular and mitochondrial metabolism and demonstrates that different phenotypes of mitochondrial function are dependent on the level of TSPO expression.
| Item Type: | Article |
|---|---|
| Uncontrolled Keywords: | PROTEIN 18 KDA; TRANSLOCATOR PROTEIN; BENZODIAZEPINE-RECEPTOR; THERAPEUTIC TARGET; MITOCHONDRIA; MACROPHAGES; EXPRESSION; MUTATIONS; MEMBRANE; LIGANDS; TSPO; mitochondria; knockdown; knockout; mitochondrial membrane potential; Ca2+ homeostasis; oxidative phosphorylation; steroid synthesis |
| Subjects: | 600 Technology > 610 Medical sciences Medicine |
| Divisions: | Medicine > Lehrstuhl für Psychiatrie und Psychotherapie Medicine > Lehrstuhl für Psychiatrie und Psychotherapie > Molekulare Neurowissenscahften |
| Depositing User: | Dr. Gernot Deinzer |
| Date Deposited: | 03 Apr 2020 12:42 |
| Last Modified: | 03 Apr 2020 12:42 |
| URI: | https://pred.uni-regensburg.de/id/eprint/26725 |
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