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CRISPR-Cas9 Mediated TSPO Gene Knockout Alters Respiration and Cellular Metabolism in Human Primary Microglia Cells

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CRISPR-Cas9 Mediated TSPO Gene Knockout Alters Respiration and Cellular Metabolism in Human Primary Microglia Cells

Vladimir M Milenkovic et al. Int J Mol Sci.

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.

Keywords: Ca2+ homeostasis; TSPO; knockdown; knockout; mitochondria; mitochondrial membrane potential; oxidative phosphorylation; steroid synthesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Translocator protein 18 kDa (TSPO) gene depletion and deletion in C20 human microglia cells. (A) TSPO gene lentiviral knockdown resulted in robust reduction of TSPO protein levels. Beta 1 tubulin was used as loading control. (B) Reduced levels of TSPO protein in TSPO knockdown (KD) cells in comparison to TSPO scramble (SCR) control were also evident in immunofluorescent co-staining using ATPB antibody. (C) Schematic diagram showing CRISPR/Cas9 guide RNA targeting sites on the exon 2 of the TSPO gene. Guide DNA targeting sites are highlighted in cyan and yellow, and start codon of TSPO gene in blue. Successful TSPO knockout in both alleles using CRISPR/Cas9 system was confirmed by DNA sequencing. (D) Western blot analysis of TSPO in C20 wt and KO1 and KO2 knockout cells reveal complete lack of TSPO expression. (E) TSPO gene deletion was further confirmed with TSPO antibody co-staining using ATPB as mitochondrial marker. Scale bars: 20 µm.
Figure 2
Figure 2
Pregnenolone synthesis in C20 and H295R cells. (A) There were no significant changes in basal pregnenolone production between C20 wt and TSPO knock down or knockout cells (-tri., without trilostane; n.s., no significance). (B) Pregnenolone synthesis in H295R steroidogenic cells, which was over 40 times higher in comparison to C20 cells under basal conditions, could be additionally upregulated in response to cAMP treatment (p < 0.001). (C) Treatment with various TSPO ligands had no effect on pregnenolone production in C20 microglia cells. (D) Basal pregnenolone production in C20 wt and KO cells was furthermore not affected by cAMP treatment.
Figure 3
Figure 3
Effects of TSPO gene deletion on mitochondrial membrane potential and intracellular calcium. (A) TSPO gene deletion resulted in significant reduction of mitochondrial membrane potential in KO cells (p < 0.001) Treating the cells (C20 wildtype) with the uncoupling agent FCCP (20 µM) reduced the fluorescence ratio to about 50%, indicating the dynamic range of the JC-1 assay. (B) JC-1 staining was not changed after lentiviral TSPO knockdown. (C) TSPO knockout cells show significantly higher cytosolic Ca2+ levels (p < 0.001), suggesting an important role of TSPO in regulating Ca2+ homeostasis. (D) Deletion of TSPO gene leads to decreased expression of VDAC1 (voltage-dependent anion channel) protein in knockout cells (p < 0.01). (E) Reduction of TSPO protein level in knockdown C20 cells was not sufficient to alter the level of intracellular calcium.
Figure 4
Figure 4
TSPO gene knockout using CRISPR/Cas9 system decreases respiration in human microglia cells. (A) Knockdown of TSPO did not alter respiration in C20 microglia cells (B) Oxygen consumption rate (OCR) was significantly reduced in several stages of respiration in TSPO KO cells (p < 0.001). (C) Basal respiration, maximal respiration, and ATP-related oxygen consumption were significantly decreased in TSPO knockout cells (p < 0.001). (D) Overexpression of TSPO in knockout cells resulted in increased respiration which did not differ from wt cells. (E) Basal respiration, maximal respiration, and ATP-related oxygen consumption which were reduced in KO cells, reached similar levels in TSPO overexpressing knockout cells.

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