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

Vladimir M Milenkovic,Stefanie Bader,Elena-Sofia Heinl,David Alvarez-Carbonell,Dounia Slim,Rainer Rupprecht,Caroline Nothdurfter,Christian H Wetzel,Victoria Koch

doi:10.3390/ijms20133359

Vladimir M Milenkovic, Stefanie Bader + Show 7 more

Open Access

https://doi.org/10.3390/ijms20133359

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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.

Highlights

The translocator protein 18 kDa (TSPO) is a highly conserved multifunctional protein residing in the outer mitochondrial membrane [1]
Different guide RNAs against the translocator protein kDa (TSPO) gene were designed (Supplementary Table S1). gRNAs were transfected into human microglial C20 cells and CRISPR/Cas9-mediated knockout of TSPO was validated by Western Blot and DNA sequencing of PCR products
We considered both the C20 TSPO knockdown as well as the TSPO knockout cell lines as a viable and promising tool to study the role of TSPO in human microglia cells

Summary

Introduction

The translocator protein 18 kDa (TSPO) is a highly conserved multifunctional protein residing in the outer mitochondrial membrane [1]. TSPO can be detected predominantly in microglia and reactive astrocytes [2], where the expression is upregulated in the context of inflammation, neurodegeneration, and malignant neoplasia. In this regard, TSPO is considered as both a diagnostic biomarker, as well as a therapeutic target [3] within the promising field of theranostics [4]. Beside its suggested role on cholesterol transport and steroid synthesis [8,10,11], TSPO has been shown to be involved in the regulation of various cellular and mitochondrial functions. TSPO is involved in regulating cellular downstream processes such as proliferation, survival, and apoptosis [3,16]

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