Year: 2018
Type: letter
Abstract: In two previous publications, we demonstrated that CRISPR/Cas9‒mediated Tspo deletion in MA-10 Leydig cells had no effect on steroidogenesis (1) but instead altered mitochondrial fatty acid oxidation without affecting mitochondrial membrane potential (Δψm) and proton leak (2). In contrast, a recent article by Fan et al. (3) reports decreases in both steroidogenic function and Δψm after Tspo disruption in the same MA-10 cells. This study lacks appropriate controls and leads to erroneous conclusions. Specifically, the authors do not demonstrate that they could rescue the observed loss of function by reintroducing TSPO into their putative knockout/mutated clones. This is a necessary burden of proof when presenting contradictory science. Without this rigor, all findings presented in this manuscript could be due to aberrant subclones with changed characteristics from a long-established cell line. We also bring to the readers’ attention that Fan et al. (3) inexplicably neglected to cite or discuss previous work on Δψm and other mitochondrial properties in TSPO knockout MA-10 cells (2). We and others have found that change in Δψm was not a consistent phenotype after TSPO deletion in different cell types [MA-10 cells, fibroblasts, hepatocytes, and microglia; discussed in Tu et al. (2)] because mitochondrial composition and the nature/extent of function differ. Previous results do not show any ultrastructural changes to mitochondria after TSPO deletion [see Tu et al. (4)]. Results in Fan et al. (3) are inconsistent with—and disregard—the current literature. To provide context, in 2013 we reported the generation of viable Tspo knockout mice that had no defects in mitochondrial cholesterol import for steroidogenesis; this was subsequently reproduced in three independent laboratories (5–7). From that time, reproducibility of results that supported the TSPO-steroidogenesis model came into question, and recent in vitro results determined that the fundamental premise underlying a mitochondrial cholesterol import function for TSPO is baseless [reviewed in Selvaraj and Stocco (8), Selvaraj et al. (9), Selvaraj and Tu (10), and Stocco et al.(11)]. This article by Fan et al. (3) follows another effort to repudiate the aforementioned in vivo Tspo knockout studies by this same research group, which we uncovered as a serious misinterpretation of mouse genetics (12). It must be noted that aside from pharmacology, all positive results specific to TSPO involvement in mitochondrial cholesterol import were reported only by this research group [reviewed in Selvaraj and Stocco (8), Selvaraj et al. (9), Selvaraj and Tu (10), and Stocco et al.(11)]. For future drug discovery, it is necessary that Tspo knockout models be used for rigorous target validation of TSPO “ligand” effects. In MA-10 cells, we found that the drug-induced steroidogenic effect was TSPO independent (1). Moving forward, we believe that efforts need to be directed at deciphering the true function of TSPO, one that is consistent and relevant across different cell types and not only in steroidogenic cells. High TSPO expression is not exclusive to steroidogenic cells; we have demonstrated similar or higher expression in brown and white adipose tissue (2). The current TSPO impasse forces us to reiterate the facts, as this topic affects a multidisciplinary scientific community and needs coherence. For readers interested in learning more, we have dissected this topic in reviews that address all TSPO findings point by point (9, 10). mitochondrial membrane potential. Financial Support: Studies by the authors referenced in this review were performed with the financial support of startup funds from Cornell University and National Institutes of Health Grant DK110059 (to V.S.) and National Institutes of Health Grant HD17481 and Robert A. Welch Foundation Grant B1-0028 (to D.M.S.). Disclosure Summary: The authors have nothing to disclose.
Source: Endocrinology
Authors: Vimal Selvaraj, Douglas M Stocco
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: bronze
OpenAlex ID: W2801827251