Response to Letter to the Editor: “Dubious Conclusions on TSPO Function”

As they did in response to our recent studies of Tspo conditional knockout mice, which provided evidence that TSPO plays an important role in steroidogenesis (1), Selvaraj and Stocco (2) commented negatively on our studies of CRISPR/Cas9-mediated Tspo mutant cell lines (3). That our results differ from theirs, however, does not make ours, or theirs, “dubious.” Selvaraj and Stocco reported in Tu et al. (4) that CRISPR/Cas9-mediated Tspo deletion had no impact on steroidogenesis in MA-10 cells. In contrast, we reported decreases in both steroidogenic function and mitochondrial membrane potential (Δψm) in two Tspo mutant cell lines (3). Selvaraj and Stocco (2) commented that our study “lacks appropriate controls and leads to erroneous conclusions.” We used the original wild-type (WT) MA-10 cells, and the selected WT cells as experimental controls so that there would be internal controls that went through the same selection process as the Tspo mutants (3). This was not done by Selvaraj and Stocco, perhaps leading to their negative results (4). We also performed transient (overnight) expression of the relevant plasmids to avoid any nonspecific plasmid genome integration (3). Results were obtained from positively selected cells from the CRISPR/Cas9-transfected cell pools. Selvaraj and Stocco used the original MA-10 cells as controls and apparently missed an appropriate control for their Tspo mutant cells with a WT genotype under the same condition(s) (4). Had they looked critically at their own studies and claims, they might have avoided their failure to detect TSPO in H295R adrenal cells, a finding that became central to their dismissal of the role of TSPO in steroidogenesis (5) but one that was disproved by three independent laboratories, including our own (6–8). We do appreciate the comment on the desirability of rescue experiments in knockout studies. There is no widely accepted approach by which to do this, but it now should be technically feasible to use CRISPR/Cas9-mediated correction of a genome mutation. The criticism that we “neglected to cite or discuss” previous work by Selvaraj and Stocco is not true (2). In our article, we presented and discussed seven of their most relevant studies as well as conflicting data from other laboratories (3). We chose to not refer to the 2016 Tu et al. (9) article. It was not our intent to “disregard current literature” in doing so (3). The article in question is based on cell lines sharing the same responses to PK 11195. Thus, any further reference would be meaningless. It should be pointed out that the results in this article are in contrast with the authors’ own previous study on the issue of Δψm (10). In these studies, the authors failed to explain how it is possible that TSPO deficiency in poor-in-TSPO fibroblasts had a dramatic effect on mitochondrial function (10) but that TSPO deficiency in rich-in-TSPO steroidogenic cells did not affect mitochondrial function (9). We were unable to understand and could not explain these results. Consequently, we were unable to relate the results presented in Tu et al. (9) to ours or to other studies in the literature. It also is not true that “the fundamental premise underlying a mitochondrial import function for TSPO is baseless” or that “all positive results specific to TSPO involvement in mitochondrial cholesterol import,” and thus steroid formation, “were reported only by [the Papadopoulos] group.” Surely, Selvaraj and Stocco know that in addition to Papadopoulos’s work, two independent laboratories provided evidence for the role of TSPO in cholesterol import for steroidogenesis (11, 12) and that another laboratory showed the use of lipoprotein-bound cholesterol in TSPO-mediated steroid formation (13). In addition, work from our laboratory and numerous others documented increased steroid production by steroid-synthesizing cells and tissues in response to TSPO drug ligands both in vitro and in vivo (see previously published reviews). We also demonstrated that TSPO has a cholesterol-interacting domain that binds cholesterol. This too has been confirmed by others (14, 15). TSPO has been shown to possess multiple functions likely linked to its mitochondrial location and ability to bind cholesterol. Given these features, TSPO affects mitochondrial membrane fluidity/permeability, membrane protein/transporter functions, and other proteins involved in steroidogenesis leading to TSPO-mediated regulation of Δψm (3). The latter could be a key basic role of TSPO across different cell types. We reported that TSPO-mediated Δψm regulates the mitochondrial import of steroidogenic acute regulatory protein (3) and likely other mitochondrial proteins as reported by other group(s) (16). There is no “TSPO impasse” but rather differences in results that cannot be dismissed by referring to them as “dubious” simply because they are not in agreement with what one wishes the scientific community to believe. wild-type mitochondrial membrane potential Disclosure Summary: The authors have nothing to disclose.

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