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The translocator protein (TSPO; 18k Da) is an evolutionarily conserved outer mitochondrial membrane (OMM) protein highly expressed in steroid-synthesizing cells and found to possess a number of physiological and drug-binding partners. Extensive pharmacological, biochemical and cell biological research over the years has led to a model of TSPO involvement in mitochondrial cholesterol transport and promotion of steroid synthesis, a model guiding the design of drugs useful in stimulating neurosteroid synthesis and alleviating psychopathological symptoms. The involvement of TSPO in these processes has been called into question; however, with the publication of TSPO-deletion mouse models which saw no changes in steroid production. Here, we review work characterizing TSPO in steroidogenesis and offer perspective to research into TSPO pharmacology and its involvement in steroid biosynthesis.

Steroidogenesis begins with cholesterol transfer into mitochondria through the transduceosome, a complex composed of cytosolic proteins that include steroidogenesis acute regulatory protein (STAR), 14-3-3 adaptor proteins, and the outer mitochondrial membrane proteins Translocator Protein (TSPO) and Voltage-Dependent Anion Channel (VDAC). TSPO is a drug- and cholesterol- binding protein found at particularly high levels in steroid synthesizing cells. Its aberrant expression has been linked to cancer, neurodegeneration, neuropsychiatric disorders and primary hypogonadism. Brain steroids serve as local regulators of neural development and excitability. Reduced levels of these steroids have been linked to depression, anxiety and neurodegeneration. Reduced serum testosterone is common among subfertile young men and aging men, and is associated with depression, metabolic syndrome and reduced sexual function. Although testosterone-replacement therapy is available, there are undesired side-effects. TSPO drug ligands have been proposed as therapeutic agents to regulate steroid levels in the brain and testis.

Inhibition of GSK-3β has been well documented to account for the behavioral actions of the mood stabilizer lithium in various animal models of mood disorders. Recent studies have showed that genetic or pharmacological inhibition of GSK-3β resulted in anxiolytic-like and pro-social behavior. In our ongoing efforts to develop GSK-3β inhibitors for the treatment of mood disorders, SAR studies on maleimide-based compounds were undertaken. We present herein for the first time that some of these GSK-3β inhibitors, in particular analogs 1 and 9, were able to stimulate progesterone production in the MA-10 mouse tumor Leydig cell model of steroidogenesis without any significant toxicity. These two compounds were tested in the SmartCube® behavioral assay and showed anxiolytic-like signatures following daily dose administration (50 mg/kg, i.p.) for 13 days. Taken together, these results support the hypothesis that GSK-3β inhibition could influence neuroactive steroid production thereby mediating the modulation of anxiety-like behavior in vivo.

Translocator protein (TSPO; 18 kDA) is a high-affinity cholesterol-binding protein that is integrally involved in cholesterol transfer from intracellular stores into mitochondria, the rate-determining step in steroid formation. Previous studies have shown that TSPO drug ligands are able to activate steroid production by MA-10 mouse Leydig tumor cells and by mitochondria isolated from steroidogenic cells. We hypothesized herein that the direct, pharmacological activation of TSPO might induce aged Leydig cells, which are characterized by reduced T production, to produce significantly higher levels of T both in vitro and in vivo. To test this, we first examined the in vitro effects of the TSPO selective and structurally distinct drug ligands N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27) and benzodiazepine 4′-chlorodiazepam (Ro5-4864) on steroidogenesis by Leydig cells isolated from aged (21-24 months old) and young adult (3-6 months old) Brown Norway rats. The ligands stimulated Leydig cell T production significantly, and equivalently, in cells of both ages, an effect that was significantly inhibited by the specific TSPO inhibitor 5-androsten-3,17,19-triol (19-Atriol). Additionally, we examined the in vivo effects of administering FGIN-1-27 to young and aged rats. In both cases, serum T levels increased significantly, consistent with the in vitro results. Indeed, serum T levels in aged rats administered FGIN-1-27 were equivalent to T levels in the serum of control young rats. Taken together, these results indicate that although there are reduced amounts of TSPO in aged Leydig cells, its direct activation is able to increase T production. We suggest that this approach might serve as a therapeutic means to increase steroid levels in vivo in cases of primary hypogonadism.

Steroids metabolically derive from lipid cholesterol, and vertebrate steroids additionally derive from the steroid pregnenolone. Pregnenolone is derived from cholesterol by hydrolytic cleavage of the aliphatic tail by mitochondrial cytochrome P450 enzyme CYP11A1, located in the inner mitochondrial membrane. Delivery of cholesterol to CYP11A1 comprises the principal control step of steroidogenesis, and requires a series of proteins spanning the mitochondrial double membranes. A critical member of this cholesterol translocation machinery is the integral outer mitochondrial membrane translocator protein (18kDa, TSPO), a high-affinity drug- and cholesterol-binding protein. The cholesterol-binding site of TSPO consists of a phylogenetically conserved cholesterol recognition/interaction amino acid consensus (CRAC). Previous studies from our group identified 5-androsten-3β,17,19-triol (19-Atriol) as drug ligand for the TSPO CRAC motif inhibiting cholesterol binding to CRAC domain and steroidogenesis. To further understand 19-Atriol's mechanism of action as well as the molecular recognition by the TSPO CRAC motif, we undertook structure-activity relationship (SAR) analysis of the 19-Atriol molecule with a variety of substituted steroids oxygenated at positions around the steroid backbone. We found that in addition to steroids hydroxylated at carbon C19, hydroxylations at C4, C7, and C11 contributed to inhibition of cAMP-mediated steroidogenesis in a minimal steroidogenic cell model. However, only substituted steroids with C19 hydroxylations exhibited specificity to TSPO, its CRAC motif, and mitochondrial cholesterol transport, as the C4, C7, and C11 hydroxylated steroids inhibited the metabolic transformation of cholesterol by CYP11A1. We thus provide new insights into structure-activity relationships of steroids inhibiting mitochondrial cholesterol transport and steroidogenic cholesterol metabolic enzymes.

Previous studies in MA-10 tumor Leydig cells demonstrated that disruption of the mitochondrial electron-transport chain (ETC), membrane potential (ΔΨ(m)), or ATP synthesis independently inhibited steroidogenesis. In contrast, studies of primary Leydig cells indicated that the ETC, ΔΨ(m), and ATP synthesis cooperatively affected steroidogenesis. These results suggest significant differences between the two systems and call into question the extent to which results from tumor Leydig cells relate to primary cells. Thus, to further understand the similarities and differences between the two systems as well as the impact of ATP disruption on steroidogenesis, we performed comparative studies of MA-10 and primary Leydig cells under similar conditions of mitochondrial disruption. We show that mitochondrial ATP synthesis is critical for steroidogenesis in both primary and tumor Leydig cells. However, in striking contrast to primary cells, perturbation of ΔΨ(m) in MA-10 cells did not substantially decrease cellular ATP content, a perplexing finding because ΔΨ(m) powers the mitochondrial ATP synthase. Further studies revealed that a significant proportion of cellular ATP in MA-10 cells derives from glycolysis. In contrast, primary cells appear to be almost completely dependent on mitochondrial respiration for their energy provision. Inhibitor studies also suggested that the MA-10 ETC is impaired. This work underscores the importance of mitochondrial ATP for hormone-stimulated steroid production in both MA-10 and primary Leydig cells while indicating that caution must be exercised in extrapolating data from tumor cells to primary tissue.

The principal site of regulation of steroid hormone biosynthesis is the transfer of cholesterol from the outer to inner mitochondrial membrane. Hormonal stimulation of steroidogenic cells promotes this mitochondrial lipid import through a multi-protein complex, termed the transduceosome, spanning the two membranes. The transduceosome complex is assembled from multiple proteins, such as the steroidogenic acute regulatory (STAR) protein and translocator protein (TSPO), and requires their targeting to the mitochondria for transduceosome function. The vast majority of mitochondrial proteins, including those participating in cholesterol import, are encoded in the nucleus. Their subsequent mitochondrial incorporation is performed through a series of protein import machineries located in the outer and inner mitochondrial membranes. Here we review our current knowledge of the mitochondrial cholesterol import machinery of the transduceosome. This is complemented with descriptions of mitochondrial protein import machineries and mechanisms by which these machineries assemble the transduceosome in steroidogenic mitochondria.

Studies of MA-10 Leydig cells have shown that intact mitochondria with active respiration are essential for LH-induced Leydig cell steroidogenesis. To further elucidate the role played by mitochondria in steroidogenesis, we examined the effects of the perturbation of the mitochondrial electron transport chain with myxothiazol (MYX) on testosterone production by primary cultures of Brown Norway rat Leydig cells. Analysis of the steroidogenic pathway revealed that cAMP production and the activities of each of 3β-hydroxysteroid dehydrogenase, 17α-hydroxylase/C17–20 lyase, and 17β-hydroxysteroid dehydrogenase were inhibited by MYX and that LH-stimulated testosterone production was suppressed. In contrast to the inhibition of LH-stimulated testosterone production by MYX, the incubation of Leydig cells with MYX in the absence of LH stimulated testosterone production. Although testosterone production was increased, steroidogenic acute regulatory protein was decreased in response to MYX, not increased as could be expected. Additional electron transport chain inhibitors had stimulatory effects on testosterone production that were similar to those of MYX, strongly suggesting that the effect of MYX on basal testosterone production is related to its effect on the mitochondrial electron transport chain. Finally, incubation of the cells with a combination of MYX and the calcium chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetracetic acid tetrakis acetoxymethyl ester suppressed MYX-mediated increased basal steroidogenesis but had no effect on hydroxycholesterol-mediated steroidogenesis. Taken together, these results indicate that inhibition of the mitochondrial electron transport chain can block LH-stimulated testosterone production through suppression of a number of steps of the steroidogenic pathway but also stimulates basal testosterone production through a calcium-mediated mechanism.