The 18 kDa translocator protein (TSPO): a new perspective in mitochondrial biology

In mammals, mitochondria are central in maintaining normal cell function and dissecting the pathways that govern their physiology and pathology is therefore of utmost importance. For a long time, the science world has acknowledged the Translocator Protein (TSPO), an intriguing molecule that, through its position and association with biological processes, stands as one of the hidden regulatory pathways in mitochondrial homeostasis. Here we aim to review the literature and highlight what links TSPO to mitochondrial homeostasis in order to delineate its contribution in the functioning and malfunctioning of this critical organelle. In detail, we will discuss: 1) TSPO localization and interplay with controlling phenomena of mitochondria (e.g. mPTP); 2) TSPO interaction with the prominent mitochondrial player VDAC; consider evidence on how TSPO relates to 3) mitochondrial energy production; 4) Ca2+ signalling and 5) the generation of Reactive Oxygen Species (ROS) before finally describing 6) its part in apoptotic cell death. In essence, we hope to demonstrate the intimate involvement TSPO has in the regulation of mitochondrial homeostasis and muster attention towards this molecule, which is equally central for both cellular and mitochondrial biology.     

The role of 18 kDa mitochondrial translocator protein (TSPO) in programmed cell death, and effects of steroids on TSPO expression

The mitochondrial 18 kDa Translocator Protein (TSPO) was first detected by its capability to bind benzodiazepines in peripheral tissues and later also in glial cells in the brain, hence its previous most common name peripheral benzodiazepine receptor (PBR). TSPO has been implicated in various functions, including apoptosis and steroidogenesis, among others. Various endogenous TSPO ligands have been proposed, for example: Diazepam Binding Inhibitor (DBI), triakontatetraneuropeptide (TTN), phospholipase A2 (PLA2), and protoporphyrin IX. However, the functional implications of interactions between the TSPO and its putative endogenous ligands still have to be firmly established. The TSPO has been suggested to interact with a mitochondrial protein complex, summarized as mitochondrial membrane permeability transition pore (MPTP), which is considered to regulate the mitochondrial membrane potential (ΔΨm). In addition, the TSPO is associated with several other proteins. The associations of the TSPO with these various proteins at the mitochondrial membranes have been attributed to functions such as apoptosis, steroidogenesis, phosphorylation, reactive oxygen species (ROS) generation, ATP production, and collapse of the ΔΨm. Interestingly, while TSPO is known to play a role in the modulation of steroid production, in turn, steroids are also known to affect TSPO expression. As with the putative endogenous TSPO ligands, the effects of steroids on TSPO functions still have to be established. In any case, steroid-TSPO interactions occur in organs and tissues as diverse as the reproductive system, kidney, and brain. In general, the steroid-TSPO interactions are thought to be part of stress responses, but may also be essential for reproductive events, embryonic development, and responses to injury, including brain injury. The present review focuses on the role of TSPO in cell death i.e. the notion that enhanced expression and/or activation of the TSPO leads to cell death, and the potential of steroids to regulate TSPO expression and activation.     

Evidence for complex binding profiles and species differences at the translocator protein (TSPO) (18 kDa)

The translocator protein (TSPO) (18 kDa) is an emerging drug target for the treatment of numerous pathologies including cancer and neurodegenerative disease. However, our limited knowledge of TSPO binding site(s) has hindered the development of TSPO ligands with potential therapeutic effects. We have synthesized a series of pyrrolobenzoxazepines (1-10) to better characterize the interaction of ligands with the TSPO across species, and to determine their functional profiles. All ligands 1-10 displaced the binding of [3H]PK 11195 to the TSPO at nanomolar concentrations, with discrepancies in binding affinity between rat and human TSPO. Interestingly, non-linear regression analysis revealed that some ligands bound to the protein with a Hill slope not equal to 1.0, suggesting possible additional TSPO binding sites with allosteric effects. However, this trend was not conserved between rat and human. When tested for their effects on pregnenolone production in rat C6 glioma cells, nitric oxide release in murine microglia, and cell proliferation in human MCF-7 breast cancer cells, the pyrrolobenzoxazepines (40 μM) displayed functional effects which did not correlate to the binding trend observed in competition assays. We propose that consideration of species differences and binding site cooperativity, plus optimization of currently accepted functional assays, will aid in the development of drugs targeting TSPO that can be used as therapeutics for human disease.     

Design, synthesis and structure-activity relationships of novel benzoxazolone derivatives as 18kDa translocator protein (TSPO) ligands

Selective 18kDa translocator protein (TSPO) ligands are expected to be therapeutic agents with a wide spectrum of action on psychiatric disorders and fewer side effects. We designed novel benzoxazolone derivatives and examined the structure-activity relationship (SAR) of a series of compounds with various substituents at the amide part and C-5 position. Although a number of the synthesized compounds showed high TSPO binding affinity, these compounds had poor drug-like properties. Further optimization of pharmacokinetic properties of these compounds led to discovery of compound 74, which exhibited anxiolytic effect in the rat Vogel conflict model.     

Structural and functional evolution of the translocator protein (18 kDa)

Translocator proteins (TSPO) are the products of a family of genes that is evolutionarily conserved from bacteria to humans and expressed in most mammalian tissues and cells. Human TSPO (18 kDa) is expressed at high levels in steroid synthesizing endocrine tissues where it localizes to mitochondria and functions in the first step of steroid formation, the transport of cholesterol into the mitochondria. TSPO expression is elevated in cancerous tissues and during tissue injury, which has lead to the hypothesis that TSPO has roles in apoptosis and the maintenance of mitochondrial integrity. We recently identified a new paralog of Tspo in both the human and mouse. This paralog arose from an ancient gene duplication event before the divergence of the classes aves and mammals, and appears to have specialized tissue-, cell-, and organelle-specific functions. Evidence from the study of TSPO homologs in mammals, bacteria, and plants supports the conclusion that the TSPO family of proteins regulates specialized functions related to oxygen-mediated metabolism. In this review, we provide a comprehensive overview of the divergent function and evolutionary origin of Tspo genes in Bacteria, Archaea, and Eukarya domains.     

The 18 kDa mitochondrial translocator protein (TSPO) prevents accumulation of protoporphyrin IX. Involvement of reactive oxygen species (ROS)

By exposing cells of the U118MG glioblastoma cell line to protoporphyrin IX (PPIX) in culture, we found that the 18 kDa mitochondrial translocator protein (TSPO) prevents intracellular accumulation of PPIX. In particular, TSPO knockdown by stable transfection of TSPO silencing siRNA vectors into U118MG cells leads to mitochondrial PPIX accumulation. In combination with light exposure, the PPIX accumulation led to cell death of the TSPO knockdown cells. In the sham control cells (stable transfection of scrambled siRNA vectors), TSPO expression remained high and no PPIX accumulation was observed. The prevention of PPIX accumulation by TSPO was not due to conversion of PPIX to heme in the sham control cells. Similar to TSPO knockdown, the reactive oxygen species (ROS) scavenger glutathione (GSH) also enhanced PPIX accumulation. This suggests that that ROS generation as modulated by TSPO activation may present a mechanism to prevent accumulation of PPIX.     

Pyrazolo[1,5-a]pyrimidine acetamides: 4-Phenyl alkyl ether derivatives as potent ligands for the 18 kDa translocator protein (TSPO)

Herein, we report the synthesis of four new phenyl alkyl ether derivatives (7, 9–11) of the pyrazolo[1,5-a]pyrimidine acetamide class, all of which showed high binding affinity and selectivity for the TSPO and, in the case of the propyl, propargyl, and butyl ether derivatives, the ability to increase pregnenolone biosynthesis by 80–175% over baseline in rat C6 glioma cells. While these compounds fit our in silico generated pharmacophore for TSPO binding the current model does not account for the observed functional activity.     

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands

Background information. TSPO (translocator protein), previously known as PBR (peripheral‐type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High‐affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. Results. Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam‐binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, ³H‐labelled PK 11195, as shown by B_max and K_d values of 10.0±0.5 pmol/mg and 4.0±1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and α‐adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K⁺, Na⁺, Cl⁻ and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. Conclusions. High‐affinity ligand binding to mitochondrial TSPO modulates neurotransmitter‐induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.     

The 18 kDa translocator protein (peripheral benzodiazepine receptor) expression in the bone of normal, osteoprotegerin or low calcium diet treated mice

The presence of the translocator protein (TSPO), previously named as the mitochondrial or peripheral benzodiazepine receptor, in bone cells was studied in vitro and in situ using RT-qPCR, and receptor autoradiography using the selective TSPO ligand PK11195.In vitro, the TSPO is highly expressed in osteoblastic and osteoclastic cells.In situ, constitutive expression of TSPO is found in bone marrow and trabecular bone, e.g., spongiosa. Mice with a reduction of bone turnover induced by a 4-day treatment of osteoprotegerin reduces [(3)H]PK11195 binding in the spongiosa (320±128 Bq x mg(-1), 499±106 Bq x mg(-1) in saline-treated controls). In contrast, mice with an increase in bone turnover caused by a 4-day low calcium diet increases [(3)H]PK11195 binding in the spongiosa (615±90 Bq x mg(-1)). Further, our study includes technical feasibility data on [(18)F]fluoride microPET imaging of rodent bone with altered turnover. Despite [(18)F]fluoride having high uptake, the in vivo signal differences were small. Using a phantom model, we describe the spillover effect and partial volume loss that affect the quantitative microPET imaging of the small bone structures in experimental mouse models. In summary, we demonstrate the expression of TSPO in small rodent bone tissues, including osteoblasts and osteoclasts. A trend increase in TSPO expression was observed in the spongiosa from low to high bone turnover conditions. However, despite the potential utility of TSPO expression as an in vivo biomarker of bone turnover in experimental rodent models, our small animal PET imaging data using [(18)F]fluoride show that even under the condition of a good biological signal-to-noise ratio and high tracer uptake, the currently achievable instrument sensitivity and spatial resolution is unlikely to be sufficient to detect subtle differences in small structures, such as mouse bone.     

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands.

BACKGROUND INFORMATION: TSPO (translocator protein), previously known as PBR (peripheral-type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High-affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium-dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. RESULTS: Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam-binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, (3)H-labelled PK 11195, as shown by B(max) and K(d) values of 10.0+/-0.5 pmol/mg and 4.0+/-1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and alpha-adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K(+), Na(+), Cl(-) and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. CONCLUSIONS: High-affinity ligand binding to mitochondrial TSPO modulates neurotransmitter-induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.     

Distribution,pharmacological characterization and function of the 18 kDa translocator protein in rat small intestine

Background information. The TSPO (18 kDa translocator protein) is a mitochondrial transmembrane protein involved in cholesterol transport in organs that synthesize steroids and bile salts. Different natural and synthetic high‐affinity TSPO ligands have been characterized through their ability to stimulate cholesterol transport, but also to stimulate other physiological functions including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. Here, we investigate the localization and functions of TSPO in the small intestine. Results. TSPO was present in enterocyte mitochondria but not in rat intestinal goblet cells. Enterocyte cytoplasm also contained the endogenous TSPO ligand, polypeptide DBI (diazepam‐binding inhibitor). Whereas intestinal TSPO had high affinity for the synthetic ligand PK 11195, the pharmacological profile of TSPO in the duodenum was distinct from the jejunum and ileum. Specifically, benzodiazepine Ro5‐4864 and protoporphyrin IX showed 5–13‐fold lower affinity for duodenal TSPO. The mRNA and protein ratios of TSPO to other mitochondrial membrane proteins VDAC (voltage‐dependent anion channel) and ANT (adenine nucleotide transporter) were significantly different. PK 11195 stimulated calcium‐dependent chloride secretion in the duodenum and calcium‐dependent chloride absorption in the ileum, but did not affect jejunum ion transport. Conclusions. The functional differences in subpopulations of TSPO in different regions of the intestine could be related to structural organization of mitochondrial protein complexes that mediate the ability of TSPO to modulate either chloride secretion or absorption in the duodenum and ileum respectively.     

Mitochondrial translocator protein (TSPO) ligands prevent doxorubicin-induced mechanical dysfunction and cell death in isolated cardiomyocytes

Contractile dysfunction and subsequent development of cardiomyopathies are well known limiting factors in the treatment of cancer with doxorubicin and have been linked to mitochondrial dysfunction. Here, using adult isolated paced cardiomyocytes, we have demonstrated that ligands of translocator protein (TSPO) 4′-chlorodiazepam and TRO40303 prevented the doxorubicin-induced alterations in contractility and improved cardiomyocyte viability. This cardioprotective effect was closely associated with both a potent reduction in reactive oxygen species production and inhibition of mitochondrial permeability transition pore opening. Thus, preventive administration of TSPO ligands may represent a novel pharmacological strategy to protect the heart during doxorubicin treatment.     

Translocator Protein 18 kDa (TSPO) Is Regulated in White and Brown Adipose Tissue by Obesity

Translocator protein 18 kDa (TSPO) is an outer-mitochondrial membrane transporter which has many functions including participation in the mitochondrial permeability transition pore, regulation of reactive oxygen species (ROS), production of cellular energy, and is the rate-limiting step in the uptake of cholesterol. TSPO expression is dysregulated during disease pathologies involving changes in tissue energy demands such as cancer, and is up-regulated in activated macrophages during the inflammatory response. Obesity is associated with decreased energy expenditure, mitochondrial dysfunction, and chronic low-grade inflammation which collectively contribute to the development of the Metabolic Syndrome. Therefore, we hypothesized that dysregulation of TSPO in adipose tissue may be a feature of disease pathology in obesity. Radioligand binding studies revealed a significant reduction in TSPO ligand binding sites in mitochondrial extracts from both white (WAT) and brown adipose tissue (BAT) in mouse models of obesity (diet-induced and genetic) compared to control animals. We also confirmed a reduction in TSPO gene expression in whole tissue extracts from WAT and BAT. Immunohistochemistry in WAT confirmed TSPO expression in adipocytes but also revealed high-levels of TSPO expression in WAT macrophages in obese animals. No changes in TSPO expression were observed in WAT or BAT after a 17 hour fast or 4 hour cold exposure. Treatment of mice with the TSPO ligand PK11195 resulted in regulation of metabolic genes in WAT. Together, these results suggest a potential role for TSPO in mediating adipose tissue homeostasis.     

In vitro catabolic effect of protoporphyrin IX in human osteoblast-like cells: possible role of the 18 kDa mitochondrial translocator protein

In several pathological conditions, when conversion of Protoporphyrin (PP)IX into heme is impaired, a toxic accumulation of PPIX might occur. PPIX has been found to have affinity to the mitochondrial Translocator Protein 18 kDa. Since it is known that TSPO is abundant in human osteoblast cells, thus we assumed that PPIX can affect cellular functions via interactions with TSPO in these cells. Therefore we aimed to study the metabolic responses of human osteoblast to a high (10^?5M) concentration of PPIX in vitro. We found that in primary culture of human osteoblast-like cells cell numbers decreased following exposure to PPIX(10^?5M). Cellular [¹⁸F]-FDG incorporation, mitochondrial mass, ATP content were suppressed, and ΔΨm collapsed. Lactate dehydrogenase activity was enhanced in culture media, indicating overall cell death, while no increase in apoptotic levels was observed. Cellular proliferation was not affected. Protein expression of TSPO, VDAC 1, and hexokinase 2 decreased, although the synthesis of mRNA for hexokinase 2 increased. Thus, PPIX(10^?5M) has a cytotoxic effect on human osteoblast-like cell in vitro. Since these cells remain viable following exposure to another TSPO ligand, PK 11195 (10^?5M), as observed previously by us, the mode of action of PPIX on osteoblast-like cells is not identical to that of PK 11195. Accordingly pathological accumulation of PPIX may cause necrosis of osteoblasts leading to bone mass loss. We show that this phenomenon is unrelated to iron overload.