Current Molecular Medicine - Volume 12, Issue 4, 2012

The need for a focused issue covering the physiology and pharmacology of the Translocator Protein (TSPO) was in demand for quite some time; this to both summarize the current knowledge on this fascinating molecule and to boost interest in experimental biologists, pharmacologists and clinicians. The leading and most representative experts on the subject have therefore contributed with enthusiasm to its realization thus generating a memorable editorial occasion that besides collecting the exploited features of TSPO also shares original data to inspire future research angles of investigation. TSPO -formerly known as the Peripheral Benzodiazepine Receptor (PBR)- is an ubiquitous 18 kDa molecule on which the synthesis of steroids depends. Located on the outer mitochondrial membrane (OMM), it is critical for the modulation of mitochondrial pathophysiology by standing -and possibly interacting with- the ‘putative’ molecules composing the mitochondrial permeability transition pore.....

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.

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 oxygenmediated 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 translocator protein (TSPO) is a potential drug target for the treatment of CNS diseases, with TSPO ligands being able to modulate steroidogenesis, apoptosis, and cell proliferation. While there exist multiple TSPO binding sites, the nature of these sites – either overlapping or allosterically linked – remains largely uncharacterized. Furthermore, while evidence suggests that microglial activation and polymerization result in changes to TSPO binding sites, these changes are poorly understood. While current pharmacophoric models can be used to synthesize TSPO ligands with high affinity and selectivity, these models are unable to predict ligands with desirable functional effects. Better characterization of TSPO binding sites in health and disease may provide insight into particular sites which mediate promising therapeutic profiles, thus refining the TSPO pharmacophore.

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.

Translocator protein (TSPO), formerly known as peripheral-type benzodiazepine receptor (PBR), has been described in several tissues and characterized as one of the main elements of steroidogenesis. However, TSPO is also involved in other pathways and cell functions, such as apoptosis regulation, protein import, membrane biogenesis, cell cycle regulation, oxygen homeostasis and mitochondrial membrane fluidity regulation. In the kidney, TSPO is normally located in the distal parts of the nephron from the thick ascending limb of the loop of Henle to the medullary collecting ducts. However when the kidney is submitted to a stress such as ischemia reperfusion injury there is a defined change in TSPO expression towards more proximal areas of the nephron, and the protein can be detected as high as proximal tubular cells and the Bowman Capsule. As the injury persists, TSPO is also located in invading mononucleated cells, in a pattern reproducing invasion by CD4+ helper Tcells, and in the damaged vessels where TSPO is expressed both in endothelial and smooth muscle cells. Herein we review the potential use of TSPO-directed treatment for ischemia reperfusion injury, particularly regarding pre-conditioning of the organ. We also detail the relationship of proximal TSPO staining with the intensity of the injury, particularly the implication of monomeric (18 kDa) TSPO and its role in hypoxiareoxygenation and apoptosis prevention. The potential implications of the protein with regeneration processes activated in response to injury and their relation with embryogenesis pathways are discussed.

The translocator protein (TSPO) is a five transmembrane domain protein localised primarily in the outer mitochondrial membrane of steroid-synthesizing tissues, including the brain. The TSPO mediates the rate-limiting step of steroidogenesis, consisting of the translocation of the substrate cholesterol from the outer to the inner mitochondrial membrane. In the recent years TSPO function has received attention in several psychiatric disorders since these diseases have been associated with unbalanced steroid levels. Accordingly, an alteration in the levels of TSPO has been found in various psychiatric disorders, including social phobia, post-traumatic stress disorder, adult separation anxiety and schizophrenia. The discovery that TSPO drug ligands are able to stimulate neurosteroid production in the brain, independently of peripheral endocrine sources, and restore neurosteroid-mediated neurotransmission, has made the TSPO an attractive drug target for treating a number of psychiatric disorders. In anxiety TSPO drug ligands have shown in vivo efficacy in pharmacologically induced anxiety models in both animals and humans. The focus of this review is to illustrate the currently available literature regarding the role of TSPO in psychiatric disorders.

Several molecular and cellular markers are currently used as prognostic indicators for diagnosis and therapeutic intervention of breast cancer. Although some of these markers have helped clinicians provide an earlier diagnosis (or prognosis), they have failed to provide adequate information about the mechanisms responsible for different stages of tumor malignancy so that more effective anticancer therapies can be developed. Recently translocator protein (TSPO), formerly known as the peripheral benzodiazepine receptor (PBR), has received attention as a potential target for anticancer drug development. It is a well-conserved protein, located at outer-inner mitochondrial membrane contact sites, and is expressed in almost all tissues, although the level of expression varies. TSPO is closely associated with the 32 kDa voltage-dependent anion channel (VDAC) and the 30 kDa adenine nucleotide translocase (ANT), considered to form the core of a mitochondria multiprotein complex [named the mitochondrial permeability transition pore (MPTP)] and plays a role in apoptotic cell death. As the major role of TSPO is steroid biosynthesis, TSPO expression is particularly high in organs involved in steroidogenesis such as the adrenals, testes, ovaries, placenta, prostate, colon, kidney, and cardiovascular system. It is well known that TSPO is over-expressed in highly aggressive tumors, especially those of the breast, and that expression correlates with advancing stages of this malignancy. TSPO expression, nuclear localization, and TSPO-mediated cholesterol transport into the nucleus are involved in breast cancer cell proliferation and aggressive phenotype expression. Hence, it can be used as a biomarker in the stage-dependent diagnosis of this cancer. In addition, cell proliferation, invasion and migration appears to be decreased when treated with high doses of TSPO ligand PK-11195, a compound that may represent a therapeutic agent for the control of breast cancer progression. Control of breast cancer development by consumption of dietary soy protein has been linked to down-regulation of the expression of TSPO-mediated angiogenic signaling molecules. This chapter provides insight into the potential of TSPO as a rational target for the development of novel therapeutics for breast cancer.

Previous studies have demonstrated the feasibility of translocator protein (TSPO) imaging to visualize and quantify human breast adenocarcinoma (MDA-MB-231) cells in vivo using a TSPO-targeted near-infrared (NIR) probe (NIR-conPK11195). This study aimed to extend the use of the TSPO-targeted probe to a more biologically relevant and clinically important tumor microenvironment as well as to assess our ability to longitudinally detect the presence and progression of breast cancer cells in the brain. The in vivo biodistribution and accumulation of NIR-conPK11195 and free (unconjugated) NIR dye were quantitatively evaluated in intracranial MDA-MB-231-bearing mice and non-tumor-bearing control mice longitudinally once a week from two to five weeks post-inoculation. The in vivo time-activity curves illustrate distinct clearance profiles for NIR-conPK11195 and free NIR dye, resulting in preferential accumulation of the TSPO-targeted probe in the intracranial tumor bearing hemisphere (TBH) with significant tumor contrast over normal muscle tissue (p<0.005 at five weeks; p<0.01 at four weeks). In addition, the TSPO-labeled TBHs demonstrated significant contrast over the TBHs of mice injected with free NIR dye (p<0.001 at four and five weeks) as well as over the TSPO-labeled non-tumor-bearing hemispheres (NTBHs) of control mice (p<0.005 at four and five weeks). Overall, TSPO-targeted molecular imaging appears useful for visualizing and quantifying breast cancer xenografts propagated in the murine brain and may assist in preclinical detection, diagnosis and monitoring of metastatic disease as well as drug discovery. Furthermore, these results indicate it should be possible to perform TSPO-imaging of breast cancer cells in the brain using radiolabeled TSPO-targeted agents, particularly in light of the fact that [11C]-labeled TSPO probes such as [11C]-PK 11195 have been successfully used to image gliomas in the clinic.

Translocator protein (TSPO) is a high affinity 18 kDa drug- and cholesterol-binding protein strongly expressed in steroidogenic tissues where it mediates cholesterol transport into mitochondria and steroid formation. Testosterone formation by Leydig cells in the testis is critical for the regulation of spermatogenesis and male fertility. Male germ cell development comprises two main phases, the pre-spermatogenesis phase occurring from fetal life to infancy and leading to spermatogonial stem cell (SSC) formation, and spermatogenesis, which consists of repetitive cycles of germ cell mitosis, meiosis and differentiation, starting with SSC differentiation and ending with spermiogenesis and spermatozoa formation. Little is known about the molecular mechanisms controlling the progression from one germ cell phenotype to the next. Here, we report that testicular germ cells express TSPO from neonatal to adult phases, although at lower levels than Leydig cells. TSPO mRNA and protein were found at specific steps of germ cell development. In fetal and neonatal gonocytes, the precursors of SSCs, TSPO appears to be mainly nuclear. In the prepubertal testis, TSPO is present in pachytene spermatocytes and dividing spermatogonia. In adult testes, it is found in a stagedependent manner in pachytene spermatocyte and round spermatid nuclei, and in mitotic spermatogonia. In search of TSPO function, the TSPO drug ligand PK 11195 was added to isolated gonocytes with or without the proliferative factors PDGF and 17β-estradiol, and was found to have no effect on gonocyte proliferation. However, TSPO strong expression in dividing spermatogonia suggests that it might play a role in spermatogonial mitosis. Taken together, these results suggest that TSPO plays a role in specific phases of germ cell development.

The pharmacological agent 1-(2-Chlorophenyl-N-methylpropyl)-3-isoquinolinecarboxamide (PK11195) is the prototypical ligand of the 18-kDa Translocator Protein (TSPO) but at μM concentrations deactivates the oncoprotein Bcl-2 increasing the efficiency of chemotherapeutic agents and promoting the Ca2+-dependent macro-autophagy (or autophagy). In this paper, we report that PK11195, in HeLa cells, modifies the mitochondria-targeted type of autophagy - hereafter referred to as mitophagy- and the associated resizing of the mitochondrial network but does so exclusively in absence of the oncoprotein Bcl-2 (Bcl-2 Kd cells). This is consequence of a “side” targeting of the mitochondrial F1Fo-ATPsynthase enzyme, since identical outcome is mimicked by the antibiotic Oligomycin, of which PK11195 matches the effect on: i) mitochondrial membrane potential (ΔΨm), ii) ATP homeostasis and iii) Reactive Oxygen Species (ROS) generation. Taken together, these data highlight a novel TSPO-independent biological effect for PK11195 and provide evidences for a hitherto uncovered Bcl-2-dependent role of the F1Fo-ATPsynthase in mitochondrial quality control.

Background and Objectives: A role for the protein that mediates the rate-limiting step of steroidogenesis, the 18 kDa Translocator Protein (TSPO), has been suggested in the pathophysiology of Adult Separation Anxiety Disorder (ASAD). It has been shown that ASAD patients have 1) low TSPO expression levels and 2) a high frequency of the allele that substitutes Ala with Thr at position 147 of TSPO. The Thr147 ASAD-associated allele has been recently related with a low pregnenolone production. The aim of the present work was to evaluate the relationship between TSPO expression levels and Ala147Thr single nucleotide polymorphism (SNP), which are the two TSPO biological parameters that we have previously examined separately. A further aim was to confirm the genetic association of Ala147Thr SNP with ASAD in an extended case-control sample and to investigate whether this SNP was related to an anxious attachment style that is thought to be connected to ASAD. Methods: TSPO expression levels were compared among patients with ASAD (n=26), without ASAD (n=26) and control samples (n=10) stratified into the two genotype groups: those with the Ala147 genotype (named “normal pregnenolone production”) and those with the Thr147 genotype (named “reduced pregnenolone production”). The case-control genetic study included patients with (n=87) or without (n=101) ASAD and 236 controls. In the patient group, the association between the Ala147Thr SNP and an anxious attachment style was analysed by stepwise logistic regression analysis. Results: The genotype with the lowest TSPO expression levels was the “normal pregnenolone production” genotype in the ASAD group. The genetic Ala147Thr SNP confirmed an excess of the Thr147 allele in ASAD patients. Stepwise logistic regression analysis did not show an association with an anxious attachment style. Conclusions: ASAD individuals who expressed normal TSPO levels exhibited the “reduced pregnenolone production” genotype. In contrast, the ASAD individuals with the “normal pregnenolone production” genotype expressed low TSPO levels. It is possible that low TSPO expression levels could compromise normal pregnenolone production. Such evidence may have therapeutic implications because it has been documented that drugs targeting TSPO increased pregnenolone production and have anxiolytic effects.

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.

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.

Renal failure due to ischemic injury is a common denominator of various clinical situations in critically ill patients. This study was designed to characterize the TPSO/Cholesterol synthesis and cell division pathways in response to different levels of ischemia. Porcine kidneys were subjected to either 60min-warm ischemia (WI) or auto-transplanted after cold storage for 24h at 4°C (CS), or both conditions (WI+CS), pathway activation and function were evaluated at 3h, 3 and 7 days after reperfusion. CS combined to WI affects renal functions indicating a high degree of injury. During the first week of reperfusion, renal levels of free and esterified cholesterol, major cellular components, increased in CS group with an attenuated production when WI was associated. CS and WI+CS groups exhibited an elevated expression of cell cycle induction markers such as PCNA and stathmin. TSPO expression was highest in groups with the lowest injury, and correlated with kidney outcome, revealing its potential for diagnosis.