Lost in translocation: the functions of the 18-kD translocator protein

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Lost in translocation: the functions of the 18-kD translocator protein Philipp Gut1, Markus Zweckstetter2,3,4, and Richard B. Banati5,6 1

Nestle´ Institute of Health Sciences, EPFL Innovation Park, Baˆtiment H, 1015 Lausanne, Switzerland Max-Planck-Institut fu¨r Biophysikalische Chemie, 37077 Go¨ttingen, Germany 3 Deutsches Zentrum fu¨r Neurodegenerative Erkrankungen (DZNE), 37077 Go¨ttingen, Germany 4 Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center, 37073 Go¨ttingen, Germany 5 Life Sciences, Australian Nuclear Science and Technology Organisation, Lucas Heights, NSW 2234, Australia 6 National Imaging Facility and Ramaciotti Centre for Brain Imaging, Brain and Mind Research Institute, Faculty of Health Sciences, University of Sydney, Sydney, NSW 2006, Australia 2

Research spanning nearly four decades has assigned to the translocator protein (18 kDa) (TSPO) a critical role, among others, in the mitochondrial import of cholesterol, the subsequent steps of (neuro)steroid production, and systemic endocrine regulation, with implications for the pathophysiology of immune, inflammatory, neurodegenerative, and psychiatric as well as neoplastic diseases. Recent knockout studies in mice unexpectedly report normal or latent phenotypes, raising doubts about the protein’s role in steroidogenesis and other previously postulated functions and challenging the validity of earlier data on the selectivity of TSPO-binding drugs. Here we provide a synthesis of the current debate from a structural and molecular biology perspective, discuss the limits of inference in loss-of-function (gene knockout) studies, and suggest new functions of TSPO. Introduction The evolutionarily conserved TSPO (see Glossary), previously named the peripheral benzodiazepine receptor (PBR), is an abundant protein found in many organs but with particularly high constitutive expression in steroidogenic tissue, including adrenal glands, gonads, placenta, and activated brain microglia [1–3]. The anatomical distribution of TSPO expression, and the early discovery that TSPO is a high-affinity binding protein of cholesterol that resides in the outer mitochondrial membrane, established an exciting link between cholesterol transport and the biosynthesis of steroids, including neurosteroids [1,4,5]. Since these initial landmark findings, a large body of evidence has gradually defined TSPO as an essential component of cholesterol transport across the mitochondrial membrane, which came to be understood as a rate-limiting step of steroid hormone production [6–9]. Eventually, a new nomenclature was introduced in 2006 to better signify TSPO’s function as a cholesterol translocator protein replacing the historical Corresponding author: Banati, R.B. ([email protected]). Keywords: neuroinflammation; neuroimaging; peripheral benzodiazepine receptor; heme metabolism; REV-ERBa; mitochondrial permeability transition pore; cholesterol. URL: http://www.tspo.info 1043-2760/ ß 2015 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.tem.2015.04.001

name that originated from the discovery of TSPO/PBR as a peripheral binding site of benzodiazepines outside the nervous system [10,11]. At the same time, the new nomenclature drew attention to the existence of other, homologous proteins thought to be involved in transmembrane (TM) signaling, the tryptophan-rich sensory proteins (TspOs) [12]. Various cell types have been shown to regulate TSPO expression in response to inflammation, cancer, obesity, nutrient status, or cellular degeneration [9,13,14], most prominently among them cells of the mononuclear–phagocyte lineages. A remarkable characteristic of TSPO is, for example, its dynamic increase of expression in microglial cells from near absence to high levels after brain or peripheral nerve injury [15]. Subsequently, TSPO radioligands have been found to be generically useful for diagnostic purposes as a biomarker of active disease or disease-related tissue remodeling [11,13]. This correlative association with pathology and its known druggability (i.e., being a biological target predicted to bind with high affinity to a drug) have identified TSPO as an attractive therapeutic target. Since the discovery of the first high-affinity TSPO-binding molecules such as the isoquinolinecarboxamide PK11195 [16], the selectivity of which has recently been confirmed in animals without TSPO [17], many more novel chemical entities with affinity for TSPO have been

Glossary Bioenergetics: cellular processes that are implicated in the transformation of energy; most commonly refers to ATP generation by carbon breakdown and oxidative phosphorylation in mitochondria. NMR spectroscopy: a spectroscopic method based on NMR that allows determination of the structure and dynamics of proteins. PK 11195: the synthetic radioligand 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide, which binds mammalian TSPO with nanomolar affinity. Protein dynamics: proteins are not rigid entities but are flexible and can respond to various external stimuli by changing their conformation. Radioligand: small synthetic molecules that are used for positron emission tomography studies. Translocator protein (18 kDa) (TSPO): an 18-kDa protein that belongs to family of TspOs. TSPO was initially described as the PBR, a secondary binding site for diazepam. It was later found that TSPO is expressed at various levels in most organs and in the brain is induced under disease conditions. TSPO thought to be primarily located in the outer mitochondrial membrane.

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Opinion reported, some of which have entered clinical trials as either diagnostic or therapeutic agents [11]. Earlier attempts to generate Tspo/ knockout mice in the mid-1990s were reported to cause embryonic lethality [4], not unexpected in view of the assumed vitally important physiological role of TSPO. In the absence of a TSPOnull model to study TSPO as a target for pharmacotherapy under normal conditions, synthetic compounds with affinity for TSPO remained the primary experimental approach for mostly in vitro competitive binding as well as functional studies. Animal models of disease that were reported to benefit from treatments with TSPO ligands include those for Alzheimer’s disease, multiple sclerosis, anxiety disorders, neuropathic pain, peripheral nerve injury, diabetes, rheumatoid arthritis, cancer, and cardiac ischemia [13,18– 24]. The rationale for at least some of these therapeutic interventions was based on the concept that neurosteroids were beneficial in the treatment of inflammation and increase cellular survival [13,18,25]. Efforts to fully develop the therapeutic potential of TSPO ligands were also based on functions of TSPO other than cholesterol transport and steroidogenesis. For example, TSPO was reported to interact with two core members of the mitochondrial permeability transition pore, the adenine nucleotide transporter (ANT), and the voltage-dependent anion channel (VDAC) [26]. A regulatory role of TSPO in the mitochondrial membrane permeability transition pore (MPTP), calcium homeostasis, the production of reactive oxygen species (ROS), and apoptosis were eventually reported and provided a rationale for the suppression of apoptosis after cardiac ischemia and the stimulation of apoptosis in cancer types characterized by high expression of TSPO [21,24,26]. Yet, recent studies of TSPO gene knockout models have not readily confirmed a role of TSPO in steroid synthesis, the regulation of the MPTP, survival, or reproduction [17,27–30]. In this review we discuss the implications of these puzzling findings. Despite all controversy and the difficulty of interpreting new data in the context of past concepts, results from various species support the notion

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that TSPO plays a direct or indirect role in the regulation of mitochondrial energy metabolism and cellular stress pathways. Structure of TSPO Analysis of the primary TSPO sequence suggested that the basic molecular architecture of TSPO is formed by five TM helices [31]. Topological and hydropathy analysis further suggested that the C terminus of TSPO is exposed to the cytoplasm with two loops found on each side of the membrane [32], with the longest loop located in the cytoplasm between TM1 and TM2 (Box 1 and Figure 1). An overall helical structure of mammalian TSPO was further supported by later work using circular dichroism as well as NMR studies on peptide fragments that corresponded to the five putative TM regions [33,34]. In addition, a bacterial homolog of TSPO, the TspO from Rhodobacter sphaeroides (RsTspO), was characterized by electron microscopy [35]. Two RsTspO molecules each comprising five TM helices were found in a homodimeric arrangement, in agreement with the pronounced tendency of RsTspO to assemble into dimers [35,36]. The dimeric arrangement as observed for RsTspO might be important for TSPO-mediated translocation of substrates in bacteria. The 3D structure for mammalian TSPO in complex with PK11195 was solved by NMR spectroscopy in 2014 [37] (Box 1). In 2015, Li et al. [38] crystallized the structure of the bacterial homolog RsTspO and of a mutant protein with an Ala138!Thr138 substitution, which might mimic the human rs6971 polymorphism [39], while Guo et al. [40] reported the crystal structure for the TspO from Bacillus cereus (BcTspO). TSPO–cholesterol interactions and translocation Key to potential TSPO-mediated cholesterol transport is the 3D structure of mammalian TSPO in complex with cholesterol. However, this is currently unavailable and only indirect evidence for the precise mechanism of the cholesterol–TSPO interaction exists. One important step toward a better understanding of the TSPO–cholesterol

Box 1. Solving the structure of the TSPO–PK11195 complex TSPO appears to have an unstable tertiary fold. This is supported by low signal dispersion in NMR spectra of TSPO in detergent micelles [33,37] and a less well-defined electron density for some of the TM helices of RsTspO in tubular helical crystals [35]. By contrast, binding of the synthetic TSPO-specific radioligand PK11195 stabilized the structure of mouse TSPO and increased the helical content [33,37], thus making it possible to solve the 3D structure of the TSPO– PK11195 complex [37]. The structure revealed a bundle of five TM helices arranged around PK11195 (Figure 1). Due to the presence of several proline residues most of the helices have kinks. When viewed from the cytosol, the five TM helices are arranged in the clockwise order TM1–TM2–TM5–TM4–TM3. The same TM topology was later found in the crystal structures of the bacterial homologs RsTspO and BcTspO [38,40]. In addition, in a structure of BcTspO obtained in the presence of PK11195, PK11195 was found in a similar hydrophobic pocket [40], as observed in the complex of PK11195 with mammalian TSPO [37], despite the 1000-fold lower affinity of PK11195 to bacterial TspOs. NMR-based dynamics measurements further showed that the five TM helices are rigidly formed, while residues 1–4 and 160–168 of mammalian TSPO remain flexible and can potentially change their 2

conformation on interaction with endogenous interaction partners [37]. The ligand-binding pocket comprises an alanine at position 147, which is substituted by threonine in the case of the rs6971 polymorphism [39]. Although the affinity of PK11195 is similar for both wild type mammalian TSPO and its A147T variant, several other small molecules, which might be used for TSPO-based imaging, have a reduced affinity to the A147 variant. The PK11195-binding pocket is further closed by the TM1–TM2 loop, which folds into a short helix and thus assumes a stable conformation (Figure 1). In agreement with the importance of the hydrophobic pocket and the TM1–TM2 loop for the recognition of small molecules, several TSPO ligands were shown to compete with PK11195 for binding to TSPO (for a review see [13]). Notably, the five TM helices are tightly packed together, such that no channel in the interior of a TSPO monomer was detected in the mouse TSPO–PK11195 complex [37] or in the crystal structures of RsTspO and BcTspO [38,40]. However, molecular dynamics simulations suggested that a single TSPO molecule could accommodate a cholesterol molecule in an interior cavity when PK11195 is not present [78], suggesting that structural changes could be induced by cholesterol in the absence of PK11195.

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cholesterol–TSPO interaction, an additional, currently unknown, mechanism would then be required to release cholesterol. Potential subsequent transport/translocation of cholesterol might occur at a TSPO oligomerization interface [35,36,38,44,45], in agreement with the ability of TSPO to form polymers in vivo [46]. Mammalian TSPO has sequence homologies with a group of TspOs that are present in many different organisms. For example, the bacterial protein RsTspO was shown to be important for photosynthetic gene expression and might act as an oxygen sensor [12]. Bacterial TspOs and mammalian TSPO share sequence similarity, with residues in TM region 1 being least conserved [44]. Consistent with these sequence differences, bacterial TSPO and mammalian TSPO differ in important functional and structural aspects [35,36,38,40]. Notably, the cholesterol recognition sequence of the mammalian protein is not conserved in bacterial TspOs, consistent with the absence of cholesterol from bacterial membranes. The functional differences are supported by structural differences, which were evidenced by the recent 3D structures of the TspOs from B. cereus and R. sphaeroides [38,40] (Box 1). Thus, further structural studies of mammalian TSPO are required, as many of the questions that are relevant for human TSPO cannot be addressed when using nonmammalian TspOs.

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Figure 1. 3D structure of mouse translocator protein (18 kDa) TSPO [Protein Data Bank (PDB) code: 2MGY] [37]. The TSPO structure is formed by five transmembrane (TM) helices in complex with PK 11195 (shown in violet). The topology of the five TM helices is TM1–TM2–TM5–TM4–TM3 when viewed from the cytosol [37]. The highly positively charged C terminus (top right), which is in direct proximity to the cholesterol-binding site, is flexible and points into the cytosol. The side chains of Y153 and R156, which are important for cholesterol binding [43], are highlighted in cyan. The side chain of A147, which is mutated to threonine in the human rs6971 polymorphism [39], is shown in green.

interaction was, therefore, the experimental demonstration that recombinantly produced TSPO binds cholesterol with nanomolar affinity in a purified system [41]. The specificity of the interaction was then supported by sitedirected mutagenesis [42] demonstrating that amino acids Y153 and R156 of TSPO are critical for the interaction with cholesterol. Y153 and R156 are located in a sequence motif at the C-terminal end of TM5 of TSPO, which was denoted as the cholesterol recognition amino acid consensus (CRAC) sequence (residues A147 to S159) [42] and is located in TM5 in the 3D structure of mouse TSPO (Box 1 and Figure 1) [37]. Notably, a cholesterol molecule can be docked to the structure in this region such that R156 interacts with the sterol hydroxyl group of cholesterol [43]. Moreover, the side chains of Y153 and R156 point to the hydrophobic environment of the membrane, suggesting that cholesterol might bind from the outside to the TSPO structure (Box 1). Taken together, current data support a model where cholesterol binds with very high affinity from the outside to residues A147–S159 of mammalian TSPO. Because of the high affinity of the

Recent data from TspoS/S knockout mice challenge an old dogma – or not? Recent publications of viable Tspo/ knockout mice by independent groups challenge the longstanding assumption that TSPO is critical for life without obvious impairment of the biosynthesis of steroids [17,27–29]. In the case of the extensively characterized Guwiyang Wurra (‘Fire Mouse’) TSPO knockout mouse model [17], comparative studies against littermate controls across the expected average lifespan of laboratory mice demonstrated that Tspo/ mice develop normally and have a healthy existence. An obvious physiologically relevant role of TSPO in cholesterol transport, steroid biosynthesis, or MPTP regulation has not been found. Thus, the lack of overt phenotypes in Tspo/ knockout mice calls for a thorough revisiting of the purported TSPO functions and a critical experimental validation of the selectivity or mechanisms of action reported in studies where TSPO ligands have been used as diagnostic or pharmacotherapeutic agents [47]. So far, two experimental paradigms have been retested using Tspo/ knockout animals and cell lines. Previously, Sileikyte et al. have shown in rat liver mitoblasts that the effects of TSPO ligands on MPTP opening are dependent on TSPO [48]. In the then absence of a TSPO-null background model, cotreatment with protoporpyhrins and subsequent photoactivation was used to trigger MPTP opening, an effect that could be inhibited by TSPO ligands. However, subsequent retesting of this experimental model using liver mitoplasts from Tspo/ knockout mice showed that the pharmacological effects were surprisingly also present in the complete absence of TSPO [27]. A further study by Tu et al. retested the induction of steroidogenesis by TSPO ligands, one of the original experiments performed in MA-10 mouse Leydig tumor cells [4], in cells 3

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Opinion with an out-of-frame deletion in exon 2 of TSPO [30]. Similar to their previous studies using Leydig cell-specific conditional knockouts of Tspo in mice, the authors showed that, at least at high concentrations, the TSPO ligand PK11195 increases progesterone synthesis despite a lack of TSPO [29]. These findings are unexpected and seem to contradict other observations that provide strong correlative support for TSPO as a steroidogenic protein, such as the selective enrichment of TSPO in steroidogenic tissues and the mitochondrial membrane. It is estimated that TSPO constitutes approximately 5–10% of all proteins in the outer membrane of steroidogenic cells [1]. A landmark study in 1985 showed, furthermore, that hypophysectomy decreased TSPO levels in the adrenal glands and testes, suggesting dependency of its expression on circulating steroidogenic hormones [5]. Based on the hypothesis that TSPO is controlled by a hormonal pathway and represents a critically important rate-limiting factor in steroid production, TSPO ligands were subsequently tested to show a steroidogenic effect in cell lines, primary cells, and mice [7,13,18,19,49,50]. The availability of viable mouse knockout models now provides a tool to validate these ‘established’ TSPO functions. While it might be tempting to interpret the recent observations in TSPO knockout animals as the unequivocal refutation of an important role of TSPO in steroidogenesis [47], caution is warranted to not dismiss previously postulated functions prematurely [51]. Many examples exist of broadly expressed proteins that biochemically appear to critically impact on mitochondrial function in cell culture settings and that present at first sight with surprisingly mild in vivo phenotypes in laboratory mice. For example, mice deficient for the mitochondrial NAD+-dependent deacetylase SIRT3 are viable and fertile and are, for most of their lives, indistinguishable from wild type littermates, despite deacetylating and functionally modifying many enzymes of b-oxidation and oxidative damage response pathways. However, under cold exposure Sirt3/ mice rapidly lose the capacity to maintain body temperature in a healthy range due to reduced boxidation, a defect that would probably render them incompatible with life in the wild [52]. Besides cold intolerance, Sirt3/ mice develop phenotypes during aging such as hearing loss or hepatocellular cancer [53,54]. An example of a disease-relevant gene whose deficiency in mice does not phenocopy the human pathophysiology is PTEN-induced putative kinase 1 (PINK1). PINK1 mutations are the second most common cause of autosomalrecessive Parkinson’s disease (PD) [55]. PINK1 is thought to play a major role in pathways of mitochondrial quality control and metabolism and often serves as a paradigmatic example of a primary mitochondrial defect that causes neurodegenerative disease. However, the loss-of-function phenotypes in mice are mild, include defects in mitochondrial bioenergetics in the striatum in the young, and are followed by a broader deficit in the aged brain. Only subtle morphological changes of mitochondrial shape could be observed. Importantly, Drosophila, but not mice, show prominent defects in mitochondrial dynamics and bioenergetics, loss of dopaminergic motor neurons, and PD-like 4

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motor deficits [56–58]. With hindsight, the history of PINK1 research shows similarities with where TSPO research is at present; that is, much of the initial work’s hypotheses were generated in immortalized cell lines and the field then was challenged by the difficulty of assigning PINK1 functions in the face of moderate phenotypes in mice [59]. TSPO has been shown in vitro to functionally interact with PINK1 [60], suggesting that similar back-up mechanisms could be in place for TSPO that circumvent the detrimental consequences of Pink1 deficiency during neurodegeneration in mice. TSPO and cellular and metabolic stress: lessons from animal models In addition to differences between species, the discrepancy between an important biochemical function in vitro and the in vivo phenotype is caused by the high capacity of mitochondria to adapt and sustain tissue functionality in response to cellular and energetic stress. For example many tissues, such as liver, brain, and kidney, are able to sustain a nearly normal respiratory rate even when Complex IV of the respiratory chain is inhibited by 80% [61]. Thus, if TSPO is critical for mitochondrial processes, functional redundancy or morphological adaptations of mitochondria could compensate for TSPO loss of function. TSPO may become phenotypically relevant when mitochondria lose the capacity to compensate during aging or in response to other stressors such as metabolic challenges. TSPO has long been known as a stress-responsive protein; that is, the shift from ‘housekeeping’-like expression to a highly abundant protein in many different cell types in response to a large variety of stressors is a widely accepted characteristic of TSPO [9]. These noxious stimuli include inflammation, malignancy, nutritional status, and aging, indicating that TSPO becomes critical only after other systems fail or have reached full compensatory capacity. Early studies have shown that TSPO ligands can modulate the rate of cellular respiration [26,62], although TSPOindependent mechanisms are likely to play a role; for, example through modulation of ATP synthase [63,64]. However, recent studies in mice, zebrafish, and fruit flies suggest that TSPO indeed impacts mitochondrial energy metabolism under stress conditions in vivo [17,23,65]. In all three experimental in vivo models, energetic deficits surfaced when the cells were experiencing situations of energetic stress. Microglia derived from Tspo/ mice show a lower baseline of mitochondrial respiration and reduced ATP levels [17]. After injury and depending on the severity, microglia undergo transition from a resident, ‘resting’ brain macrophage to an ‘activated’, highly proliferative cell type. This switch toward rapid proliferation requires energy for growth and cellular division, but most importantly requires efficient breakdown of large amounts of carbon sources to generate acetyl-coenzyme A (CoA) as building blocks for new membranes [66]. The activation as well as the expansion of the microglial pool appeared to be normal in the Guwiyang Wurra Tspo/ mice [17]. However, more detailed data about the post-lesion proliferation kinetics of activated microglia over a longer period are not yet available and the theoretical existence of another constitutively TSPO-negative

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Opinion subpopulation of microglia that is able to compensate in vivo cannot be excluded. Nevertheless, a fruitful experimental strategy should be to probe TSPO functions during the switch from quiescent to rapidly proliferating cell types such as microglia or immune cells. To investigate the role of TSPO in regulating energy balance at an organismal level, Tspo/ mice can be challenged by a high-fat diet. Phenotyping methods such as the use of metabolic chambers to measure oxygen consumption and physical activity, dual-energy X-ray absorptiometry (DEXA) measurements to estimate fat and lean mass, and glucose and insulin tolerance tests can be applied to determine whether TSPO is a critical component for organismal energy homeostasis [67]. To this end, Tspofl/fl mice are an important addition to the tool kit since they allow the cell type-specific deletion of TSPO in tissues of interest and could, for example, be used to test the role of TSPO in adipocytes or the liver in a cell-autonomous fashion. Zebrafish are amenable to in vivo drug testing, making them an attractive model to probe the selectivity and pharmacological effects of TSPO ligands. A recent screen has demonstrated that the synthetic TSPO ligands PK 11195 and Ro5-4864 act as enhancers of a fasting response and protect mice against glucose intolerance and hepatic steatosis [23]. Importantly, TSPO ligands had a significant metabolic effect only when zebrafish or mice were exposed to fasting, indicating that modulation of TSPO affects

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energy metabolism in the context of energetic stress. Targeted disruption of the tspo gene, the CRAC domain, or single nucleotides that are important for synthetic or endogenous ligand binding will yield insights into gene function and pharmacology. A study using morpholinomediated knockdown of tspo, which transiently suppresses tspo expression during the first days of development, showed a selective defect in erythropoiesis [68]. Whether this defect is due to TSPO functions in the mitochondria of erythroblasts or a mitochondria-independent function in the membranes of mature erythrocytes is currently unknown, but it further illustrates the use of zebrafish to identify novel functions of TSPO. Drosophila is amenable to dietary, genetic, and pharmacological interventions. A recent study shows that loss of dTSPO extends lifespan, is protective against neurodegeneration, and impairs mitochondrial cellular respiration [65]. An interesting finding of this study is that expression of dTSPO is increased in the fly during aging. Old, but not young, dTSPO-deficient flies showed a marked decrease of mitochondrial respiration, which again indicates that dTSPO has conserved functions when cells are exposed to stressors such as cellular aging. An important advantage of fruit flies as research tools is that a genome-wide gene knockdown repository is available, which allows epistasis experiments and the dissection of gene networks that are associated with metabolic functions of TSPO.

Box 2. Studying endogenous ligands as a window into TSPO function An intriguing characteristic of TSPO is its ability to bind different endogenous and synthetic small molecules with high (nanomolar to micromolar) affinity [9]. However, the biological purpose of heme, porphyrin, or cholesterol binding to TSPO remains largely undefined. Many intracellular metabolites are well known to feed back on homeostatic mechanisms of metabolic control; for example, on transcription through chromatin modifiers or on enzyme function through allosteric effects [79]. Studying the functional relationship of ligand binding to TSPO may yield valuable insights into physiological processes that rely on TSPO. Here we speculate on the TSPO–heme interaction and how it could merge with known functions of REVERBa, another high-affinity heme-binding protein; that is, heme is known to act in a classical feedback loop that limits its own biosynthesis through binding of the nuclear receptor REV-ERBa [80]. Levels of free heme have to be tightly regulated based on energy substrate flux, as excess heme can cause oxidative stress and damage cells when reacting with molecular oxygen. REV-ERBa is a critical component of the circadian clock core machinery that adapts cellular metabolism to circadian rhythms and acts a transcriptional suppressor of gluconeogenesis and lipid metabolism [80,81]. When the demand for heme increases during fasting, the transcriptional coregulator peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC1a) induces expression of Alas1, which encodes the rate-limiting enzyme of heme biosynthesis, as well as that of the gluconeogenic genes Pck1 and G6pc in the liver [82,83]. REV-ERBa, by contrast, in a heme-dependent manner, suppresses the expression of gluconeogenic genes thereby relaying the information of a cellular high-energy level to the nucleus and limiting the production of excess heme by reducing Alas1 expression [81]. The speculation to connect TSPO with REV-ERBa and regulatory networks is based on the common characteristic that both proteins are high-affinity heme binders and that an overlap exists for downstream metabolic pathways; that is, modulation of TSPO with synthetic ligands activates a transcriptional network in zebrafish that opposes REV-ERBa function, including increased expression of the heme biosynthetic gene alas1 and the fasting-inducible gluconeogenic genes pck1 and g6pc [23]. Figure I depicts how heme binding to

TSPO may be able to complement the control of transcriptionmediated control of energy metabolism and oxidative damage by REV-ERBa.

Heme availability, oxidave damage

Heme/porphyrins PK 11195 Gluconeogenesis, fasng metabolism, heme biosynthesis, oxidave damage

TSPO ROS

ATP

PK 11195

G6pc Pck1

Alas1

REV-ERBα Mitochondria

Nucleus

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Figure I. Translocator protein (18 kDa) (TSPO) and REV-ERBa are high-affinity heme-binding proteins with overlapping functions in heme and energy metabolism. On heme binding, REV-ERBa suppresses expression of Deltaaminolevulinate synthase 1 (Alas1), the rate-limiting enzyme of heme synthesis, thereby limiting heme excess and oxidative damage. In addition, heme–REVERBa signals a high nutrient status and suppresses expression of the gluconeogenic genes phosphoenolpyruvate carboxykinase (Pck1) and glucose6-phosphatase (G6pc) in the liver [82,83]. TSPO is known to catalyze the degradation of free porphyrins and to be essential for respiration and ATP production in TSPO-deficient microglia and aged Drosophila [17,40,65,69]. Thus, TSPO may complement the nuclear signaling axis of REV-ERBa to reduce oxidative damage and to adapt energy metabolism in mitochondria. TSPO ligands, such as PK 11195, oppose REV-ERBa signaling and increase expression of alas1, pck1, and g6pc. PK 11195 also reduces ATP levels and increases reactive oxygen species (ROS) [9,62,84], further suggesting that TSPO ligands oppose REV-ERBa signaling. Future studies will show whether TSPO has physiologically relevant roles in a mitochondrial pathway that limits ROS damage through heme degradation and that modulates ATP production in response to fluctuations of intracellular heme levels. Dashed arrows indicate hypothetical links; unbroken lines represent experimentally established connections.

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Opinion Lastly, unicellular organisms such as bacteria and yeast provide an opportunity to examine evolutionarily ancient functions of TSPO, particularly related to heme transport and catabolism [40,69]. For example, a screen for synthetically lethal interactions could generate knowledge about conserved interaction partners that contribute to functional redundancy. With the right tools and a selection of in vivo models with distinct advantages, a more precise picture of TSPO functions in cellular metabolism, stress response pathways, and other functions should emerge in the near future. The study of endogenous ligands may further elucidate the role of TSPO in integrating cytosolic cues of energy state with mitochondrial or metabolic homeostasis (Box 2). Emerging TSPO functions It is a popular assumption that ancient, evolutionarily highly conserved genes should naturally be essential in the regulation of well-definable, fundamental physiological process; hence, one should expect embryonic lethal outcomes or at least an obvious phenotype from the loss of such a gene, as has been reported for TSPO [4]. However, phylostratigraphic analysis of the human genome suggests that ancient genes predominate among the disease-associated but ‘nonessential’ genes [70,71]. At the same time, there are indications that disease genes might be more frequently involved in stress response pathways; that is, complex multipathway processes for which it might currently be difficult to assign a function in simple physiological terms [72]. Thus, while the evolutionary dynamics behind the preservation of the ancient TSPO is likely to be more complex than presently understood [73], an importance as a disease-modifying gene regulated in cellular stress or conditions of pathological challenge or adaptation should be considered. Current evidence in humans is sparse but intriguing, in as far as the known rs6971 polymorphism in the TSPO gene [74] may relate to complex mental health phenotypes; notably, to different metabolic responses as seen in differences in weight gain under antipsychotic treatment [75]. A more fundamental reason for the limits of inference in gene knockout studies is the fact that simple explanatory reductionism ignores that ‘functions’ generally emerge from more complex interactions between different proteins. In other words, and with specific reference to the Tspo/ knockout models, for as long as it is unestablished whether and how the absence of TSPO results in adaptive changes, it is difficult to exclude that at least some of the TSPO emergent functions in normal animals include regulatory effects on steroid synthesis. In the context of emergent properties of microglia, one of the cell types characterized by high abundance of inducible TSPO [76] has made the case that emergence at its core means that ‘the interaction is an entity of the system’ and thus cannot be meaningfully reduced into other entities. Observations in Guwiyang Wurra Tspo/ mice after peripheral nerve injury [17] have revealed no obvious failure in the activation of perineuronal microglia typically found around the somata of the injured neurons. Microglial activation was determined by the upregulation of microglial CD11b (Mac-1a, Mb2 integrin, also known as the C3 complement 6

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receptor), a highly sensitive and commonly used method, indicating the presence of neuroinflammation. Thus, the state change of microglial activation does not appear to critically depend on the actions of TSPO. It remains to be established whether the absence of TSPO impairs the further transformation of microglia into inflammatory macrophages as the altered mitochondrial function in isolated Tspo/ microglia might suggest [17]. As a complement to the now ongoing loss-of-function studies, gainof-function studies should be of value; that is, an approach where the interactions are not removed but enhanced or altered. Concluding remarks and future perspectives The availability of viable TSPO knockout mice provides powerful tools to study under better-controlled in vivo conditions the selectivity of TSPO-binding drugs and their therapeutic potential in a range of pathologies, both prerequisites to reducing the risk of late-stage failure of clinical trials. As part of future research (Box 3), it is important that, first, some of the critical correlative observations on the augmentation of (neuro)steroid production and associated beneficial health effects reported after treatment with TSPO ligands are revisited in TSPO-null animals. Second, the absence of an essential function of TSPO in living animals (at least in mice) requires a detailed analysis of the functional networks that operate together with TSPO. Finally, the future research agenda for TSPO needs to clarify terms such as ‘translocation’ and ‘neuroinflammation’ [11,77] and make use of the evolving conceptual tools

Box 3. Outstanding questions TSPO structure  Is TSPO able to dynamically adopt its conformation or oligomerization state in response to different physiological conditions and interaction partners?  Would these, as-yet-unidentified, dynamics support a role in cholesterol or heme transport? TSPO function  Would a fundamental role in mitochondrial energy metabolism also explain effects on steroid metabolism?  What is the consequence of decreased ATP production for microglial proliferation and ‘neuroinflammatory’ function of activated microglial cells?  Under which conditions does TSPO become an essential component of mitochondrial energy balance?  Which metabolic or cellular stress responses critically require TSPO function in vivo? TSPO ligands  Which therapeutically beneficial effects of TSPO can truly be explained by actions via TSPO?  Which TSPO-binding molecules, peptides, or antibodies are specific and selective enough for robust experimentation and clinical trials?  In view of the observation that TSPO is expressed in various functional states of cell activation and vastly differing pathologies from slowly progressive noninflammatory to acutely inflammatory, what is the exact rationale behind therapeutic use of TSPO ligands? Is there a common pathway and is the therapeutic modulation of TSPO context dependent?  What are the minimum standards for regarding a TSPO drug as of validated specificity and selectivity?

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Opinion of systems biology, which are better able to capture complex functions in the context of health and disease. The basis for reaching a better validated understanding of TSPO’s function, however, is the availability and sharing of tools and protocols for laboratory or clinical experimentation that allow independent replication, such as TSPObinding compounds and antibodies with comprehensively validated specificity and selectivity. We expect the near future to reveal novel and exciting functions that will refine the rationale for TSPO as a regulator of mitochondrial function and thus a therapeutic target for the amelioration of a broad range of diseases. Acknowledgments M.Z. thanks Dr Stefan Becker, Dr Łukasz Jaremko, and Dr Mariusz Jaremko for insightful discussions. M.Z. was supported by the Deutsche Forschungsgemeinschaft Collaborative Research Center 803, Project A11, and the European Research Council (grant agreement number 282008). R.B.B. gratefully acknowledges Dr Guo-Jun Liu and Dr Ryan Middleton for their contribution to the development of the Guwiyang Wurra (‘Fire Mouse’) TSPO knockout mouse model (PCT/AU2014/000250). R.B.B. received support from the Deutsche Forschungsgemeinschaft, the European Framework Programme (FP6), the Medical Research Council of the UK, the Australian Research Council, and the Australian Nuclear Science and Technology Organisation.

Disclaimer statement P.G. is an employee of Nestle´ Institute of Health Sciences SA, part of Nestle´ Group.

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