Genetic analysis of dTSPO,an outer mitochondrial membrane protein,reveals its functions in apoptosis,longevity, and Aβ42‐induced neurodegeneration

The outer mitochondrial membrane (OMM) protein, the translocator protein 18 kDa (TSPO), formerly named the peripheral benzodiazepine receptor (PBR), has been proposed to participate in the pathogenesis of neurodegenerative diseases. To clarify the TSPO function, we identified the Drosophila homolog, CG2789/dTSPO, and studied the effects of its inactivation by P‐element insertion, RNAi knockdown, and inhibition by ligands (PK11195, Ro5‐4864). Inhibition of dTSPO inhibited wing disk apoptosis in response to γ‐irradiation or H₂O₂ exposure, as well as extended male fly lifespan and inhibited Aβ42‐induced neurodegeneration in association with decreased caspase activation. Therefore, dTSPO is an essential mediator of apoptosis in Drosophila and plays a central role in controlling longevity and neurodegenerative disease, making it a promising drug target.     

arouser reveals a role for synapse number in the regulation of ethanol sensitivity

A reduced sensitivity to the sedating effects of alcohol is a characteristic associated with alcohol use disorders (AUDs). A genetic screen for ethanol sedation mutants in Drosophila identified arouser (aru), which functions in developing neurons to reduce ethanol sensitivity. Genetic evidence suggests that aru regulates ethanol sensitivity through its activation by Egfr/Erk signaling and its inhibition by PI3K/Akt signaling. The aru mutant also has an increased number of synaptic terminals in the larva and adult fly. Both the increased ethanol sensitivity and synapse number of the aru mutant are restored upon adult social isolation, suggesting a causal relationship between synapse number and ethanol sensitivity. We thus show that a developmental abnormality affecting synapse number and ethanol sensitivity is not permanent and can be reversed by manipulating the environment of the adult fly.     

An Inexpensive,Scalable Behavioral Assay for Measuring Ethanol Sedation Sensitivity and Rapid Tolerance in Drosophila

Alcohol use disorder (AUD) is a serious health challenge. Despite a large hereditary component to AUD, few genes have been unambiguously implicated in their etiology. The fruit fly, Drosophila melanogaster, is a powerful model for exploring molecular-genetic mechanisms underlying alcohol-related behaviors and therefore holds great promise for identifying and understanding the function of genes that influence AUD. The use of the Drosophila model for these types of studies depends on the availability of assays that reliably measure behavioral responses to ethanol. This report describes an assay suitable for assessing ethanol sensitivity and rapid tolerance in flies. Ethanol sensitivity measured in this assay is influenced by the volume and concentration of ethanol used, a variety of previously reported genetic manipulations, and also the length of time the flies are housed without food immediately prior to testing. In contrast, ethanol sensitivity measured in this assay is not affected by the vigor of fly handling, sex of the flies, and supplementation of growth medium with antibiotics or live yeast. Three different methods for quantitating ethanol sensitivity are described, all leading to essentially indistinguishable ethanol sensitivity results. The scalable nature of this assay, combined with its overall simplicity to set-up and relatively low expense, make it suitable for small and large scale genetic analysis of ethanol sensitivity and rapid tolerance in Drosophila.     

The genetic relationships between ethanol preference, acute ethanol sensitivity, and ethanol tolerance in Drosophila melanogaster

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.     

The genetic relationships between ethanol preference,acute ethanol sensitivity,and ethanol tolerance in Drosophila melanogaster

The relationship between alcohol consumption, sensitivity, and tolerance is an important question that has been addressed in humans and rodent models. Studies have shown that alcohol consumption and risk of abuse may correlate with (1) increased sensitivity to the stimulant effects of alcohol, (2) decreased sensitivity to the depressant effects of alcohol, and (3) increased alcohol tolerance. However, many conflicting results have been observed. To complement these studies, we utilized a different organism and approach to analyze the relationship between ethanol consumption and other ethanol responses. Using a set of 20 Drosophila melanogaster mutants that were isolated for altered ethanol sensitivity, we measured ethanol-induced hyperactivity, ethanol sedation, sedation tolerance, and ethanol consumption preference. Ethanol preference showed a strong positive correlation with ethanol tolerance, consistent with some rodent and human studies, but not with ethanol hyperactivity or sedation. No pairwise correlations were observed between ethanol hyperactivity, sedation, and tolerance. The evolutionary conservation of the relationship between tolerance and ethanol consumption in flies, rodents, and humans indicates that there are fundamental biological mechanisms linking specific ethanol responses.     

Peripheral, central and behavioral responses to the cuticular pheromone bouquet in Drosophila melanogaster males

Pheromonal communication is crucial with regard to mate choice in many animals including insects. Drosophila melanogaster flies produce a pheromonal bouquet with many cuticular hydrocarbons some of which diverge between the sexes and differently affect male courtship behavior. Cuticular pheromones have a relatively high weight and are thought to be -- mostly but not only -- detected by gustatory contact. However, the response of the peripheral and central gustatory systems to these substances remains poorly explored. We measured the effect induced by pheromonal cuticular mixtures on (i) the electrophysiological response of peripheral gustatory receptor neurons, (ii) the calcium variation in brain centers receiving these gustatory inputs and (iii) the behavioral reaction induced in control males and in mutant desat1 males, which show abnormal pheromone production and perception. While male and female pheromones induced inhibitory-like effects on taste receptor neurons, the contact of male pheromones on male fore-tarsi elicits a long-lasting response of higher intensity in the dedicated gustatory brain center. We found that the behavior of control males was more strongly inhibited by male pheromones than by female pheromones, but this difference disappeared in anosmic males. Mutant desat1 males showed an increased sensitivity of their peripheral gustatory neurons to contact pheromones and a behavioral incapacity to discriminate sex pheromones. Together our data indicate that cuticular hydrocarbons induce long-lasting inhibitory effects on the relevant taste pathway which may interact with the olfactory pathway to modulate pheromonal perception.     

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.     

A Pair of Dopamine Neurons Target the D1-Like Dopamine Receptor DopR in the Central Complex to Promote Ethanol-Stimulated Locomotion in Drosophila

Dopamine is a mediator of the stimulant properties of drugs of abuse, including ethanol, in mammals and in the fruit fly Drosophila. The neural substrates for the stimulant actions of ethanol in flies are not known. We show that a subset of dopamine neurons and their targets, through the action of the D1-like dopamine receptor DopR, promote locomotor activation in response to acute ethanol exposure. A bilateral pair of dopaminergic neurons in the fly brain mediates the enhanced locomotor activity induced by ethanol exposure, and promotes locomotion when directly activated. These neurons project to the central complex ellipsoid body, a structure implicated in regulating motor behaviors. Ellipsoid body neurons are required for ethanol-induced locomotor activity and they express DopR. Elimination of DopR blunts the locomotor activating effects of ethanol, and this behavior can be restored by selective expression of DopR in the ellipsoid body. These data tie the activity of defined dopamine neurons to D1-like DopR-expressing neurons to form a neural circuit that governs acute responding to ethanol.     

Regulation of the Mitochondrial Permeability Transition Pore by the Outer Membrane Does Not Involve the Peripheral Benzodiazepine Receptor (Translocator Protein of 18 kDa (TSPO))

Translocator protein of 18 kDa (TSPO) is a highly conserved, ubiquitous protein localized in the outer mitochondrial membrane, where it is thought to play a key role in the mitochondrial transport of cholesterol, a key step in the generation of steroid hormones. However, it was first characterized as the peripheral benzodiazepine receptor because it appears to be responsible for high affinity binding of a number of benzodiazepines to non-neuronal tissues. Ensuing studies have employed natural and synthetic ligands to assess the role of TSPO function in a number of natural and pathological circumstances. Largely through the use of these compounds and biochemical associations, TSPO has been proposed to play a role in the mitochondrial permeability transition pore (PTP), which has been associated with cell death in many human pathological conditions. Here, we critically assess the role of TSPO in the function of the PTP through the generation of mice in which the Tspo gene has been conditionally eliminated. Our results show that 1) TSPO plays no role in the regulation or structure of the PTP, 2) endogenous and synthetic ligands of TSPO do not regulate PTP activity through TSPO, 3) outer mitochondrial membrane regulation of PTP activity occurs though a mechanism that does not require TSPO, and 4) hearts lacking TSPO are as sensitive to ischemia-reperfusion injury as hearts from control mice. These results call into question a wide variety of studies implicating TSPO in a number of pathological processes through its actions on the PTP.     

An evolutionary conserved role for anaplastic lymphoma kinase in behavioral responses to ethanol

Anaplastic lymphoma kinase (Alk) is a gene expressed in the nervous system that encodes a receptor tyrosine kinase commonly known for its oncogenic function in various human cancers. We have determined that Alk is associated with altered behavioral responses to ethanol in the fruit fly Drosophila melanogaster, in mice, and in humans. Mutant flies containing transposon insertions in dAlk demonstrate increased resistance to the sedating effect of ethanol. Database analyses revealed that Alk expression levels in the brains of recombinant inbred mice are negatively correlated with ethanol-induced ataxia and ethanol consumption. We therefore tested Alk gene knockout mice and found that they sedate longer in response to high doses of ethanol and consume more ethanol than wild-type mice. Finally, sequencing of human ALK led to the discovery of four polymorphisms associated with a low level of response to ethanol, an intermediate phenotype that is predictive of future alcohol use disorders (AUDs). These results suggest that Alk plays an evolutionary conserved role in ethanol-related behaviors. Moreover, ALK may be a novel candidate gene conferring risk for AUDs as well as a potential target for pharmacological intervention.     

KCNQ Channels Show Conserved Ethanol Block and Function in Ethanol Behaviour

In humans, KCNQ2/3 channels form an M-current that regulates neuronal excitability, with mutations in these channels causing benign neonatal familial convulsions. The M-current is important in mechanisms of neural plasticity underlying associative memory and in the response to ethanol, with KCNQ controlling the release of dopamine after ethanol exposure. We show that dKCNQ is broadly expressed in the nervous system, with targeted reduction in neuronal KCNQ increasing neural excitability and KCNQ overexpression decreasing excitability and calcium signalling, consistent with KCNQ regulating the resting membrane potential and neural release as in mammalian neurons. We show that the single KCNQ channel in Drosophila (dKCNQ) has similar electrophysiological properties to neuronal KCNQ2/3, including conserved acute sensitivity to ethanol block, with the fly channel (IC₅₀ = 19.8 mM) being more sensitive than its mammalian ortholog (IC₅₀ = 42.1 mM). This suggests that the role of KCNQ in alcohol behaviour can be determined for the first time by using Drosophila. We present evidence that loss of KCNQ function in Drosophila increased sensitivity and tolerance to the sedative effects of ethanol. Acute activation of dopaminergic neurons by heat-activated TRP channel or KCNQ-RNAi expression produced ethanol hypersensitivity, suggesting that both act via a common mechanism involving membrane depolarisation and increased dopamine signalling leading to ethanol sedation.     

Mitochondrial Translocator Protein (TSPO) Function Is Not Essential for Heme Biosynthesis

Function of the mammalian translocator protein (TSPO; previously known as the peripheral benzodiazepine receptor) remains unclear because its presumed role in steroidogenesis and mitochondrial permeability transition established using pharmacological methods has been refuted in recent genetic studies. Protoporphyrin IX (PPIX) is considered a conserved endogenous ligand for TSPO. In bacteria, TSPO was identified to regulate tetrapyrrole metabolism and chemical catalysis of PPIX in the presence of light, and in vertebrates, TSPO function has been linked to porphyrin transport and heme biosynthesis. Positive correlation between high TSPO expression in cancer cells and susceptibility to photodynamic therapy based on their increased ability to convert the precursor 5-aminolevulinic acid (ALA) to PPIX appeared to reinforce this mechanism. In this study, we used TSPO knock-out (Tspo^−/−) mice, primary cells, and different tumor cell lines to examine the role of TSPO in erythropoiesis, heme levels, PPIX biosynthesis, phototoxic cell death, and mitochondrial bioenergetic homeostasis. In contrast to expectations, our results demonstrate that TSPO deficiency does not adversely affect erythropoiesis, heme biosynthesis, bioconversion of ALA to PPIX, and porphyrin-mediated phototoxic cell death. TSPO expression levels in cancer cells do not correlate with their ability to convert ALA to PPIX. In fibroblasts, we observed that TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential (ΔΨm) indicative of a cellular metabolic shift, without a negative impact on porphyrin biosynthetic capability. Based on these findings, we conclude that mammalian TSPO does not have a critical physiological function related to PPIX and heme biosynthesis.     

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.     

TSPO over-expression increases motility, transmigration and proliferation properties of C6 rat glioma cells

Gliomas are one of the most malignant cancers. The molecular bases regulating the onset of such tumors are still poorly understood. The translocator protein (TSPO), formerly known as the peripheral-type benzodiazepine receptor, is a mitochondrial permeability transition (MPT)-pore protein robustly expressed in gliomas and involved in the regulation of apoptosis and cell proliferation. TSPO expression levels have been correlated with tumor malignancy. Here we describe the production of C6 rat glioma cells engineered to over-express the TSPO protein with the aim of providing the first direct evidence of a correlation between TSPO expression level and glioma cell aggressiveness. We observed that TSPO potentiates proliferation, motility and transmigration capabilities as well as the ability to overcome contact-induced cell growth inhibition of glioma cells. On the whole, these data demonstrate that TSPO density influences metastatic potential of glioma cells. Since several data suggest that TSPO ligands may act as chemotherapeutic agents, in this paper we also demonstrate that TSPO ligand-induced cell death is dependent on TSPO density. These findings suggest that the use of TSPO ligands as chemotherapeutic agents could be effective on aggressive tumor cells with a high TSPO expression level.     

Bacterial Ortholog of Mammalian Translocator Protein (TSPO) with Virulence Regulating Activity

The translocator protein (TSPO), previously designated as peripheral-type benzodiazepine receptor, is a protein mainly located in the outer mitochondrial membrane of eukaryotic cells. TSPO is implicated in major physiological functions and functionally associated with other proteins such as the voltage-dependent anionic channel, also designated as mitochondrial porin. Surprisingly, a TSPO-related protein was identified in the photosynthetic bacterium Rhodobacter sphaeroides but it was initially considered as a relict of evolution. In the present study we cloned a tspO gene in Pseudomonas fluorescens MF37, a non-photosynthetic eubacterium and we used bioinformatics tools to identify TSPO in the genome of 97 other bacteria. P. fluorescens TSPO was recognized by antibodies against mouse protein and by PK 11195, an artificial ligand of mitochondrial TSPO. As in eukaryotes, bacterial TSPO appears functionally organized as a dimer and the apparent Kd for PK 11195 is in the same range than for its eukaryotic counterpart. When P. fluorescens MF37 was treated with PK 11195 (10⁻⁵ M) adhesion to living or artificial surfaces and biofilm formation activity were increased. Conversely, the apoptotic potential of bacteria on eukaryotic cells was significantly reduced. This effect of PK11195 was abolished in a mutant of P. fluorescens MF37 deficient for its major outer membrane porin, OprF. The present results demonstrate the existence of a bacterial TSPO that shares common structural and functional characteristics with its mammalian counterpart. This protein, apparently involved in adhesion and virulence, reveals the existence of a possible new inter kingdom signalling system and suggests that the human microbiome should be involuntarily exposed to the evolutionary pressure of benzodiazepines and related molecules. This discovery also represents a promising opportunity for the development of alternative antibacterial strategies.     

Regulation of the inner membrane mitochondrial permeability transition by the outer membrane translocator protein (peripheral benzodiazepine receptor)

We studied the properties of the permeability transition pore (PTP) in rat liver mitochondria and in mitoplasts retaining inner membrane ultrastructure and energy-linked functions. Like mitochondria, mitoplasts readily underwent a permeability transition following Ca(2+) uptake in a process that maintained sensitivity to cyclosporin A. On the other hand, major differences between mitochondria and mitoplasts emerged in PTP regulation by ligands of the outer membrane translocator protein of 18 kDa, TSPO, formerly known as the peripheral benzodiazepine receptor. Indeed, (i) in mitoplasts, the PTP could not be activated by photo-oxidation after treatment with dicarboxylic porphyrins endowed with protoporphyrin IX configuration, which bind TSPO in intact mitochondria; and (ii) mitoplasts became resistant to the PTP-inducing effects of N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and of other selective ligands of TSPO. Thus, the permeability transition is an inner membrane event that is regulated by the outer membrane through specific interactions with TSPO.     

A novel conjugable translocator protein ligand labeled with a fluorescence dye for in vitro imaging

A conjugable analogue of the benzodiazepine 4' '-chlorodiazepam (Ro5-4864), C6Ro5-4864 was synthesized to probe the binding sites of translocator protein (18 kDa; TSPO), previously known as the peripheral benzodiazepine receptor for molecular imaging. The amino group in this analogue allows universal conjugation to signaling molecules. Lissamine-C6Ro5-4864, synthesized from C6Ro5-4864 and a lissamine fluorescence dye, was investigated in this study. This imaging agent exhibited micromolar binding affinity (Ki = 2.6 microM) to TSPO and was successfully imaged in TSPO rich glioma and breast cancer cell lines. These findings suggest that C6Ro5-4864 may provide opportunities in imaging disease states where TSPO levels are affected, such as cancer and neurologic diseases.     

Modification of Male Courtship Motivation by Olfactory Habituation via the GABAA Receptor in Drosophila melanogaster

A male-specific component, 11-cis-vaccenyl acetate (cVA) works as an anti-aphrodisiac pheromone in Drosophila melanogaster. The presence of cVA on a male suppresses the courtship motivation of other males and contributes to suppression of male-male homosexual courtship, while the absence of cVA on a female stimulates the sexual motivation of nearby males and enhances the male-female interaction. However, little is known how a male distinguishes the presence or absence of cVA on a target fly from either self-produced cVA or secondhand cVA from other males in the vicinity. In this study, we demonstrate that male flies have keen sensitivity to cVA; therefore, the presence of another male in the area reduces courtship toward a female. This reduced level of sexual motivation, however, could be overcome by pretest odor exposure via olfactory habituation to cVA. Real-time imaging of cVA-responsive sensory neurons using the neural activity sensor revealed that prolonged exposure to cVA decreased the levels of cVA responses in the primary olfactory center. Pharmacological and genetic screening revealed that signal transduction via GABA_A receptors contributed to this olfactory habituation. We also found that the habituation experience increased the copulation success of wild-type males in a group. In contrast, transgenic males, in which GABA input in a small subset of local neurons was blocked by RNAi, failed to acquire the sexual advantage conferred by habituation. Thus, we illustrate a novel phenomenon in which olfactory habituation positively affects sexual capability in a competitive environment.     

The translocator protein (peripheral benzodiazepine receptor) mediates rat-selective activation of the mitochondrial permeability transition by norbormide

We have investigated the mechanism of rat-selective induction of the mitochondrial permeability transition (PT) by norbormide (NRB). We show that the inducing effect of NRB on the PT (i) is inhibited by the selective ligands of the 18kDa outer membrane (OMM) translocator protein (TSPO, formerly peripheral benzodiazepine receptor) protoporphyrin IX, N,N-dihexyl-2-(4-fluorophenyl)indole-3-acetamide and 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one; and (ii) is lost in digitonin mitoplasts, which lack an intact OMM. In mitoplasts the PT can still be induced by the NRB cationic derivative OL14, which contrary to NRB is also effective in intact mitochondria from mouse and guinea pig. We conclude that selective NRB transport into rat mitochondria occurs via TSPO in the OMM, which allows its translocation to PT-regulating sites in the inner membrane. Thus, species-specificity of NRB toward the rat PT depends on subtle differences in the structure of TSPO or of TSPO-associated proteins affecting its substrate specificity.