TSPO participates in many essential mitochondria-based physiological processes, including metabolism and cellular bioenergetics, mitochondrial respiration, cholesterol transport and steroidogenesis, immunomodulation, porphyrin transport, and heme biosynthesis [
20‐
24]. It has also been suggested that TSPO may play critical roles in cell proliferation, tumorigenesis, and apoptosis [
24‐
26]. In order to discern and evaluate the function of any protein, particularly one like TSPO that seems to be multifunctional, one must consider specific characteristics that could provide clues to the possible roles it may play. The characteristics that need to be investigated should include its (i) tissue, cellular, and subcellular localization, (ii) characteristics and effects of endogenous and exogenous ligands, (iii) molecular structure and cellular functions, (iv) genetics and genetic models, and (v) evolution.
Tissue, cellular, and subcellular localization
TSPO was first characterized for its ability to bind with specificity and high-affinity various classes of chemicals such as benzodiazepines, isoquinoline carboxamides, indole acetamides, pyrazolopyrimidines, and aryloxyanilides, as well as endogenous ligands including porphyrins, the endozepine diazepam binding inhibitor [
20‐
24,
27‐
33]. Radioligand binding and later on immunodetection studies revealed that the distribution pattern of TSPO between rodents and humans is similar; secretory and glandular tissues were particularly rich in TSPO. These studies also indicated that although TSPO is present in most tissues in most species at various levels of expression, it is most abundant in steroid-synthesizing adrenal and gonadal tissues. The heart and kidney express intermediate levels of TSPO, while lower levels are found in the liver and brain. Interestingly, when considering the mitochondrial content of each tissue, there is not always a clear correlation between TSPO levels and mitochondrial content. This finding suggests that tissue- and cell-specific factors regulating
TSPO gene expression are driving TSPO content rather than factors driving mitochondria formation.
Steady-state mRNA profiling shows that
TSPO mRNA is present in all tissues and correlates well with reported protein expression levels [
25]. Moreover, the expression patterns of mouse TSPO were found to be well-correlated and similar to that reported for human
TSPO [
34,
35].
TSPO levels were found to be elevated in cancer cell lines and numerous cancers suggesting a role for TSPO in cell proliferation and carcinogenesis [
24,
25,
29,
36‐
38]. Increased TSPO levels in cancer are due to gene amplification; Sp1, Sp3, and Sp4 transcription factor regulation of constitutive TSPO expression; and epigenetic modifications of the proximal promoter and first intron [
25,
39,
40].
TSPO is an integral outer mitochondrial membrane protein spanning the membrane through its 5 α-helical domains [
41‐
54]. While TSPO is a nuclear encoded protein, unlike most mitochondrial proteins, TSPO does not possess a mitochondrial targeting sequence, although it contains information on the C-terminus that is essential for its mitochondrial import [
55]. After integration into the OMM, TSPO forms dimers and sometimes polymers [
20,
56,
57] at the outer and inner mitochondrial membrane contact sites where it becomes part of a larger protein complex [
58]. This complex includes the OMM voltage-dependent anion channel 1 (VDAC1); ATPase family AAA domain-containing protein 3 (ATAD3), a protein that spans across the mitochondrial membranes, and in steroidogenic cells; and the inner mitochondrial membrane cytochrome P450 side-chain cleavage enzyme (CYP11A1), among others [
58,
59]. In addition, cytosolic, endoplasmic reticulum and Golgi proteins have been shown to associate with TSPO to form functional complexes [
59‐
61]. When assembled together, these proteins function as a signal transduction complex, or “transduceosome” mediating the transmission of information to mitochondrial TSPO.
Although 95% of TSPO is found in the mitochondria, the protein can be found in intracellular locations other than mitochondria, such as the (peri)nuclear region and plasma membrane, likely playing different functions. Nevertheless, non-mitochondrial TSPO [
43,
62] has received little attention so far.
Characteristics and effects of endogenous and exogenous ligands
TSPO is involved primarily in the mitochondria of steroid synthesizing cells. Steroidogenesis in the mitochondria begins with the transport of substrate cholesterol from intracellular stores into the mitochondria. Therefore, the role or roles of TSPO in mitochondrial steroidogenesis and cholesterol transport, in particular, were investigated.
With the availability of high-affinity TSPO ligands, the function of TSPO in various tissues was explored, aiming to assess whether these ligands could affect mitochondrial function, including steroid production. Several TSPO ligands were found to affect mitochondrial respiration [
63] and increase oxygen consumption [
64] and ATP synthesis [
65]. At the same time, detailed studies demonstrated the ability of these ligands to induce cholesterol transport into mitochondria and steroid formation in all steroidogenic cells in vitro and in vivo [
56,
66,
67]. These studies were later extended to neurosteroid synthesizing glia cells in the brain [
68‐
71]. TSPO ligands were also shown to affect intracellular cholesterol trafficking and lipid droplet accumulation, a function that may not be related to steroidogenesis [
72,
73].
However, there are ligand-specific differences as well as off-target effects. These differences may be explained by the tissue and cell-specific microenvironment and the presence of endogenous ligands, e.g., porphyrins and endozepines, in specific tissues/cells that may compete with the exogenous ligand. Moreover, the fact that TSPO exists within large protein complexes suggests that TSPO ligand selectivity may be governed by the protein-complex composition and not only by the interaction with TSPO alone [
74]. In addition, it was recently shown that TSPO ligands have different occupancy times for TSPO and this affects their ability to induce steroid formation [
75,
76]. Concerning the off-target effects, most of the time these are linked to the use of high concentrations of TSPO ligands, thousands of time higher than the affinity of these compounds for TSPO. Indeed, lipophilic TSPO ligands used at high concentrations are likely to interact with membranes or other not yet identified targets resulting in off-target effects [
66,
77]. In addition, TSPO ligands were found to exert cell-type specific effects raising again the question of the role of the microenvironment, ligand residence time, and the presence of endogenous ligands [
78,
79].
Over the years, the effects of TSPO drug ligands with various mitochondrial activities/functions have also been shown, including changes in VDAC1, F-ATP synthase and ANT activities, modulation of reactive oxygen species (ROS) production, and calcium levels and effects on mitochondrial membrane potential and permeability transition pore (MPTP) [
51,
52,
59,
80‐
85]. These effects were found to be tissue- and cell-specific and sometimes ligand-specific or observed only in cell lines. However, in some cases, the effects were observed in the presence of micromolar concentrations of TSPO ligands, far beyond the affinity of the protein for the compounds. The complex formed by the mitochondrial TSPO in association with VDAC1 has been suggested to have a role in apoptosis, possibly through MPTP opening, and cholesterol transport. TSPO drug ligands have been found to exert both proliferative and antiapoptotic effects, as well as antiproliferative properties, acting in a biphasic manner [
24,
25,
29,
36‐
38,
86].
Molecular structure and cellular functions
The drug ligand binding domains of TSPO have been mapped [
87] and it was subsequently shown that TSPO is a high-affinity cholesterol binding protein containing a conserved cholesterol recognition amino acid consensus domain in the C-terminus [
88,
89]. The drug and cholesterol binding domains were found to be in distinct domains of the protein results confirmed by NMR [
87,
88,
90]. Moreover, these findings were further confirmed in structural studies by NMR and crystallography studies that reported the atomic structure of TSPO [
91‐
96]. These studies also proposed that the functional TSPO is a dimer, that ligand binding to TSPO can promote cholesterol movement, and that cholesterol is an allosteric regulator of TSPO [
91,
93,
94,
97].
The ability of TSPO to bind drug ligands and cholesterol is its two major intrinsic properties and mostly likely the ones determining its function. We summarized above the reported effects of TSPO ligands on mitochondrial function. Although in steroidogenic and liver cells the role of a cholesterol binding protein segregating the steroidogenic pool of cholesterol from structural cholesterol and facilitating its import into the mitochondria for steroid and formation is obvious, for other cells it is not so clear. However, cholesterol transfer in the inner mitochondrial membrane is needed for biogenesis of mitochondrial membranes during cell proliferation and/or repair. TSPO may also function as a sink for cholesterol which when free could be toxic for the cells. It is also possible that TSPO may be facilitating the movement of free cholesterol from the mitochondria to other organelles, as shown in astrocytes [
72], fibroblasts [
72], macrophages [
98], retinal cells [
99], and the steroidogenic Leydig cells [
73]. Moreover, TSPO-mediated accumulation of free cholesterol in the mitochondria may affect mitochondrial membrane fluidity/permeability, fission/fusion processes, membrane protein/transporter function(s), and/or membrane potential [
72,
83,
100‐
102].
TSPO was also shown to regulate mitophagy [
59,
103]. TSPO, by binding to VDAC1, reduces mitochondrial coupling and promotes an overproduction of ROS that counteracts Parkin-mediated ubiquitination of proteins. These data suggested TSPO as an element in the regulation of mitochondrial quality control by autophagy. Further studies showed that TSPO deregulates mitochondrial Ca
2+ signaling, leading to a parallel increase in the cytosolic Ca
2+ pools that activate the Ca
2+-dependent NADPH oxidase, thereby increasing ROS [
104]. The inhibition of mitochondrial Ca
2+ uptake by TSPO is a consequence of the phosphorylation of VDAC1 by PKA, which is recruited to the mitochondria by ACBD3, VDAC1, ACBD3, PKA, and all transduceosome components recruited at TSPO. This is proposed as a novel OMM-based pathway to control intracellular Ca
2+ dynamics and redox transients in cytotoxicity [
104].
Genetics and genetic models
A series of articles came out in the last 15 years assessing the direct role of TSPO in various cellular pathways. First, the role of TSPO in opening the MPTP in liver mitochondrial function was investigated in an animal model depleted of liver TSPO [
105]. The data obtained showed that the absence of TSPO does not affect liver MPTP function. Then, studies in rodents with genetic depletion of
TSPO led to conflicting results including no effect on steroid synthesis [
106‐
108], reduced steroid output, inhibition of corticosteroid response to adrenocorticotropic hormone, changes in lipid homeostasis in Leydig cells and reduction of circulating testosterone levels, and suppression of neurosteroid formation [
109‐
112].
In addition, discordant data was reported on MA-10 mouse Leydig cells. Knockdown of
TSPO expression using antisense oligonucleotides or antisense RNA reduces the ability of the cells to form steroids, while CRISPR/Cas9-guided
TSPO deletion has either no effect or abolishes steroid synthesis [
113‐
116]. These differences have been discussed in detail in other reviews [
66,
67].
Among all these studies, it seems that there is consistency between laboratories on the role of TSPO in neurosteroid formation where genetic deletion of TSPO led to reduced neurosteroid synthesis [
110,
112]. These results suggest that the role of TSPO in steroid formation may be primary and rate-determining in cells where steroid formation is independent on hormonal control, e.g., brain, compared to the classical peripheral steroid forming gonads and adrenal where pituitary hormones control the massive steroid production. In peripheral steroidogenic organs, TSPO may play a secondary role or play a role in cases where the cells do not respond to pituitary hormones, as in male hypogonadism where TSPO ligands can recover the drug- or age-induced reduction in androgen formation [
66].
Numerous biochemical, pharmacological, and clinical data in the field of photodynamic therapy in oncology have demonstrated the role of ability of TSPO to bind porphyrins and its role in porphyrin and heme transport and synthesis [
117‐
119]. Using the same mice as before [
107], the same group failed to show a role for TSPO in porphyrin and heme biosynthesis or transport [
120].
TSPO deficiency decreased the oxygen consumption rate and mitochondrial membrane potential in mouse fibroblasts [
120], MA-10 mouse Leydig cells [
116], and C20 human microglia cells where it also reduced respiratory function [
121]. Mitochondrial membrane potential depends on the flux of respiratory substrates adenosine triphosphate, adenosine diphosphate, and Pi through VDAC. Adenine nucleotide translocator also plays a role in maintenance of the membrane potential [
116]. Therefore, TSPO likely controls cellular and mitochondrial metabolism via regulation of the mitochondrial membrane potential and affects OMM permeability and/or outer and inner membrane contacts/fusion.
Interestingly, lack of TSPO was shown to affect mitochondrial respiration and increase oxygen consumption in some cell and animal
Tspo KO models, but not in others [
63,
65,
79,
107,
108,
120,
121]. More recent studies also failed to show a direct link of TSPO to F-ATP synthase, which was shown to form the MPTP [
122].
The differences underlying the disparate results from these genetic animal and cell models are not well understood. However, they clearly indicate differences between the pharmacology of TSPO and its intrinsic cellular functions. It is also likely that species differences, the presence of external or intrinsic stimuli, as well as differences in age, sex, and metabolic status of the species used may control the expression of TSPO. Considering that TSPO is one of the evolutionarily oldest proteins (see below), we proposed that it serves as the basis for fundamental functions and, thus, in case of its absence, compensatory mechanisms may have evolved. Moreover, even if its absence may not always affect animal phenotype, its presence, concentrated at the OMM, plays a regulatory role in mitochondrial function and associated tissue-specific phenotypes. Moreover, its presence provides us with a molecular target able to modulate mitochondrial and cell functions.
TSPO genetics in humans provide some of the most important information on the function of this protein. No humans have been identified lacking TSPO. In humans, the presence of a number of polymorphisms have been identified in the
TSPO gene, including rs6971 [
123]. This polymorphism causes a non-conservative amino acid substitution, Ala147Thr, resulting in altered binding affinity of TSPO for specific ligands [
123]. The presence of this
TSPO polymorphism has been linked to the function of the hypothalamic-pituitary-adrenal axis, predisposing carriers to psychiatric disorders [
124‐
127], and potentially impairing the response of patients to anxiolytic TSPO drug ligands [
128,
129]. The presence of this
TSPO polymorphism was linked to reduced pregnenolone [
130] and adrenocorticotropic hormone (ACTH)-induced corticosteroid levels [
110] and shown to be associated with dysregulated cortisol rhythms and consequent clinical exacerbations in bipolar disorders [
131]. This finding provides clear evidence of the link between TSPO, cholesterol binding, and steroid formation under normal and stress conditions.
Evolution
TSPO is an evolutionary conserved 3.5-billion-year-old protein [
132]. TspO, named for its high tryptophan content and apparent role in the regulation of the transition between photosynthesis and respiration, is the mammalian TSPO ortholog in the photosynthetic bacterium
Rhodobacter [
133], a close living relative of mitochondria [
57]. Detailed evolutionary studies indicated that the
Tspo gene family has been expanded by gene duplications from a bacterial environmental sensor or signal transducer to a functional bioregulator adapted to organism-, tissue-, cell-, and organelle-specific needs. Interestingly, the mammalian protein is able to rescue the phenotypes of bacterial
TspO KO suggesting a conserved function [
134] and that one compensates for the loss of oxygen sensing function that occurs when the other is depleted.
An additional Tspo family member, Tspo2, has been characterized [
135]. Comparative analysis of Tspo1, the first family member to be identified, and Tspo2 structure and function indicates that TSPO2 was characterized by the loss of diagnostic drug ligand binding, but retention of cholesterol binding properties, and is involved in cholesterol redistribution during erythropoiesis [
135]. Whether there are additional family members in mammals or humans remains to be determined. However, the highly conserved sequence would seem to indicate that such expansion in members was not needed to support the rich expansion of cellular functions.
Pharmacological and structural evidence supports TSPO functioning in tetrapyrrole biosynthesis, porphyrin transport, heme metabolism, cholesterol transport/trafficking, steroid formation, control of ROS levels, and the protection of mitochondria from free radical damage. All evolutionarily conserved functions are linked to mitochondria and affected by changes in mitochondrial membrane potential, a function dependent on the presence of TSPO. Few years ago, we proposed that the central role of TSPO throughout evolution is in oxygen-mediated metabolism. This central function has diversified roles in tissue- and cell-specific signaling, metabolism, cholesterol trafficking, immunological responses, apoptosis, steroid synthesis, and host-defense response to disease and injury, all oxygen-mediated pathways [
22,
132].