117 TSPO expression in healthy and diseased brain respiration62, increase oxygen consumption63 and ATP synthesis64. 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 vivo55,65,66. These studies were later extended to neurosteroid synthesizing glia cells in the brain67-70. TSPO ligands were also shown to affect intracellular cholesterol trafficking and lipid droplet accumulation, a function that may not be related to steroidogenesis71,72. 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 proteincomplex composition and not only by the interaction with TSPO alone73. In addition, it was recently shown that TSPO ligands have different occupancy times for TSPO and this affects their ability to induce steroid formation74,75. 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 effects65,76. In addition, TSPO ligands were found to exert celltype specific effects raising again the question of the role of the microenvironment, ligand residence time, and the presence of endogenous ligands77,78. 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)50,51,58,79-84. 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 anti-apoptotic effects, as well as anti-proliferative properties, acting in a biphasic manner23,24,28,35-37,85. Molecular structure and cellular functions The drug ligand binding domains of TSPO have been mapped86 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-terminus87,88. The drug and cholesterol binding domains were found to be in distinct domains of the protein results confirmed by NMR86,87,89. Moreover, these findings were further confirmed in structural studies by NMR and crystallography studies that reported the atomic structure of TSPO90-95. 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 TSPO90,92,93,96. 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
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