The Translocator Protein

TSPO FUNCTIONAL ROLES

The best characterized function of TSPO is the regulation of cholesterol translocation through mitochondrial membranes, which is the rate-determining step in steroid biosynthesis (11). Once in the mitochondria, cholesterol is converted to pregnenolone via an oxidative cleavage of its side chain by cytochrome P450_SCC (2). TSPO ligands such as cholesterol are able to initiate steroidogenesis by binding to the protein.

The presence of TSPO at the MPTP also implicates the protein in the regulation of apoptotic and necrotic cell death, with ligands being able to cause opening of the MPTP, resulting in induction of apoptosis (11). TSPO ligands also inhibit cell proliferation in cancer cell lines, causing an accumulation of cells in the G₁/G₀ phase of the cell cycle, ultimately inhibiting the progression of cells to the S and G₂/M phase, in which cell proliferation occurs (12). Effects on cell proliferation may be due to a small proportion of the protein being expressed within the cell nucleus. However, the effects of TSPO ligands on apoptosis and cell proliferation vary depending on ligand concentration, with antiproliferative and proapoptotic actions at micromolar concentrations but proproliferative effects through stimulation of mitosis and antiapoptotic effects at nanomolar concentrations (11,13).

Because the TSPO is expressed on microglia and other immune cells, this protein also plays a role in immune regulation. Ligands that bind to TSPO are able to exert neuroprotective effects by modulating cytokine production (1,2,14). However, whereas neuroprotective effects of TSPO ligands are mediated at micromolar concentrations, more recent studies suggest that nanomolar concentrations mediate different functional profiles (14).

TSPO BINDING SITES

Although research suggests that there exist multiple TSPO binding sites, the nature of these sites and their functional significance is poorly understood. Two ligands have been essential for characterizing the TSPO: the benzodiazepine Ro 5-4864 and the isoquinoline carboxamide PK11195 (Fig. 2), both of which are selective for the TSPO and display nanomolar binding affinity. Although these ligands exhibit saturable binding and reciprocal competition in radioligand binding assays, results are not consistent across species and can be modified separately in both rats and humans (2). Furthermore, site-directed mutagenesis studies suggest certain residues in the first putative loop of TSPO are important for the binding of Ro 5-4864 but not PK11195. Thus, it is thought that PK11195 and Ro 5-4864 bind to heterogeneous sites at TSPO, either overlapping or allosterically coupled.

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FIGURE 2.

Saturation and competition binding profiles of various TSPO ligands using brain samples from different species in vitro. *Competition binding experiments conducted using ³H-PK11195 as radioligand. ^†Where binding was best-fit to 2-site model using Scatchard analysis. H = high-affinity site; L = low-affinity site.

Studies also describe PK11195 binding to multiple sites, which contradicts the initial finding that it bound to a single population of saturable sites. Scatchard analysis of ³H-PK11195 binding to Ehrlich tumor cells revealed 2 independent binding sites (13). Ehrlich tumor cells are a murine ascitic cell line, possessing high concentrations of polymeric TSPO. Alternatively, the induction of a high-affinity TSPO binding site may be related to an increase in steroid formation (15). Thus, it is possible that conformation of TSPO is altered to activate cholesterol delivery to the inner mitochondrial membrane. Considering that TSPO polymerization is associated with steroidogenesis, it is possible that polymerization results in a conformational change to TSPO binding sites, potentially with allosteric effects.

More recently, studies in the human brain using various TSPO PET radioligands have revealed that TSPO binding sites vary across individuals and tissue type (16). For example, whereas ³H-PBR28 binds competitively to the ³H-PK11195 site in the brains of rhesus monkeys (17), ³H-PBR28 binds to multiple sites in postmortem human brains, with different affinities across a range of patients (Fig. 2) (18). Saturation data depict ³H-PBR28 binding to a low-affinity TSPO binding site, a high-affinity binding site, or a population of mixed-affinity binding sites, whereby data best fit a 2-site model (18). Conversely, ³H-PK11195 binds in the same manner in brain samples across all patients (18). Interestingly, however, differences in PK11195 binding across patients can be observed in some peripheral tissue (heart and lungs), correlating to the changes seen in the binding of PBR28 in brain (16). Since this discovery, several additional ligands that are commonly used for PET have been shown to display variable binding profiles across human subjects, with some ligands binding to multiple binding sites in select patients (Fig. 2) (19). This is despite the observation of a simple 1-site binding interaction when initially screened in the rat or rhesus monkey (Fig. 2).

It is possible that the variability in binding across human subjects could be a consequence of the level and nature of microglial activation, or the dose at which TSPO ligands are evaluated. Nevertheless, the concept of multiple TSPO binding sites and variable conformational states of the protein needs to be considered when developing and evaluating new PET TSPO ligands. This will aid in the development of adequate methods for quantitative analysis and a better understanding of what form of TSPO PET ligands are imaging.