Translocator protein (TSPO) and PET

The translocator protein 18kDa (TSPO), earlier called peripheral benzodiazepine receptor (PBR) and mitochondrial benzodiazepine receptor, is a five transmembrane domain protein. TSPO is mainly situated in the outer mitochondrial membrane, but it is also present in Golgi apparatus, lysosomes, peroxisomes, nucleus, and on plasma membrane, also in mature human red cells (Batarseh & Papadopoulos, 2010). In mitochondria, TSPO may have role in regulating energy and ROS homeostasis, and transporting cholesterol into mitochondria, and TSPO concentrations are high in steroid producing tissues. Depending on the tissue, TSPO is involved in apoptosis, cell proliferation and differentiation (Batarseh & Papadopoulos, 2010). Mitochondrial dysfunction or abnormal microglia autophagy may be seen as reduced TSPO expression (Zürcher et al., 2020). TSPO has been found to be upregulated in certain tumours. TSPO ligands modulate mitochondrial and cytosolic Ca²⁺ dynamics.

Secretory and glandular tissues, such as adrenal glands, pineal gland, salivary glands, and olfactory epithelium contain high levels of TSPO; intermediate levels in renal and myocardial tissues, and low levels in the brain and liver (Gavish et al., 1999; Batarseh & Papadopoulos, 2010). TSPO is expressed in macrophages, and could be targeted in inflammation imaging.

In the brain parenchyma TSPO is located in glial cells (microglia and astrocytes), and has thus been used as a biomarker of activated glial cells (Chen & Guilarte, 2008; Alam et al., 2017; Nomura et al., 2021). Rather than activation phenotype, TSPO expression may predominantly reflect cell density (Nutma et al., 2021). TSPO is present also in the neurovascular unit, including endothelial cells, smooth muscle cells, and vascular red blood cells (Wimberley et al., 2021).

PET radioligands

[¹¹C]-(R)-PK11195 is the most used PET radioligand for imaging TSPO. Because of the high lipophilicity of PK11195, a relatively high fraction of measured tissue uptake is due to nonspecific binding. Other tracers with better target-to-background ratio have been developed (Dollé et al., 2009; Kreisl et al., 2010; Luus et al., 2010; Boutin et al., 2015; Fujita et al., 2017; Alam et al., 2017; Keller et al., 2017, 2018, and 2019; Yan et al., 2023).

Although the PET radioligands exhibit saturable binding and reciprocal competition in binding assays, results are not consistent across species (Kreisl et al., 2010; Scarf & Kassiou, 2011). Different PET tracers may bind to heterogeneous sites at TSPO, either overlapping or allosterically coupled, for example caused by TSPO polymerization (Scarf & Kassiou, 2011).

TSPO radioligands are mainly used for imaging neuroinflammation, for instance in MS and AD. [¹¹C]DPA-713 has shown wide-spread glial activation in the brains of patients with post-treatment Lyme disease symptoms (Coughlin et al., 2018), and [¹¹C]PBR28 in patients with fibromyalgia (Albrecht et al., 2019).

TSPO ligands are being studied for imaging of atherosclerotic plaques. [¹⁸F]FEMPA uptake is increased in plaques with high macrophage content, but no difference between atherosclerotic and healthy mice was observed (Hellberg et al., 2017).

Detection of mitochondria-rich target tissues

TSPO ligands shown promise in detecting brown adipose tissue because of its high content of mitochondria (Ran et al., 2018; Hartimath et al., 2020; Oh et al., 2020).

Mitochondrial dysfunction leads to reduced TSPO radioligand uptake.

rs6971 polymorphism

In humans, rs6971 polymorphism in the TSPO gene affects the binding of cholesterol and the rate of steroid synthesis (Owen et al., 2017), and is associated with bipolar disorder (Colasanti et al., 2013) and plasma cholesterol level (Kim et al., 2018).

The rs6971 polymorphism leads to three TSPO radioligand binding profiles: high-affinity binders (HAB), low-affinity binders (LAB), and mixed-affinity binders (MAB) which express both TSPO types in equal proportion (Owen et al, 2011 and 2012; Kreisl et al., 2010 and 2013a; Yoder et al, 2013). [³H]PK11195 was found to bind similarly in brain samples across all patients (Owen et al., 2010), but in heart and lungs differences in PK11195 across patients were seen (Kreisl et al., 2010). With other ligands than PK11195, the variable binding affinity to the three identified human sub-populations (Owen et al., 2011) is the main source of variability in brain PET results. This has been specifically shown for PBR28 (Owen et al., 2012) and [¹⁸F]FEPPA (Mizrahi et al., 2012).

Radioligand uptake in LAB group is generally too low to be reliably quantified with PET; therefore genotyping must be done prior to PET to omit the LAB subjects. It has been speculated that non-binders might not have been identified with [¹¹C]-(R)-PK11195 because of its poor specific-to-nonspecific binding ratio (Fujita et al., 2008; Kreisl et al., 2010; Fujita et al., 2017). Of four TSPO tracers [¹¹C]-(R)-PK11195, [¹¹C]PBR28, [¹⁸F]DPA-713, and [¹¹C]ER176, the only one that can be used to study all three binding groups is [¹¹C]ER176 (Ikawa et al., 2017; Fujita et al., 2017).

PET radioligands which are less affected by TSPO polymorphism are being developed (Tiwari et al., 2015; Di Grigoli et al., 2015; Fujita et al., 2017; Kim et al., 2020; MacAskill et al., 2021a; Ramakrishnan et al., 2021). [¹⁸F]LW223 binding is not susceptible to human rs6971 polymorphism, and is able to detect inflammation in rat myocardial infarction model (MacAskill et al., 2021a and 2021b). [¹⁸F]FEBMP is insensitive to the polymorphism, and is selective for glial TSPO over vascular TSPO (Ji et al., 2021).

Quantification of TSPO using PET

Binding of currently available TSPO PET radioligands is reversible, and 1- or 2-tissue compartmental models and multiple-time graphical analysis for reversible binding have been used to analyse the PET data. Spectral analysis can be used to produce high-quality V_T images (Veronese et al., 2021).

In the brain the simplified reference tissue model (SRTM) can be applied, although an ideal reference tissue (containing no TSPO) is not available. A clustering method has been introduced to produce a reference curve for brain [¹¹C]-(R)-PK11195 studies (Turkheimer et al., 2007). Wimberley et al (2021) have reviewed the brain TSPO quantification methods and their limitations.

Small animal studies are usually analysed by SUV or brain tissue-to-cerebellum ratio, for instance in the mice studies using [¹⁸F]GE-180 (López-Picón et al., 2018; Hellberg et al., 2018). In certain animal models of brain diseases, for instance the EAE animal model of MS, the healthy hemisphere can be used as reference region (Airas et al., 2015; Vainio et al., 2019 and 2022).

Vascular binding

TSPO level in the normal brain is very low, and thus the blood vessel uptake of many TSPO ligands becomes predominant. In case of [¹¹C]-(R)-PK11195, also the nonspecific binding hinders the visualization of low levels of glial activation. [¹⁸F]FEBMP and [¹¹C]Ac5216 are relatively selective for glial TSPO over vascular TSPO (Ji et al., 2021).

In AD brain, vascular fibrosis decreases the size of lumens, reduces blood volume, and leads to loss of vascular TSPO, particularly in smooth muscles, as shown by immunohistochemistry (Tomasi et al., 2008).

Vascular component can be included in SRTM as a linear term for both the target and reference region, increasing the estimated BP_ND difference between AD patients and control subjects (Tomasi et al., 2008).

Sink effect

The abundance of TSPO in peripheral organs affects the availability of PET radioligands for binding in the brain (Turkheimer et al., 2007). Displacement studies with unlabelled PK11195 have even resulted in increased uptake of [¹¹C]-(R)-PK11195 in the brain, and blocking studies of [¹⁸F]FEPPA and [¹¹C]PBR28 based on SUV have failed because of increased tracer availability with higher mass (Wilson et al., 2008). This emphasizes the need for full quantification with proper input functions (arterial plasma or reference tissue input), instead of semi-quantitative methods like SUV.

Higher levels of BMI and insulin resistance are associated with higher cerebral [¹¹C]-(R)-PK11195 distribution volume ratio (Ekblad et al., 2023), which supposedly is not affected by the sink effect.

Assessment of peripheral TSPO level may be less affected by the mass effect: a blocking study in atherosclerosis model in mice demonstrated decreased tissue SUV of [¹⁸F]GE-180, even with increased retention of activity in the blood (Hellberg et al., 2018).

Plasma protein binding

An additional factor which may add to the variance is the binding of TSPO ligands to plasma proteins. PK11195 has been shown to bind strongly to AGP, levels of which vary during infection and inflammatory diseases, and AGP could be locally synthesized at the sites of brain injury (Lockhart et al., 2003).

Literature

Airas L, Rissanen E, Rinne JO. Imaging neuroinflammation in multiple sclerosis using TSPO-PET. Clin Transl Imaging 2015; 3: 461-473. doi: 10.1007/s40336-015-0147-6.

Alam MM, Lee J, Lee S-Y. Recent progress in the development of TSPO PET ligands for neuroinflammation imaging in neurological diseases. Nucl Med Mol Imaging 2017; 51: 283-296. doi: 10.1007/s13139-017-0475-8.

Batarseh A, Papadopoulos V. Regulation of translocator protein 18 kDa (TSPO) expression in health and disease states. Mol Cell Endocrinol. 2010: 327: 1-12. doi: 10.1016/j.mce.2010.06.013.

Chauveau F, Becker G, Boutin H. Have (R)-[¹¹C]PK11195 challengers fulfilled the promise? A scoping review of clinical TSPO PET studies. Eur J Nucl Med Mol Imaging 2021; 49: 201-220. doi: 10.1007/s00259-021-05425-w.

Chen M-K, Guilarte TR. Translocator protein 18 kDa (TSPO): molecular sensor of brain injury and repair. Pharmacol Therapeutics 2008; 118: 1-17. doi: 10.1016/j.pharmthera.2007.12.004.

Gavish M, Bachman I, Shoukrun R, Katz Y, Veenman L, Weisinger G, Weizman A. Enigma of the peripheral benzodiazepine receptor. Pharm Rev. 1999; 51(4): 629-650. PMID: 10581326.

Guo Q, Owen DR, Rabiner EA, Turkheimer FE, Gunn RN. A graphical method to compare the in vivo binding potential of PET radioligands in the absence of a reference region: application to [¹¹C]PBR28 and [¹⁸F]PBR111 for TSPO imaging. J Cereb Blood Flow Metab. 2014; 34: 1162-1168. doi: 10.1038/jcbfm.2014.65.

Hinz R, Boellaard R. Challenges of quantification of TSPO in the human brain. Clin Transl Imaging 2015; 3: 403-416. doi: 10.1007/s40336-015-0138-7

Largeau B, Dupont A-C, Guilloteau D, Santiago-Ribeiro M-J, Arlicot N. TSPO PET imaging: from microglial activation to peripheral sterile inflammatory diseases? Contrast Media & Mol Imaging 2017; 6592139.

Luus C, Hanani R, Reynolds A, Kassiou M. The development of PET radioligands for imaging the translocator protein (18 kDa): what have we learned? J Label Compd Radiopharm. 2010; 53: 501-510. doi: 10.1002/jlcr.1752.

Owen DRJ, Gunn RN, Rabiner EA, Bennacef I, Fujita M, Kreisl WC, Innis RB, Pike VW, Reynolds R, Matthews PM, Parker CA. Mixed-affinity binding in humans with 18-kDa translocator protein ligands. J Nucl Med. 2011; 52: 24-32. doi: 10.2967/jnumed.110.079459.

Scarf AM, Kassiou M. The translocator protein. J Nucl Med. 2011; 52: 677-680. doi: 10.2967/jnumed.110.086629.

Turkheimer FE, Rizzo G, Bloomfield PS, Howes O, Zanotti-Fregonara P, Bertoldo A, Veronese M. The methodology of TSPO imaging with positron emission tomography. Biochem Soc Trans. 2015; 43: 586-592. doi: 10.1042/BST20150058.

Van Camp N, Lavisse S, Roost P, Gubinelli F, Hillmer A, Boutin H. TSPO imaging in animal models of brain diseases. Eur J Nucl Med Mol Imaging 2021; 49: 77-109. doi: 10.1007/s00259-021-05379-z.

Wimberley C, Lavisse S, Hillmer A, Hinz R, Turkheimer F, Zanotti-Fregonara P. Kinetic modeling and parameter estimation of TSPO PET imaging in the human brain. Eur J Nucl Med Mol Imaging 2021; 49: 246-256. doi: 10.1007/s00259-021-05248-9.

Tags: TSPO, Inflammation, Glial cells, Mitochondria, Cholesterol

Updated at: 2023-08-02
Created at: 2012-09-14
Written by: Vesa Oikonen