What does the Peripheral Benzodiazepine Receptor do?

After 30+ years of research, one would think we should know by now.

During my research career, my lab demonstrated the structural homology between benzodiazepine class molecules and thyroid hormones/metabolites. We were the first to describe the inhibition of the membrane thyroid hormone transporter by benzodiazepines and used a detailed Structure Activity Relationship to illustrate its potential pharmacophore.

These molecular interactions prompted us to look for interactions at other cellular sites. One series of investigations included the Peripheral Benzodiazepine Receptor, distinctly different from the central CNS site. It is found most commonly in the mitochondrial outer membrane. When isolated, it turned out to be a highly conserved five transmembrane domain 18 kDa protein, putatively proposed to translocate cholesterol. Thus was named, the mitochondrial translocator protein (TSPO). However, over the years, research ‘progress’ never fully clarified the role, but the name stuck.

Three-dimensional structures of representative TSPO ligands. (a) endogenous ligands, such as protoporphyrin IX, diazepam binding inhibitor (DBI), and phospholipase A2 (PLA2); (b) classical synthetic ligands, such as 7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and PK 11195; (c) novel ligands, such as N,N-di-n-hexyl 2-(4-fluorophenyl)indole-3-acetamide (FGIN-1-27), 7-chloro-N,N,5-trimethyl-4-oxo-3-phenyl-3,5-dihydro-4H-pyridazino[4,5-b]-indole-1-acetamide (SSR180575) and 3,5-Seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303).

Commonly used to treat anxiety and insomnia, benzodiazepines chronic use may cause cognitive impairment and increase the risk for dementia. A recent study reported that diazepam impairs the structural plasticity of dendritic spines, causing cognitive impairment in mice model. Diazepam induced these deficits via the mitochondrial 18 kDa translocator protein, rather than classical γ-aminobutyric acid type A receptors. Changes to microglial morphology, and phagocytosis of synaptic material, altered synaptic plasticity. When the diazepam treatment was discontinued, synaptic effects were eventually reversible.

Thyroid Hormone / Benzodiazepine Interactions

I wrote a comprehensive literature review in 1993, focussed on neuropsychiatric aspects of benzos and thyroid hormones, and where they might interact. The PBR/TSPO site was included in that analysis.

from Kragie 1993 Neuropsychiatric Implications of Thyroid Hormone and Benzodiazepine Interactions.

Then, in 1994, my lab looked at the affinity and density of the PBR/TSPO sites in rat liver and cardiac tissues, comparing hypothyroid and euthyroid states.

We measured the density and affinity of the peripheral benzodiazepine receptor (PBR) ligand, [3H]4′-Cl-diazepam, in cardiac ventricular and liver homogenates from thyroidectomized (TX) Holtzman adult male rats, and compared these data to sham-operated controls. When data from hypothyroid tissues were compared to those of controls, the densities of PBRs were decreased in cardiac ventricles but not in liver tissue. This reduction in cardiac PBR density is opposite to what has been reported for ventricular calcium channel density in hypothyroidism. PBR affinity for the ligand was increased in both the liver and ventricular homogenates from the hypothyroid tissues, but this difference was not seen in membranes prepared from the liver homogenates. Although 4′-Cl-diazepam affinity is reported to vary between tissues from different species, this is the first report of an in vivo hormone treatment induced change in the benzodiazepine type PBR affinity. Liver tissues from both groups failed to show any interaction when radiolabeled [3H]4′-Cl-diazepam was tested against competing concentrations of thyroid hormone analogs. - Kragie, Smiehorowski 1994 Altered peripheral benzodiazepine receptor binding in cardiac and liver tissues from thyroidectomized rats.

So we established that thyroid hormone status, but not the hormones directly, did affect the PBR/TSPO. Still did not know how it changed its function.

Current Proposed Models of PBR Functions:

A recent review of all the literature came out in J of Endocrinology, in particular its role in steroidogenesis and central neurosteroidogenesis. Four alternative functional roles were proposed. 1. The response to redox stress and the generation of reactive oxygen species from the mitochondria. 2. Porphyrins are ligands for the receptor, so it may be involved in their degradation. 3. Certainly related to thyroid status, it may have a role in regulating oxygen consumption/energy generation. 4. Involvement in lipid transport, metabolism, energy generation.

Perspectives for TSPO action in cells. (1) TSPO as a regulator of redox homeostasis. It has been suggested that TSPO could interact with VDAC1 or NOX2 to induce production of ROS. (2) TSPO as an enzyme for protoporphyrin IX degradation. Chemical catalysis of PPIX degradation by bacterial TSPO has been proposed as a function for TSPO. The reaction rate was dependent on availability of light and oxygen. (3) TSPO as a regulator of oxygen consumption. (4) TSPO as a regulator of lipid metabolism. TSPO deletion could increase fatty acid oxidation in steroidogenic cells. Potential interaction of TSPO with Acyl-CoA binding protein/ACBP or CPT1A could affect import of fatty acids into mitochondria for β-oxidation. 10.1530/JOE-16-0241

Heart

But what role does it play in the cardiac tissues? Ischemia followed by reperfusion induces reactive oxygen species (ROS) generation. The increase of ROS production can lead to oscillations in mitochondrial membrane potential affecting the inner membrane anion channels (IMACs), triggering arrhythmia. The burst of ROS activates signaling kinases and transcription factors related to cardiac hypertrophy. IMACs influence the mitochondrial permeability transition pore (mPTP), leading to the release of cytochrome c and activation of caspase cascade.

Ischemia/reperfusion rapidly induces reactive oxygen species (ROS) production from the electron transport chain, and ROS are released to mitochondrial matrix. A local burst of mitochondrial ROS leads to the increase of ROS production and oscillations in mitochondrial membrane potential, which can destabilize neighboring mitochondrial membrane by inducing the release of ROS. The collapses of are regulated by inner membrane anion channels (IMACs) opening, which is key in the genesis of arrhythmia. The burst of ROS in cellular matrix activates a broad variety of signaling kinases and transcription factors related to cardiac hypertrophy. The imbalance of IMAC influences the opening of mitochondrial permeability transition pore (mPTP), leading to the release of cytochrome c and activation of caspase cascade, and contributes to myocardial infarction. Targeting TSPO can reverse the above changes. - Xiaolong Qi et al

Liver

Does it have a similar role in liver tissue? A recent paper looked at bile synthesis in fatty-fibrotic disease states. TSPO levels increase in parallel with the evolution of simple steatosis (SS) to nonalcoholic steatohepatitis (NASH) in nonalcoholic fatty liver disease (NAFLD). They induced lipid accumulation in vitro by treating Huh7 cells or primary mouse hepatocytes with either Acetylated Human Low Density Lipoprotein to increase triacylglycerol or AcLDL+58035 to induce free cholesterol accumulation in the presence or absence of TSPO. In this experimental model:

• TSPO deficiency inhibits Acyl-coenzyme A: cholesterol acyltransferase leading to unesterified free cholesterol accumulation

• Loss of TSPO in hepatocytes leads to unesterified free cholesterol accumulation that promotes simple steatosis

• Loss of TSPO attenuates liver fibrosis via downregulation of bile acid production

  

Thyroid hormones and bile acid membrane transporters do share characteristics. And they also decrease bile production via the related CYP enzymes. Hmmmm…. maybe another site of interaction?

We’ll just have to see what the next thirty years of research susses out.

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REFERENCES

Vimal Selvaraj, Lan N Tu. Current status and future perspectives: TSPO in steroid neuroendocrinology Journal of Endocrinology. Volume 231: Issue 1: R1–R30. Oct 2016 DOI: https://doi.org/10.1530/JOE-16-0241

Laura Kragie. Neuropsychiatric Implications of Thyroid Hormone and Benzodiazepine Interactions. March 1993. Endocrine Research 19(1):1-32 https://www.researchgate.net/publication/14814307_Neuropsychiatric_Implications_of_Thyroid_Hormone_and_Benzodiazepine_Interactions

Yuan Shi et al, Long-term diazepam treatment enhances microglial spine engulfment and impairs cognitive performance via the mitochondrial 18 kDa translocator protein (TSPO), Nature Neuroscience (2022). DOI: 10.1038/s41593-022-01013-9

Laura Kragie, Rich Smiehorowski 1994 Altered peripheral benzodiazepine receptor binding in cardiac and liver tissues from thyroidectomized rats. February Life Sciences 55(24):1911-8 DOI:10.1016/0024-3205(94)00523-0 https://www.researchgate.net/publication/15207893_Altered_peripheral_benzodiazepine_receptor_binding_in_cardiac_and_liver_tissues_from_thyroidectomized_rats

Xiaolong Qi et al. Translocator Protein (18 kDa): A Promising Therapeutic Target and Diagnostic Tool for Cardiovascular Diseases. Oxid Med Cell Longev. 2012: 162934. doi: 10.1155/2012/162934

Yuchang Li et al Cholesterol-binding translocator protein TSPO regulates steatosis and bile acid synthesis in nonalcoholic fatty liver disease. iScience. Volume 24, ISSUE 5, 102457, May 21, 2021 DOI:https://doi.org/10.1016/j.isci.2021.102457