The translocator protein (18kDa) (TSPO) is an outer mitochondrial membrane protein formerly known as peripheral benzodiazepine receptor . TSPO was first identified as a peripheral tissue diazepam binding site and since then it has been implicated in many cellular processes. Amongst these are steroid biosynthesis, protein import, heme biosynthesis, immunomodulation, cellular respiration and oxidative processes . This literary review focuses on TSPO mediated cholesterol transport and mitochondrial permeability transition regulation. These processes are associated with a variety of disorders for which TSPO ligands are being developed to treat. This promising molecular target will also be discussed in context of its cardio and neurological protective properties through knowledge arisen from TSPO ligands.
Subcellular localisation and structure of TSPO
TSPO is an outer mitochondrial membrane (OMM) protein enriched at inner mitochondrial membrane (IMM)-OMM contact sites . TSPO was first isolated in a ternary receptor complex with the voltage-dependent anion channel (VDAC) and the adenine nucleotide transporter (ANT) . This multimeric complex has been observed to encompass 4-16 TSPO 18kDa receptors which can transition between monomeric to polymeric states in response to signals such as reactive oxygen species (ROS) which alters its affinity to specific ligands . In freeze fractured proteoliposomes, 50% of TSPO receptors were present as dimers under physiological conditions, suggesting that this is the most representative state of TSPO.
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Several lines of evidence have proposed a mammalian TSPO structural arrangement which allows the binding as well as transport of certain ligands. Firstly, hydropathy experiments have shown a pentahelical TSPO structure featuring five transmembrane (TM) α-helical domains that completely traverse the outer mitochondrial membrane . Circular dichroism and NMR experiments have given further insight into the unstable TSPO tertiary fold made up of 5 independently folded TM domains, which become stabilized upon drug ligand binding . Furthermore, the TSPO primary sequence of 169 amino acids has 80% homology between species, the structure of which is highly conserved in nature . Therefore, cryo-EM structures of the bacterial homologue tryptophan rich sensory protein (TspO), a dimer of five α helices packed into a ten helix bundle supports previous structural and polymerisation evidence .
Cholesterol transport and steroid biosynthesis
Steroidogenesis is the process by which steroids are produced from cholesterol. The most widely studied role of TSPO is cholesterol transport and has been demonstrated using various experimental techniques .
A 240kDa mitochondrial complex consisting of TSPO, protein kinase A (PKA) RIα subunit, PKARIα associated protein 7 (PAP7), steroidogenic acute regulatory protein (STAR) and VDAC was identified by immunoblot analysis and is now better known as the transduceosome . This complex facilitates the transport of cholesterol from a cytosolic donor or an intracellular store through TSPO and VDAC contact sites in the OMM to the IMM (Figure 1) . The formation of the transduceosome is initiated by hormonal stimulation and subsequent cAMP signalling cascade . In a cAMP dependent manner, PAP7 is transported from the trans Golgi to the mitochondria where TSPO redistribute so that PAP7 and PKARIα can interact with TSPO . PAP7 transduces the elevated cAMP concentration signal to the rest of the transduceosome by bridging PKARIα and TSPO which, in combination with the arrival of STAR form a STAR-PKARIα-PAP7-TSPO complex to transport cholesterol . TSPO is responsible for transporting cholesterol through the OMM and IMM into the matrix, the rate determining step of steroidogenesis . As cholesterol reaches the mitochondrial matrix it can interact with the cytochrome P450 enzyme CYP11A1 which converts cholesterol to pregnenolone . Pregnenolone can then leave the mitochondria so to be converted into steroids such as progesterone by the endoplasmic reticulum resident enzyme 3β-hydroxysteroid dehydrogenase .
Figure 1 The transduceosome complex facilitates steroidogenesis. Adapted from
Hormonal stimulation promotes assembly of the inner and OMM spanning transduceosome complex. PAP7 is recruited from the Golgi to scaffold cytosolic PKA-RIα subunits and transduce the cAMP signal to the rest of the transduceosome proteins. STAR is activated by PKA phosphorylation and allows the formation of the STAR-PKARIα-PAP7-TSPO transduceosome complex to transport cholesterol to the matrix. Cholesterol is converted into pregnenolone by the cytochrome p450 enzyme CYP11A1 .
TSPO has a high affinity for cholesterol
Cholesterol, the sole precursor molecule for steroidogenesis, has a high affinity for TPSO as a ligand . It is transported into mitochondria by binding onto a cholesterol recognition amino acid consensus (CRAC) domain within TSPO . The model of TSPO cholesterol binding was illustrated by isolating a TSPO recombinant protein which demonstrated its ability to bind cholesterol at the carboxyl 150-156 TSPO amino acids . A direct role for TSPO in cholesterol transport was presented using SV-3T3 fibroblasts transfected with pCMV523 vectors to express mammalian TSPO which caused an increase in cholesterol uptake directly linked to an increase in TSPO receptors present in the cell .
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The current literature suggests stimulatory or inhibitory effects on cholesterol transport can be exerted by different TSPO ligands and by the precise polymeric conformation of the TSPO receptor. Flunitrazepam, an antagonist TSPO ligand can bind to a TSPO sites (other than the CRAC domain) to inhibit the ability of TSPO to bind and transport cholesterol . On the other hand enhanced cholesterol binding rate and uptake in a ligand dependent manner was observed by adding the TSPO ligand PK1110 to the dimeric state of TSPO . The effect of cholesterol transport exerted by TSPO ligands was shown to be specific to the OMM as TSPO ligand stimulated translocation of cholesterol into the mitochondria was observed in mitochondria and not in mitoplasts . However TSPO ligands have not been observed to increase cholesterol transport beyond the signalling pathways offered by hormones and cAMP .
Mitochondrial Permeability transition
Mitochondrial permeability transition (mPT) is characterised by a sudden increase in the permeability of the IMM due to the opening of a nonspecific mitochondrial permeability transition pore (mPTP) in response to factors such as elevated Ca2+ matrix levels or oxidative stress . When the IMM becomes permeable to molecules smaller than 1.5 kDa, ions and water enter the matrix and cause swelling, OMM rupture and subsequent release of proapoptotic factors such as cytochrome c and apoptosis inducing factor (AIF) into the cytosol .
To date, the molecular identity of the mPTP remains uncertain . The latest model of mPTP opening involves calcium induced pore formation which can be facilitated by the peptidyl-prolyl cis-trans isomerase activity of cyclophilin-D (CyP-D) and induces a conformational change on PiC enhanced by the close association of ANT . This overall mechanism can be regulated by components of the OMM such as VDAC and TSPO .
However, it is unlikely that cyclophilin D and ANT are components of the pore itself as under strong enough conditions, mPTP opening can still occur in the absence of these two components . Therefore, recent literature has shifted towards investigating whether the mitochondrial phosphate carrier (PiC) is the pore forming component (figure 2). Coimmunoprecipitation between PiC and ANT demonstrated a close association between the two components, both of which can also bind modulators of mPTP opening such as phenylarsine oxide . Furthermore, knockdown of PiC in HeLa cells increases resistance to apoptotic cell death triggered by staurosporine, the effect of which is induced when PiC is overexpressed .
Figure 2 The mitochondrial permeability transition pore complex structure. Adapted from .
Mitochondria permeability transition is primarily triggered by Ca2+ and ROS overload. Pore formation can be facilitated by CyP-D which induces a conformational change on PiC enhanced by the close association of ANT. Cyclosporine A (CsA) reduces the activity of CyP-D and can therefore inhibit opening of the mPTP. Other mitochondrial membrane proteins can exert regulatory roles such as VDAC and TSPO.
Association of TSPO with mPTP
The biochemical co-purification of TSPO with ANT and VDAC provided the first insight into a possible association between TSPO and proteins which comprise an active or regulatory role in the mPTP complex . There is sufficient evidence to suggest TSPO has a regulatory role in mPTP opening. This has been shown using TSPO antibodies and ligands. In the presence of an antibody specific to TSPO, a delay in Ca2+ induced mPTP opening was observed . This suggests that TSPO is most likely in a pore inducing pre activated form, possibly as a result of bound endogenous ligands or its polymeric state . However, mPT can occur in the absence of an intact OMM (containing TSPO) in mitoplasts. This raises questions over the current model whereby the mPTP spans both the OMM and IMM . This also implies the OMM exhibits a regulatory rather than a fundamental role in pore opening, possibly through other proteins in contact with the IMM such as TSPO.
Regulation of the mPTP by TSPO
TSPO may have a crucial role in determining cell death fate either by apoptosis or necrosis as mPTP opening is a key initiator of apoptosis through the release of proapoptotic factors. Furthermore, if the mPTP can be closed again, once opened, it may prevent necrotic cell death .
The effect TSPO exerts on the mPTP is not fully understood. However, three main roles for the TSPO have been suggested; ligand binding, ligand transport and phosphorylation mediated mPTP regulation.
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Firstly, regulation of the mPT can be exerted by different TSPO ligands which may induce or inhibit mPTP opening . The well characterised PK11195 TSPO ligand induces mPTP opening in isolated cardiac mitochondria consistent with mitochondrial swelling, oxidative phosphorylation uncoupling and cytochrome c loss in a calcium independent and cyclosporin A sensitive manner . In contrast the TSPO ligand Ro5-4864 has been observed to delay Ca2+ induced mPTP opening in vivo . Although, there are contradictions in the literature on the consistent effect TSPO specific ligands have on mitochondrial pore opening. Ro5-4864 has been shown to have the opposing effect on mPTP opening in rat cardiac mitochondria where Ro5-4864 induced mPT without any calcium addition . It is possible that the effects of TSPO ligands on TSPO, including the consequences for mPTP, are ligand and cell type specific. However, by looking thoroughly through the literature it has been noticed that when TSPO ligands such as PK11195 and Ro5-4864 are used in concentrations close to their nano-molar affinity for TSPO they prevent mPTP opening. High concentrations of these TSPO ligands may therefore be exerting pro-apoptotic effects via non TSPO mediated mechanisms when used at higher concentrations .
Secondly, TSPO can regulate the mPT by facilitating the diffusion of molecules to the IMM or matrix; allowing the molecules to fulfil their mPTP-dependent function. For example, TSPO facilitated porphyrin transport leads to an inhibition in mPTP opening .
Finally, TSPO can regulate the mPT could be mediated by phosphorylation of phosphoproteins associated with the mPTP. The increase in protein phosphorylation observed upon mPTP opening was inhibited by using a TSPO specific antibody; this consequently lead to the observation of delayed mPTP opening .
Cardio protective properties of TSPO (figure 3)
Ischemia reperfusion injury (see review )
Myocardial ischaemia is defined as the deprivation of blood and oxygen supply to the heart. This is common in diseases such as atherosclerosis, myocardial infarction or during heart surgery. The condition leads to decreased mitochondrial oxidative phosphorylation, pH, ATP and thus ATP dependent repair pathways which can lead to necrotic cell death. Short term ischaemia can be reversible, however, upon reperfusion a burst of ROS from the electron transport chain and xanthine oxidase increase cell damage which causes necrotic and apoptotic cell. The increased damage reperfusion exerts on ischaemia is called ischaemia reperfusion injury. The conditions which induce mPTP opening such as Ca2+ and ROS overload are prominent in myocardial ischaemia-reperfusion injury. It is further exaggerated by the loss of adenine nucleotides and increase of Pi (inorganic phosphate) during ischaemia. Oxidative stress and neutrophil activation are two other underpinning processes of ischaemia reperfusion injury. They act to create an inflammatory response and increases the damage to the surrounding heart cells .
Evidence supporting the mPTP as a cardio protective target
To reduce cell death from ischaemia reperfusion injury, the proton motive force must be recreated across the mitochondria by closing the mPTP. Therefore the mPTP is a target for cardio protection by limiting mitochondrial membrane permeabilization . Although the mPTP identity is not fully understood, the components currently believed to be part of the pore have been targeted on myocardial cells exposed to ischaemic and reperfusion conditions to investigate possible cardioprotective effects. Infarct size in Cyclophilin D deficient (knockout) mice subjected to cardiac ischaemia reperfusion injury were found to have 40% reduced infarct size in comparison to wild type mice . The effectiveness of suppressing the mPTP as a cardio protective technique was also investigated with known drug inhibitors of the mPTP such as cyclosporin A. This led to improved cell survival, from 62% to 91%, in human atrial tissue from the onset of reoxygenation. . This technique has also been shown to be cardio protective at the time of reperfusion in human cardiac muscle by using cyclosporine A to reduce necrotic cell death . Although cyclosporine A does protect from reperfusion by inhibiting mPTP opening it has harmful side effects. The search for a cardio protective drug which inhibits opening of the mPTP without any detrimental side effects is a key strategy to treat ischaemia reperfusion injury.
TSPO ligands reduces mPTP opening in the heart
Although TSPO is primarily present in steroid synthesising tissues, it is also present in non steroidogenic tissues such as the heart . Certain TSPO ligands have been observed to inhibit mPTP opening when used close to their nano-molar affinity for TSPO. Therefore, the regulatory role of TSPO on the mPTP could theoretically be exploited using TSPO drug ligands during ischaemia reperfusion injury to increase cell survival. The TSPO ligand Ro5-4864 was observed to reduce myocardial infarct size and protect against ischaemic and reperfusion insults by inhibiting OMM permeabilization . The cardioprotective effects Ro5-4864 exerts are proposed to be due to reorganising the balance of Bcl-2 family proteins to promote cell survival and reduce the release of intermembrane proapoptotic proteins such as cytochrome c and AIF . However, the above compounds bind to a nonspecific TSPO site which affects steroidogenesis and appears to prevent apoptotic cell death rather than necrotic cell death, the most prevalent during ischaemia reperfusion injury.
3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) is a new compound modified from a neuroprotective drug which binds specifically to the CRAC domain on TSPO. Although, the ligand competes with cholesterol, clinical trials suggest steroidogenesis is not affected, possibly due to the lower affinity of TRO40303 for TSPO than cholesterol .
TRO40303 reduced cell mortality by 38% upon reperfusion by delaying mPTP and reducing AIF release upon Ca2+ stimulation. However, in comparison to CsA, the inhibitory effect TRO40303 exerts on mPTP opening may be a combination of two cardioprotective properties; inhibiting mPTP opening and reducing ROS production . TRO40303 has entered phase 2 clinical trials to treat cardiac ischaemia-reperfusion injury in acute myocardial infarction by intra venous infusion in patients prior to reperfusion by angioplasty.
The above TSPO ligands which maintain the mitochondrial membrane potential, integrity and protect the heart from ischaemia reperfusion injury are strong cardioprotective therapeutic targets.
However due to the close interaction between TSPO, components of the mPTP and other mitochondrial proteins in the OMM, the above effects may not be specific to TSPO.
TSPO decreases inflammatory responses after ischaemia reperfusion injury
The majority of cell death in cardiac ischaemia reperfusion injury occurs by necrosis. Neutrophils are recruited to the damaged area, create an inflammatory response and further damage heart tissue . There is evidence to suggest TSPO ligands can be cardio protective by decreasing the damage caused by necrosis mediated inflammatory responses, although this process is not fully understood. It has been proposed that TSPO ligands, inhibit the action of inflammatory cells by a mechanism that is possibly regulated by glucocorticoid synthesis . In this case, certain TSPO ligands may facilitate TSPO meditated cholesterol transport, the rate limiting step in glucocortid synthesis. Furthermore, a strong link between TSPO expression and inflammation caused by ischaemia reperfusion was also shown in monocytes and B/T lymphocytes . However, reduced inflammation upon TSPO ligand administration is most likely an additive effect to the cardioprotective properties of targeting TSPO considering that when there has been no contribution from platelets or neutrophils in isolated fluid-perfused hearts, infarct size was observed to reduce with SSR180575 .
TSPO ligands maintain oxidative phosphorylation and decrease ROS levels
TSPO receptors are present in close contact to the components of the electron transport chain, therefore, TSPO is suggested to have a role within mitochondrial respiration and oxygen homeostasis . Preserving oxidative phosphorylation has been shown to decrease ischaemic and reperfusion induced infarct size as well as contractile dysfunction. It was also shown to increase cardiac recovery in isolated rabbit hearts, possibly due to the availability of energy for ATP dependent repair pathways. ROS were used to dissipate the membrane potential and decrease oxidative phosphorylation, both of which were preserved in the presence of the TSPO ligand SSR180575 . The mechanism by which TSPO ligands can preserve the activity of respiratory chain components is not known. However, in a recent paper, the TSPO ligand 4'-chlorodiazepam has shed light onto a plausible mechanism. It is thought to revolve around the control of ROS within the electron transport chain. In ischaemia reperfusion injury ROS are produced by the mitochondria, this initiates the triggering events which have both a direct and indirect effects on cell death . NADH dehydrogenase (complex I of the respiratory chain) and the ubiquinone-cytochrome b within complex II are ROS causative agents under decreased oxidative capacity conditions, such as during reperfusion . Interestingly, 4'-chlorodiazepam has been shown to increase the activity of both complex I and complex II, resulting in reduced ROS generation and maintenance of the mitochondrial electron transport chain . The irreversibly bound TSPO ligand SSR180575 was also shown to prevent in vivo apoptosis after ischaemia reperfusion by reducing oxidative stress. It has been suggested that this was achieved by protecting the mitochondria from exogenous ROS and also by regulating the mitochondrial generation of ROS . TSPO may therefore act as an oxygen sensor, that in response to oxygen, preserves the mPT and thus prevents the release of pro-apoptotic factors and so reducing apoptosis . Xanthine oxidase and NADPH oxidase are other key producers of ROS in cardiac tissue when the activities of these are increased during reperfusion . 4'-chlorodiazepam was also observed to decrease the activity of xanthine oxidase and NADPH oxidase and thus reduce ROS levels.
Figure 3 Summary of the cardioprotective pathways TSPO can exert in the presence of certain ligands
Ischaemia reperfusion injury leads to mPTP opening. TSPO ligands have been shown to inhibit ROS production in a variety of ways. A) TSPO can increase cholesterol transport and glucocortid synthesis. Glucocortid decreases the inflammatory response and the subsequent damage such a response could inflict on healthy cardiac tissue surrounding the site of injury. B) The activity of Complex I and III can be increased by TSPO which in turn preserves the membrane potential producing the energy required for ATP dependent cell repair processes. C) TSPO can decrease the ROS production of Xanthine oxidase and NADPH oxidase which decreases cell damage and mPTP opening. D) TSPO ligands have also been shown to improve the mPTP resistance to higher [Ca2+] and therefore reduce the duration of pore opening, thus decreasing necrotic as well as apoptotic cell death.
Neuroprotective properties of TSPO (figure 4)
Within the brain TSPO is mostly present in the mitochondria of cerebellar and cortical neurons as well as glial and neuroblastoma cells . Following brain injury such as stroke, hypoglycaemia or traumatic brain injury a range of cellular signals propagate a cell death response . These include the dissipation of the transmembrane potential needed for oxidative phosphorylation and the permeabilization of the OMM which results in the release of cytochrome c and caspase activation . Mitochondrial damage is a common pathway between these events following neurotrauma . Furthermore TSPO ligands have shown to protect from the above effects and also appear to have additional neuroprotective mechanisms by either glial proliferation or by the production of neurotropic factors that lead to increased neuronal survival .
TSPO neurological expression
Typical TSPO expression levels in the brain are low, but these are up regulated in locations where damage has occurred . The increased levels of TSPO receptors have been shown to help the functional recovery of these cells, however the precise mechanism exploited by TSPO to generate this effect is largely unknown .
Kianic acid induced traumatic brain injury in rats was observed to increase TSPO expression fivefold in the hippocampus . TSPO density may be observed to increase due to microglia invasion at the site of injury, the microglia themselves express large numbers of TSPO . Alternatively, microglia could be increasing the number of TSPO receptors within their cells . Regardless of the mechanism, TSPO ligands can be used as sensitive neuroimaging markers for brain damage in vivo by using techniques such as single photon emission computed tomography .
TSPO reduces apoptosis
Experimental techniques have used a variety of TSPO ligands to show how TSPO, through its regulatory role on the mPTP, can decrease apoptotic cell death. For many years, the neuro-protective effect of diazepam was believed to be hypothermic and GABAA receptor mediated. However, diazepam has also been shown to suppress stress induced cytochrome c release and therefore apoptosis by targeting TSPO . Pre-treatment with Ro5-4864 was observed to increase the number of surviving neurons and decrease caspase 3 and 9 activity which is dependent on cytochrome c release . Cholest-4-en-3-one (TRO19622) was identified as a neuroprotective drug for Amyotrophic lateral sclerosis and stopped the progressive cell death of cortical and spinal motor neurons by reducing cytochrome c release . However, TRO19622 binds to both TSPO and VDAC, therefore it is possible that the drug exerts its effects through additional mechanisms to those associated with TSPO .
Another mechanism by which TSPO may regulate apoptosis was observed with pre-treatment with SSR180575. Although the pathway by which TSPO may exert this effect is largely unknown, apoptosis was observed to decrease by up regulating anti-apoptotic Bcl-2 and down regulating pro apoptotic Bax proteins .
Apoptotic cell death was observed to decrease proportionally to an increase in TSPO expression caused by an increase in cerebral oxygenation levels . This suggests that TSPO may also convey an oxygen dependent regulation on apoptosis.
TSPO induce apoptosis
On the other hand there is evidence suggesting a pro apoptotic function of TSPO can be induced with the binding of certain ligands. Colchine is a microtubule disturbing drug which induces apoptosis, upon the addition of TSPO ligands such as PK11195 , Ro5-4864 and Diazepam, cytochrome c and AIF release was amplified and thus an increase in caspase 3 activity was observed . The concentrations of TSPO ligands required to induce apoptosis by mPTP opening were in the micro molar range when these have nano-molar affinities for TSPO, therefore it is unclear whether this is a direct effect on TSPO . It is possible that TSPO ligands can only exert pro-apoptotic activity in the presence of an external pro apoptotic agent, such as Colchine but, they may be unable to exhibit this behaviour on their own.
TSPO is neuroprotective via steroid biosynthesis
TSPO ligands have been shown to not only reduce cell death but also encourage neuronal renewal as observed with TRO19622 which promoted neuronal outgrowth, branching and overall higher neuronal density . TSPO ligands that allow the translocation of cholesterol into the matrix exert neuroprotective qualities which other TSPO ligands are not capable of achieving. In the same experiment Ro5-4864 prevented kianic acid induced loss of neurons and hippocampal damage whereas PK11195 binds to a different site in the TSPO receptor and does not translocate cholesterol into the mitochondrial matrix in nervous tissue did not. This suggests that Ro5-4864 may facilitate neuronal recovery because it facilitates TSPO aided transport of cholesterol to initiate steroidogenesis . Similarly, SSR180575 enhances pregnenolone concentration in the brain and possibly activates neuroprotective steroid biosynthesis in microglial cells that can increase neuronal protection, survival and repair . SSR180575 was observed to penetrate the brain well and have a long effective duration of up to 12 hours of action at doses which occupied 50 to 70% of TSPO receptors in vivo studies. The neuroprotective effects of SSR180575 were further investigated (ClinicalTrial.gov NCT00502515) in patients with diabetic and mild peripheral neuropathy in order to assess the effect of the TSPO ligand on the rate of nerve regenerations. The steroid metabolic pathways involved in decreasing motor neuron death have not been identified nor has the process that inhibits the release of anti-apoptotic factors through the mPTP which inhibits apoptosis in glial or neuronal cells . Collectively the current evidence supports a model where stimulating the formation of neurosteroids, such as pregnenolone by glia cells in response to TSPO ligands in vivo induces neurogenesis.
Figure 4 Summary of neuroprotective pathways TSPO can exert in the presence of certain ligands
TSPO density is increased at the site of brain injury due to microglia recruitment and possibly increased TSPO expression in brain cells. A) In glial cells TSPO can increase the cholesterol transported into the mitochondria leading to an increase in the synthesis of Neurosteroids. Neurosteroids are neuroprotective by interacting with neurosteroids receptors on glial or neuronal cells. Otherwise neurosteroids can modulate the production of cytokines from the CNTF family in glial cells. B) Some TSPO ligands have been shown to reduce mPTP opening and therefore reduce apoptotic cell death by reducing the release of cytochrome c form the mitochondrial matrix and therefore diminish the caspase activity of caspase 3 and 9.
Under physiological conditions TSPO is most likely involved in the transport of cholesterol and protoporphyrin species. Under stressful conditions such as Ca2+ and ROS overload TSPO most likely rearranges into a higher polymeric state to enhance mPTP opening.
Different TSPO ligands will be present at different concentrations depending on the cell's physiological state and are likely to compete with each other to regulate the above effects. TSPO ligands have been shown to promote the recovery of neuronal and cardiac cells predominantly by stimulating cholesterol transport and reducing mPTP opening. However, the understanding behind the broad range of biological processes TSPO is associated with heavily relies on the effects TSPO ligands exert and these may not necessarily reflect the true cellular function of TSPO.