Translocator protein (18 kDa) (Tspo) in the retina and implications for ocular diseases

Translocator protein (18 kDa) (Tspo), formerly known as peripheral benzodiazepine receptor is a highly conserved transmembrane protein primarily located in the outer mitochondrial membrane. In the central nervous system (CNS), especially in glia cells, Tspo is upregulated upon inflammation. Consequently, Tspo was used as a tool for diagnostic in vivo imaging of neuroinflammation in the brain and as a potential therapeutic target. Several synthetic Tspo ligands have been explored as immunomodulatory and neuroprotective therapy approaches. Although the function of Tspo and how its ligands exert these beneficial effects is not fully clear, it became a research topic of interest, especially in ocular diseases in the past few years. This review summarizes state-of-the-art knowledge of Tspo expression and its proposed functions in different cells of the retina including microglia, retinal pigment epithelium and Müller cells. Tspo is involved in cytokine signaling, oxidative stress and reactive oxygen species production, calcium signaling, neurosteroid synthesis, energy metabolism, and cholesterol efflux. We also highlight recent developments in preclinical models targeting Tspo and summarize the relevance of Tspo biology for ocular and retinal diseases. We conclude that glial upregulation of Tspo in different ocular pathologies and the use of Tspo ligands as promising therapeutic approaches in preclinical studies underline the importance of Tspo as a potential disease-modifying protein.


Introduction
The translocator protein (18 kDa) (Tspo) was first identified in 1977 as a benzodiazepine binding site in peripheral tissues that was distinct from the central benzodiazepine receptor (CBR) as it was not coupled to gamma-aminobutyric acid (GABA) receptors (Braestrup and Squires, 1977).Although initially termed peripheral-type benzodiazepine receptor (PBR) in order to distinguish it from CBR, multiple aliases were used to refer to this protein including mitochondrial benzodiazepine receptor (Anholt et al., 1986), mitochondrial diazepam-binding inhibitor receptor complex (Romeo et al., 1993) or pk18 (Blahos et al., 1995).In 2006, PBR has been renamed to Tspo due to its proposed role in translocation of cholesterol across the outer mitochondrial membrane (OMM) which represents the rate-limiting step of hormone-induced steroid formation (Mukhin et al., 1989;Papadopoulos et al, 1997Papadopoulos et al, , 2006)).However, findings from various research groups analyzing steroidogenesis in conditional steroidogenic-specific or global Tspo knockout (KO) mice have challenged the previous literature and refuted the involvement of Tspo in steroid hormone biosynthesis (Banati et al., 2014;Morohaku et al., 2014;Tu et al., 2014).
Interestingly, a role for Tspo as a component of the MPTP has been recently questioned as it was shown that Tspo-deficient mitochondria still undergo permeability transition (Sileikyte et al., 2014).In fact, it was shown that the MPTP is formed by dimers of F 0 F 1 ATP synthase and that Tspo ligands can bind to and inhibit this complex, suggesting that the observed effects on MPTP are indeed Tspo independent (Cleary et al., 2007;Giorgio et al., 2013).Of note, most of the functions that were attributed to Tspo were evidenced by means of synthetic Tspo ligands, which were proven to have off-target effects.Thus, further experiments are needed to conclude direct involvement of Tspo in specific functions.
Despite the lack of a precise molecular function, Tspo has emerged as an important biomarker for gliosis in the central nervous system (CNS) including the retina (Daugherty et al., 2013;Scholz et al., 2015b;Wang et al., 2014).Thus, various Tspo positron emission tomography (PET) ligands have been developed to monitor the dynamics of neuroinflammation (Banati et al., 2014).The fact that several studies have also demonstrated their ability to mitigate inflammation and to promote neuronal survival highlighted Tspo as a potential disease-modifying protein and thus as an attractive target for therapeutic interventions (Ferzaz et al., 2002;Scholz et al., 2015b;Veiga et al., 2005).Although the function of Tspo and how its ligands exert these beneficial effects is not fully clear, it became a research topic of interest, especially in ocular pathologies including rare genetic retinal diseases, age related macular degeneration (AMD), diabetic retinopathy and glaucoma.However, the significance of Tspo induction and especially its exact role in these diseases still remain elusive.
In this review, we comprehensively summarize the current status of Tspo expression and discuss proposed functions in the eye.Furthermore, we comment on recent developments in preclinical models targeting Tspo and summarize the relevance in ocular and retinal diseases.

Evolution and structure of Tspo
In the human genome, the TSPO gene is located on chromosome 22q13.31(Chang et al., 1992) while the murine Tspo homolog is found on chromosome 15 (Sileikyte et al., 2014).Both genes are composed of four exons, while exon 1, a small part of exon 2 and half of exon 4 remain untranslated (Casalotti et al., 1992;Lin et al., 1993) (Fig. 1 A).Both mRNAs translate a nuclear encoded five α-helical transmembrane protein composed of 169 amino acids that is primarily located in the OMM (Anholt et al., 1986) (Fig. 1 B).Our current understanding of the Tspo structure at molecular level is based on the nuclear magnetic resonance (NMR) structure of mouse Tspo (Jaremko et al., 2014) and two crystal structures of the bacterial homologs from Cereibacter sphaeroides and Bacillus cereus (Guo et al., 2015;Li et al., 2013).From the cytosolic view, the five transmembrane helices (TM) of Tspo are packed together in a clockwise order TM1-TM2-TM5-TM4-TM3, with the longest loop located in between TM1 and TM2 (Jaremko et al., 2015).The C-terminal part in TM5 resides in the cytoplasm and harbors a cholesterol-recognition amino acid consensus (CRAC) motif (residues 147-159) that binds cholesterol at nanomolar concentrations (Fig. 1 B) (Jamin et al., 2005;Li et al., 2001).Beside its ability to bind cholesterol, several endogenous Tspo ligands exist such as heme, porphyrins (Verma et al., 1987), diazepam binding inhibitor (DBI) and its proteolytic products octadecaneuropeptide (ODN) and triakontatetraneuropeptide (TTN) (Costa and Guidotti, 1991).However, based on the recent high-resolution NMR structure of mouse Tspo, the previously assumed channel-like core formation of Tspo for cholesterol translocation (Rupprecht et al., 2010) was refuted, as the side chains of the CRAC motif are located on the outside pointing towards the membrane (Jaremko et al., 2014).
Mouse Tspo was mainly reported to be monomeric in detergent systems but there are also indications that some fractions of Tspo could exist as oligomers in lipid bilayers (Jaipuria et al., 2017;Papadopoulos et al., 1994;Teboul et al., 2012).However, a recent study showed that the lipid-mimetic system which is used to solubilize mouse Tspo for NMR studies, destabilizes the protein thermodynamically, introduces structural perturbations and also alters the characteristics of ligand binding (Xia et al., 2019).Interestingly, in the retina and retinal pigment epithelium (RPE)/choroid higher molecular weight bands of Tspo were identified after laser injury, whose precise composition and role still remain elusive (Wolf et al., 2020).This suggests a cell-type specific reorganization of Tspo that may be associated with different functions.Therefore, the precise role of these Tspo monomers, dimers or oligomers deserves further studies.
Tspo is a highly conserved protein found in many Archaea, Bacteria and Eukarya (Balsemão-Pires et al., 2011;Fan et al., 2012).Both human and mouse Tspo share an 81.1% sequence homology (Selvaraj et al., 2015) (Fig. 1C).Together with the fact that mammalian TSPO can compensate for the loss of function of the Tspo homolog in the proteobacterium Cereibacter sphaeroides (formerly known as Rhodobacter sphaeroides), suggests that its functions are, at least in part, evolutionarily conserved (Yeliseev et al., 1997).
Notably, a paralog of Tspo, Tspo2, was recently identified in humans, rats and mice.In contrast to Tspo, expression of Tspo2 is restricted to late erythroblasts where it has been suggested to play a role in intracellular cholesterol redistribution during erythropoiesis (Fan et al., 2009;Kiatpakdee et al., 2020).

Transcriptional regulation of Tspo
The expression pattern of Tspo is highly conserved among different species but the magnitude of its expression is tissue-and cell-specific (Nutma et al., 2021).Interestingly, the human TSPO mRNA profile is relatively similar to that reported for mouse Tspo (Bribes et al., 2004;Wang et al., 2012).While Tspo has been extensively studied in the context of ligand binding, its transcriptional regulation, especially during neuroinflammation, is not fully understood.The first studies that characterized the mouse Tspo promoter were performed in steroidogenic and non-steroidogenic cell lines such as MA-10 Leydig cells and NIH-3T3 cells, respectively (Giatzakis and Papadopoulos, 2004).It was shown that the Tspo promoter, similar to housekeeping genes, lacks TATA and CCAAT boxes but harbors four well-conserved GC boxes and various transcription factor binding sites for specificity protein 1/specificity protein 3 (Sp1/Sp3), v-ets erythroblastosis virus E26 oncogene homolog (Ets), and activator protein 1 (Ap1) (Giatzakis and Papadopoulos, 2004).Functional analysis of the cloned promoter region, revealed that two distal Sp1/Sp3 sites are crucial for basal promoter activity while steroidogenic cells rely on additional areas upstream of the core promoter (Giatzakis et al., 2007;Giatzakis and Papadopoulos, 2004).These findings imply that different promoter regions drive Tspo expression in cells with constitutive expression compared to cells with inducible expression.
In the eye, Tspo shows a high and constitutive expression in the RPE, while glial cells, in particular microglia, strongly upregulate its expression from a very low baseline during neurodegeneration and inflammation (Karlstetter et al., 2014;Wang et al., 2014;Wolf et al., 2020).Recently, our group showed that the first 150 bp of the Tspo promoter contain all core promoter elements required to drive constitute TSPO expression in ARPE-19 cells, while this region showed only little activity in BV-2 microglia (Rashid et al., 2018) (Fig. 2A).Indeed, mutations in the core binding motifs of the four GC boxes in the minimal promoter region or siRNA-mediated knockdown of the GC box binding proteins Sp1, Sp3 and Sp4 significantly reduced TSPO promoter activity and TSPO expression in ARPE-19 cells.However, ubiquitous transcription factors such as Sp1 and Sp3 cannot confer microglia-specific Tspo expression.In fact, the minimal promoter region extends − 845 bp upstream of the transcription start site (TSS) in BV-2 microglia cells, which is sufficient to drive strong Tspo expression (Fig. 2B and C).In addition, deletion mutagenesis of − 593 to − 520 sequences that harbor binding motifs for Pu.1, Ap1 (cJun/cFos) and Sp1/3/4 as well as siRNA-mediated knockdown of these factors significantly decreased basal and LPS-induced Tspo promoter activity in microglia.Notably, LPS stimulation enhanced the recruitment of Pu.1, Ap1 (cJun/cFos), Sp1, Sp3 and Sp4 to the Tspo promoter with Ap1 (cJun/cFos) as a key driver of Tspo expression in activated BV-2 cells (Rashid et al., 2018) (Fig. 2B  and C).To date, transcriptional regulation in other retinal cells that can express Tspo such as Müller cells, vascular cells and endothelial cells (ECs) has not been investigated so far and deserves further studies.

Expression of Tspo
Tspo is expressed in many different tissues including heart, liver, lung, kidney, brain, and retina, whereas highest expression levels are found in steroidogenic tissues such as placenta, testis, gonads and adrenal glands (Anholt et al., 1986).Although the expression of Tspo in the healthy CNS is extremely weak, it increases predominantly in reactive glia upon aging and neuropathological conditions.Indeed, in the developing and mature murine retina the expression levels of Tspo are low while increasing upon aging (Fig. 3A).Also in the human aged retina, high TSPO levels were detected in microglia (Fig. 4A) (Karlstetter et al., 2014).
However, in the RPE Tspo is constitutively expressed during adulthood and significantly decreases in mice older than 70 weeks (Fig. 3B  and 4B) (Biswas et al., 2017).
In the eye, expression of Tspo is not only confined to microglia and RPE but is also found in other retinal cells such as Müller cells and ECs (Fig. 4B) (Mages et al., 2019;Wang et al., 2014).Tspo in microglia shows a very low baseline expression in the healthy retina, which is strongly increased upon light-induced retinal degeneration (Fig. 4C) (Klee et al., 2019b;Scholz et al., 2015b) or endotoxin-induced inflammation (Wang et al., 2014).Also, in the laser-induced mouse model of neovascular AMD (nAMD), accumulating Cx3cr1 GFP -positive retinal phagocytes within the laser lesions of the retina and RPE/choroid show strong Tspo expression three days after laser injury (Fig. 4D).
Increased Tspo expression specifically in Cx3cr1 GFP -positive cells was also reported in other retinal injury models including optic nerve crush, N-methyl-D-aspartate (NMDA)-induced excitotoxicity, and subretinal hemorrhage (Wang et al., 2014).While no injury or inflammation-induced expression of Tspo in Müller cells was reported in the abovementioned models, a recent study showed that Tspo increased in Müller cells with some delay following microgliosis (Mages et al., 2019).In the brain, Tspo expression is high in both microglia and astrocytes during neuroinflammation (Tournier et al., 2020), whereas retinal astrocytes do not expressed Tspo (Wang et al., 2014).
Initial subcellular localization studies showed that Tspo is mainly located in the OMM, whereas other studies also noted that Tspo can be found at the plasma membranes, in the cytoplasm and the nucleus.In mouse adrenal cortex, Tspo was localized to the plasma membrane and cytoplasm (Oke et al., 1992), while MA-10 Leydig cells showed Tspo localization to mitochondria and cytoplasm (Garnier et al., 1993;Liu et al., 2006).Moreover, in the human breast cancer cell line MDA-MB-231 and in human breast cancer biopsies, TSPO was primarily found in the nucleus (Hardwick et al., 1999).

Functional role of Tspo in retinal cells
In the eye, Tspo is expressed at different levels in microglia, RPE; Müller cells and ECs, while it is absent in retinal neurons (Mages et al., 2019;Wang et al., 2014).Although Tspo expression in the brain was also found in astrocytes (Gui et al., 2019), pericytes (Gui et al., 2019) as well as in choroidal and perivascular macrophages (Ricigliano et al., 2022), no data are available about Tspo expression in these cell types in the eye.
To date, Tspo has been linked to a variety of cellular functions in retinal cells including cytokine signaling, oxidative stress and ROS production, calcium (Ca 2+ ) signaling, neurosteroid synthesis, energy metabolism and cholesterol efflux.In this section of the manuscript, the proposed functions and roles of Tspo in different retinal cells will be summarized (Fig. 5A-E).

Endothelial cells
In the human and rodent brain, Tspo is constitutively expressed by ECs in the blood vessels (Gui et al., 2019;Vicente-Rodríguez et al., 2021).However, the role of Tspo and its expression in retinal ECs has not been studied in detail so far.The retina harbors two classes of ECs: vascular ECs in the inner retina and choroidal ECs in the outer retina.In both types of ECs, Tspo expression has been described.Tspo was detected in the inner retinal vasculature from adult mouse retinas (Mages et al., 2019;Wang et al., 2014).In addition, cell type-specific expression analysis from retinal cells using magnetic-activated cell sorting (MACS) revealed highest Tspo expression in vascular ECs (Mages et al., 2019).Interestingly, Tspo levels in ECs did not increase upon retinal damage such as transient retinal ischemia (Mages et al., 2019).In contrast, increased TSPO expression was observed in phorbol-12-myristate-13-acetate (PMA)-activated human umbilical vein endothelial cells (HUVECs) as an adaptive compensatory response (Joo et al., 2015).Adenoviral overexpression of TSPO inhibited PMA-induced vascular cell adhesion protein 1 (VCAM-1) expression and ROS production while siRNA-mediated silencing abolished these effects (Joo et al., 2015).However, the exact role of Tspo in retinal vascular ECs still remains elusive.
So far, only one in vitro study investigated the function and expression of Tspo in choroidal ECs.Using the chorioretinal endothelial cell line RF/6A, Tspo expression was found to be co-localized with the mitochondrial network (Biswas et al., 2018).RF/6A cells treated with the Tspo ligands Etifoxine or XBD173 showed increased cholesterol efflux that was accompanied by higher expression of cholesterol transporter genes while the biosynthesis of cholesterol and phospholipids were reduced (Biswas et al., 2018).Of note, the eligibility to use RF/6A cells as surrogates for choroidal ECs has been challenged.A study by Makin et al. validated different commercially available RF/6A cells based on their genetic, transcriptomic and functional profiles and compared these cells to primary ECs.RF/6A cells seem to lack several classical endothelial cell markers and various functional characteristics (Makin et al., 2018).Therefore, the expression and function of Tspo in choroidal ECs needs further evaluation for example using suitable cell-specific Tspo KO models.

Retinal pigment epithelium (RPE)
The RPE shows a constitutive expression of Tspo which remains unaltered upon inflammation (Scholz et al., 2015a;Wang et al., 2014).Notably, we and others have shown that in aged mice the expression of Tspo is significantly reduced (Biswas et al., 2017).In 20-month-old mice, reduced Tspo expression in the RPE was accompanied by increased cholesterol accumulation in these cells, while the expression of cholesterol transporter genes such as ATP binding cassette transporter A1 (Abca1) and ATP-binding cassette transporter G1 (Abcg1) were decreased (Biswas et al., 2017).
In line with this, a CRISPR/Cas9-mediated TSPO KO in human ARPE-19 cells revealed impaired cholesterol efflux (Biswas et al., 2017) and lipid metabolism (Alamri et al., 2019).Liquid chromatography mass spectrometry (LC/MS) of TSPO KO cells showed increased levels of fatty acids and glycerophospholipids (Alamri et al., 2019).In addition, RPE-specific Tspo KO mice (Tspo flox/flox x BEST1 Cre ) showed normal retinal morphology but the presence of droplet-like structures in the RPE increased with age, indicating accumulation of lipids (Klee et al., 2018).This was also observed in the RPE of full-body Tspo KO mice, where levels of cholesterol, triglycerides and phospholipids were higher compared to wild type (WT) littermates (Farhan et al., 2021).
Oxidative stress has long been considered to have a major influence on RPE function and health.The RPE is responsible for the turnover and recycling of shed photoreceptor outer segments (POS) that are phagocytosed in a diurnal fashion, a process where ROS are generated (Miceli et al., 1994;Tate et al., 1995).Tspo has been suggested to be involved in the regulation of ROS production.Treatment with oxidized low-density Images modified from (Wolf et al., 2020).lipoprotein (oxLDL) resulted in increased ROS production in TSPO-deficient ARPE-19 cells compared to WT cells (Biswas et al., 2017).Also, it was shown that the TSPO ligands XBD173, PK11195 and Ro5-4864 reduced intracellular ROS levels in ARPE-19 cells co-cultured with supernatants from reactive human SV-40 microglia and LLOMe.At the same time, TSPO ligands induced the expression of genes involved in the nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) pathway, an important antioxidant defense system (Rashid et al., 2020).
However, both studies used the fluorescent dye 2′,7′-dichlorohydrofluorescein diacetate (DCFHDA) to analyze intracellular ROS levels which is relatively unspecific in terms of compartmentalization of ROS production (Dikalov and Harrison, 2014;Hempel et al., 1999).Indeed, the ROS sources and its subspecies as well as the compartmentalization of ROS production are important factors that determine if it acts as a signaling molecule or toxin (Kaludercic et al., 2014;Ushio-Fukai, 2009).
Therefore, the effects of TSPO ligands or TSPO-KO in RPE cells need to be evaluated specifically for ROS derived either from the mitochondria matrix, from extracellular/phagosomal or cytosolic sources.
Autophagy is a natural, protective, homeostatic mechanism that removes dysfunctional cellular components and is associated with ROS and oxidative damage (Szumiel, 2010).In the aging RPE, autophagy has a critical role to maintain its homeostasis and mice with RPE-specific disruption of this process (Rb1cc flox/flox x BEST1 Cre ) develop a retinal phenotype reminiscent of age-related degeneration (Yao et al., 2015).A role for Tspo in mitochondrial autophagy (mitophagy) has been reported in mouse embryonic fibroblasts (MEFs), where increased levels of Tspo resulted in increased intracellular ROS and decreased efficiency of mitophagy (Gatliff et al., 2014).Although stimulation of ARPE-19 cells with LLOMe or conditioned media from reactive human SV-40 microglia induced autophagy, treatment with TSPO ligands had no effect on this process (Rashid et al., 2020).

Microglia
Microglia reactivity represents a common hallmark of many retinal degenerative diseases.Microglia are rapidly alerted by various triggers associated with degeneration of retinal neurons.This is accompanied by a strong upregulation of Tspo expression from very low baseline levels (Karlstetter et al., 2015;Wang et al., 2014).Thus, Tspo represents a biomarker for gliosis and Tspo-dependent effects on microglia functions are relatively well studied.In this context, Tspo was shown to modulate cytokine and chemokine signaling, Ca 2+ and ROS signaling, neurosteroid synthesis and energy metabolism.
Notably, tamoxifen-induced conditional deletion of Tspo in resident microglia using Tspo flox/flox x Cx3cr1 CreERT2 mice or targeting the protein with XBD173 revealed reduced secretion of pro-inflammatory cytokines and attenuated phagocyte reactivity in the retina and RPE/ choroid after laser injury (Wolf et al., 2020).Interestingly, a mouse study on parkinsonism also reported that XBD173-specific transcriptional changes includes pathways related to cytokine production (Gong et al., 2019).

Calcium and ROS
Ca 2+ is an important second messenger that regulates a variety of cellular functions and is responsible for the activation of ROS-generating enzymes (Berridge, 2012;Gordeeva et al., 2003).The role of Tspo in microglial Ca 2+ homeostasis is highly discussed.
Increased Ca 2+ levels have been shown to activate ROS-generating enzymes and the formation of free radicals (Gordeeva et al., 2003).The enzyme family of NADPH oxidases (Nox) is mainly responsible for regulated ROS production and consists of the membrane-bound enzymes Nox1, Nox2, Nox3, Nox4, Nox5 (only expressed in humans) and the dual oxidases Duox1 and Duox2.All isoforms transfer electrons from NADPH across biological membranes to molecular oxygen generating superoxide (O 2 .- ) and subsequently H 2 O 2 (Bedard and Krause, 2007).Nevertheless, mitochondria may also generate oxidants under certain conditions (Herb et al., 2019).
Interestingly, a Tspo-dependent regulation of Nox enzymes was described before (Choi et al., 2011;Guilarte et al., 2016;Loth et al., 2020).Tspo is suggested to be associated with the Nox2 subunits gp91 phox and p22 phox and thus to be involved in its maturation and ROS production in primary microglia (Loth et al., 2020).Increased ROS production was shown in primary microglia exposed to Tspo ligands that was abrogated by Nox2 inhibitors (Choi et al., 2011).
Of note, the fact that the membrane-bound subunit p22 phox is not only required to assemble Nox2 but also Nox1, Nox3 and Nox4 (Kawahara et al., 2005;Nakano et al., 2008) must be considered when interpreting these data.
The identification of specific Nox isoforms in tissues or cells by antibodies is challenging as proper antibody validations including appropriate positive and negative controls are often missing (Diebold et al., 2019), Animal studies with genetically modified Nox enzymes in eye diseases are scarce and most of the research on microglia was performed with cell lines (Chan et al., 2016;Yokota et al., 2011).Here, Nox enzymes were either knocked down (Cheng et al., 2018;Gatliff et al., 2017) or ROS production was chemically inhibited with diphenyleneiodonium or apocynin (Choi et al., 2011;Gatliff et al., 2017;Hou et al., 2018).While commonly termed specific Nox inhibitors, these compounds show many side effects and their specificity is highly questioned (Altenhofer et al., 2015).
Recently, our group postulated a critical role for Tspo in Nox1dependent ROS production in the retina.Microglia deficient for Tspo or treated with various Tspo ligands were not able to produce extracellular and phagosomal ROS upon stimulation.Using different Nox KO mice, we could show that isolated microglia produce extracellular and phagosomal ROS exclusively via Nox1, while other Nox isoforms were dispensable.Tspo seems to be crucial for the increase of cytosolic Ca 2+ levels that induce Nox1-derived ROS (Wolf et al., 2020).The exact mechanisms regulating the entry of Ca 2+ into the cytosol that is required for Nox1 activation remain still elusive.Additionally, cytosolic ROS and ROS produced in the mitochondrial matrix were not detected in stimulated microglia nor affected by Tspo KO or XBD173 treatment (Wolf et al., 2020).In contrast, KD of Tspo in BV-2 cells resulted in increased intracellular ROS levels (Wang et al., 2014).Also, treatment of primary rat microglia with Tspo ligands PK11195 and Ro5-4864 was shown to increase intracellular ROS production (Choi et al., 2011).
This discrepancy could be due to the different cells, methodological approaches and ROS detection probes used in these studies.Likewise, cytosolic ROS was often measured with 2′,7′-dichlorohydrofluorescein diacetate (H2DCFDA) which is relatively unspecific in terms of compartmentalization of ROS production (Dikalov and Harrison, 2014;Hempel et al., 1999).

Neurosteroids
One of the most discussed functions attributed to Tspo is the synthesis of neurosteroids.Due to its ability to bind cholesterol, Tspo is thought to facilitate the transport of cholesterol from the cytosol to the mitochondrial matrix, the rate-limiting step in neurosteroidogenesis. Cholesterol is then converted to pregnenolone, the main precursor of steroid hormones, by cytochrome P450scc (Rupprecht et al., 2010).Studies on BV-2 cells treated with Tspo ligands XBD173 or Ro5-4864 revealed increased pregnenolone levels in cell supernatants, while Tspo KD resulted in significantly lower levels (Bader et al., 2019;Karlstetter et al., 2014).In addition, this Tspo ligand-induced pregnenolone synthesis was absent in Tspo-deficient cells (Bader et al., 2019).
However, immortalized human C20 microglia cells stimulated with different Tspo ligands such as XBD173, Ro5-4864, PK11195, or Etifoxine did not increase pregnenolone levels.Also, a CRISPR/Cas9-mediated KO as well as lentiviral-mediated Tspo knockdown in these cells did not affect pregnenolone synthesis (Milenkovic et al., 2019).In contrast, a recent study using C20 cells reported that Tspo knockdown resulted in lower pregnenolone levels while treatment with XBD173 resulted in increased levels.Of note, other Tspo ligands such as Ro5-4864, PK11195 and Etifoxine failed to enhance pregnenolone synthesis (Germelli et al., 2021).
Interestingly, the effect of PK11195 on steroid production in Tspo-deficient Leydig cells was not mediated through Tspo but rather represents off-target effects (Tu et al., 2015).In line with this, the human adrenocortical cell line H295R, which has no endogenous Tspo expression, is still capable of producing steroids (Tu et al., 2014).These controversial results together with the fact that recent studies failed to detect differences in steroid synthesis in Tspo KO mice challenged its role in steroidogenesis (Banati et al., 2014;Tu et al., 2014).However, experiments using isolated retinal microglia from conditional microglia-specific Tspo KO mice would be helpful in order to decipher the role of Tspo and its ligands in microglial neurosteroid synthesis.

Energy metabolism
Tspo has been recently linked to basic mitochondrial functions and energy production (Gatliff et al., 2014;Liu et al., 2017).Tspo KO in human C20 microglia resulted in a decreased mitochondrial membrane potential (MMP) and respiratory rate (Milenkovic et al., 2019).Reduced Tspo levels in BV-2 microglia increased non-mitochondrial respiration and proton leakage followed by decreased spare respiratory capacity.Although treatment with Tspo ligands XBD173 and PK11195 resulted in decreased MMP in WT and Tspo KD cells, it was increased upon Ro5-4864 stimulation (Bader et al., 2019).Hence, the effect of Tspo ligands on MMP are at least partially independent of Tspo.
Studies on isolated primary microglia from neonatal brains showed reduced oxygen consumption rate in Tspo-deficient microglia compared to WT cells (Banati et al., 2014;Yao et al., 2020).In contrast, analysis of MMP, mitochondrial morphology and ATP levels in isolated microglia from adult microglia-specific Tspo KO mice showed no differences (Wolf et al., 2020).This discrepancy could be due to differences in endogenous Tspo expression levels in immortalized cell lines and primary microglia.Nevertheless, studies on liver-specific Tspo KO mice showed neither mitochondrial ultrastructural alterations nor MMP or ATP level differences (Sileikyte et al., 2014).Also, experiments using Tspo-deficient MA-10 Leydig cells (Tu et al., 2016) or isolated mitochondria from ventricles of cardiac-specific Tspo KO mice showed no signs of dysfunction (Thai et al., 2018).Therefore, the exact role of Tspo in mitochondrial integrity and energy metabolism in the retina needs further validation.

Müller cells
Müller cells, together with microglia mediate and shape the magnitude of retinal immune responses trough reciprocal interactions.Microglia-derived factors either enhance or inhibit the release of neurotrophic factors from Müller cells to support photoreceptor survival or mediate apoptosis (Harada et al., 2003;Wang and Wong, 2014).Conversely, activated Müller cells express and secrete the endogenous Tspo ligand DBI, which negatively regulates aspects of microglial reactivity (Wang et al., 2014).Apart from that, Müller cells can also express Tspo at high levels (Mages et al., 2019).In a mouse model of transient retinal ischemia, increased Tspo expression in Müller cells was observed 7 days post-surgery, while microglia showed the strongest increase already after three days (Mages et al., 2019).In contrast, other retinal injury models or mouse models for retinal degeneration showed no injury-induced increase of Tspo in Müller cells apart from microglia (Scholz et al., 2015b;Wang et al., 2014).Interestingly, analysis of isolated Müller cells from postischemic XBD173-treated retinas revealed reduced Müller cell gliosis concomitant with increased DBI expression.Here, neuron-supportive functions such as cell volume regulation, stable expression of glutamine synthetase (Gs), a key enzyme involved in the glutamate-glutamine cycle, were preserved upon XBD173 treatment (Mages et al., 2019).
Of note, in the ocular hypertension mouse model for glaucoma, Müller cell gliosis preceded microgliosis.Tspo expression in reactive microglia was decreased when Müller cell activation was suppressed by intravitreal injection of 2-methyl-6-(phenylethynyl)pyridine (MPEP), a metabotropic glutamate receptor 5 (mGluR5) antagonist (Hu et al., 2021).The use of Müller cell-specific Tspo mice and the analysis of Tspo-deficient Müller cells could provide a valuable insight into Tspo functions in these glial cells.

Importance of Tspo for ocular diseases
An increase in Tspo levels has been observed in various neurodegenerative diseases and this was mainly attributed to reactive microglia.Although Tspo ligands have been originally developed for noninvasive diagnostic imaging in vivo, several studies have highlighted their ability to dampen microglia reactivity and to promote neuronal survival (Ferzaz et al., 2002;Ryu et al., 2005).The Tspo ligands Ro5-4864 and XBD173 have been shown to attenuate neuroinflammation as well as neuropathology and behavioral impairments in mouse models mimicking Alzheimer's disease (AD) (Barron et al., 2013) and parkinsonism (Gong et al., 2019).Noteworthy, Tspo does not only represent a biomarker for gliosis in the brain but also in the retina.In the retina, Tspo regulates the magnitude of microglia responses together with its Müller cell-derived endogenous ligand DBI (Wang et al., 2014).Hence, Tspo is relevant as a disease-modifying gene and represents an attractive target for therapeutic interventions.In this section, we will summarize recent developments in preclinical model systems targeting Tspo and its relevance in human ocular and retinal diseases based on existing data sets.

Age-related macular degeneration (AMD)
AMD is a complex genetic and progressive chronic disease of the central retina that leads to severe vision loss among the elderly in the western world (Swaroop et al., 2009).Clinically, early stages of AMD are characterized by pigmentary changes in the macula and the accumulation of insoluble extracellular material, called drusen, in the subretinal region of the macula (Fritsche et al., 2014).Late-stage or advanced AMD can manifest either as geographic atrophy (GA) (dry form) or as the neovascular form characterized by choroidal neovascularization (CNV) (McLeod et al., 2009).Although dry and neovascular AMD are clinically very different, both forms are not mutually exclusive and are likely to be bilateral (Joachim et al., 2017).Angiogenic growth factors such as vascular endothelial growth factor A (VEGF-A) promote the formation of abnormal leaky blood vessels and thus the treatment of nAMD currently relies on intravitreal injections of anti-VEGF inhibitors (Ba et al., 2015;Witmer, 2003).However, these therapies have significant limitations such as the burden of frequent injections and treatment resistance (Yang et al., 2016).Recently, the two drugs Izervay® (Avacincaptad Pegol) and SYFOVRE® (Pegcetacoplan) were FDA approved to treat patients with GA.Both drugs are complement inhibitors, with pegcetacoplan acting on complement factor C3 (Liao et al., 2020) and avacincaptad pegol on C5 (Jaffe et al., 2021).While the etiology of AMD is still not well understood, genome-wide association studies and preclinical model systems have unequivocally shown dysregulated innate immune responses in AMD involving the complement system and mononuclear phagocytes, including local microglia (Akhtar-Schafer et al., 2018;Fritsche et al., 2014).
Increased Tspo expression has been shown in the light-damaged retina (Table 1) (Chen et al., 2023b;Scholz et al., 2015b;Tabel et al., 2022), a mouse model that mimics several features of dry AMD including innate immune activation and selective photoreceptor cell death (Grimm et al., 2000).In addition, targeting Tspo with the synthetic ligand XBD173 counter-regulates microgliosis and ameliorates light-induced retinal degeneration (Scholz et al., 2015b).This neuroprotective effect of Tspo ligands has also been validated in a mouse model of focal blue light damage (Table 1) (Scholz et al., 2015b).However, light-exposed mice with a full body Tspo KO showed no differences in microglia reactivity or retinal degeneration compared to WT mice (Table 1) (Klee et al., 2019a).Beside microglia, Tspo is also expressed in Müller cells, ECs and RPE while its magnitude of expression and function differs.Thus, Tspo deficiency could be more detrimental in retinal cells that show high expression levels than cells with low Tspo abundance.Also, a number of mouse studies have revealed absent or reduced phenotypic differences in global KO compared to conditional KO mice due to a phenomenon known as genetic compensation (El-Brolosy and Stainier, 2017).Notably, our study using the laser-induced CNV mouse model, a system to study key aspects of nAMD such as local inflammation and CNV formation (Lambert et al., 2013), showed that targeting Tspo with XBD173 or microglia-specific genetic deletion of Tspo attenuates phagocyte reactivity and limits vessel leakage and CNV (Table 1) (Wolf et al., 2020).
Interestingly, increased TSPO expression has also been documented in retinas from AMD patients.Here, 3' RNA-seq analysis of treatmentnaïve subretinal CNV membranes extracted during vitreoretinal surgery   from nAMD patients revealed increased TSPO expression compared to control tissue without AMD (Schlecht et al., 2020) (Table 2).Also, a study using single-cell RNA-Seq of cells from the macula and peripheral regions from donor retinas that included control, early and late AMD stages showed increased TSPO expression especially in the macula of late AMD patients (Lyu et al., 2021) (Table 2).Of note, in this study the late AMD stages were not further categorized into GA or nAMD.
Recently, a study using induced pluripotent stem cells (iPSC)-derived RPE cells generated from patients with GA identified increased TSPO levels in transcriptomics and proteomics approaches compared to control cells (Senabouth et al., 2022) (Table 2).In contrast, single-cell RNA-Seq of human macular RPE/Choroids derived from donors with GA or nAMD did not detect altered TSPO expression compared to control samples (Voigt et al., 2022) (Table 2).

Diabetic retinopathy (DR)
Diabetic retinopathy (DR) is the most common complication of diabetes mellitus.Clinically, DR is divided into two forms, an early nonproliferative form (NPDR) and an advanced stage of PDR.Both forms are characterized by increased vascular permeability, vessel occlusions and diabetic macular edema (DME) as a direct consequence of the breakdown of blood-retinal-barrier (BRB).In addition, PDR is characterized by pathological neovascularization (Duh et al., 2017).Although, the intravitreal administration of anti-VEGF agents represents the gold standard therapy for DR and DME (Diabetic Retinopathy Clinical Research et al., 2015), nearly half of the patients fail to show significant clinical improvement (Duh et al., 2017).Interestingly, mounting evidence from human patients and mouse models show that inflammation, including microglia reactivity contributes significantly to the pathogenesis of DR (Kinuthia et al., 2020).Notably, several studies have demonstrated that modulation of microglia can improve disease outcome in murine DR models (Al-Dosary et al., 2017).Thus, targeting Tspo in order to modulate microglia-mediated inflammation could represent a potential therapeutic approach.Indeed, a study using streptozotocin (STZ)-induced diabetic rats showed increased levels of Iba1 and Tspo in retinas during the early disease phase (Table 1) (Jiang et al., 2022).Another study involving STZ-induced diabetic rats also validated increased Iba1 and Tspo expression at early disease stages.In addition, PET imaging with Tspo ligand [ 18 F]-DPA-714 revealed an increased uptake and biodistribution in the eyes of early diabetic rats (Chen et al., 2023a).However, a different study using the same DR model observed a decreased [ 18 F]-DPA-714 uptake and biodistribution as well as reduced Tspo expression 12 weeks after STZ-induced diabetes (Zhou et al., 2020).Of note, this study did not assess microglia reactivity apart from Tspo expression.
Increased levels of Tspo and its endogenous ligand DBI were also reported in a mouse model of oxygen-induced retinopathy (OIR).Interestingly, OIR mice treated with anti-Vegf showed increased expression of DBI and decreased Tspo levels (Table 1) (Gao et al., 2022).
High TSPO levels have also been detected in peripheral blood mononuclear cells (PBMCs) isolated from DR patients.Among them, TSPO expression was highest in PDR patients compared with NPDR group (Table 2) (Guo et al., 2022).Also, a study analyzing sera from NPDR and PDR patients revealed increased TSPO levels in both forms with PDR showing the highest TSPO expression (Table 2) (Trotta et al., 2022).Here, increased TSPO levels positively correlated with IBA1 and glucose transporter 5 (GLUT5) levels (Trotta et al., 2022).

Inherited retinal diseases
Inherited retinal diseases (IRDs) represent a heterogenous group of rare blinding conditions characterized by progressive retinal degeneration that can lead to complete blindness.There have been tremendous advances in understanding the genetics of IRDs with more than 300 causal genes identified to date (RetNet, https://sph.uth.edu/retnet/,accessed November 2023).
Retinitis pigmentosa (RP) is one of the most prevalent forms of IRD and is clinically and genetically heterogeneous.Initial symptoms include night-blindness and a gradual constriction of peripheral vision due to loss of rod photoreceptors which is often followed by secondary loss of cones that lead to impairment of central vision and ultimately legal blindness (Ayuso and Millan, 2010).Of note, microglia reactivity is a common and early hallmark of IRDs and broadly independent of the underlying genetic defect (Karlstetter et al., 2015).Interestingly, Fam161a gene trap mice, a mouse model for autosomal recessive RP, showed increased Tspo expression in microglia in the outer retinal layer.Also, retinal degeneration (rd8) mice, that carry a mutation in the crumbs homolog 1 (Crb1) gene, showed increased Tspo expression in microglia associated with retinal rosettes (Table 1) (Karlstetter et al., 2015).Rd10 mice carry a spontaneous mutation of the rod-phosphodiesterase (Pde6b) gene that causes autosomal recessive RP.Here, increased expression of Tspo was associated with photoreceptor degeneration and microglia reactivity (Table 1) (Klee et al., 2019b;Zhao et al., 2015).Systemic Tspo KO in Rd10 mice did not affect microglia reactivity nor the progression of retinal degeneration (Klee et al., 2019a).However, the lack of phenotype in global Tspo KO studies could be due to compensatory mechanisms (El-Brolosy and Stainier, 2017).Mutations in the membrane-type frizzled related protein (Mfrp) gene are also known to cause autosomal recessive RP.Single cell RNA-Seq of Mfrp KI/KI mouse retinas revealed an increased Tspo expression specifically in microglia compared to WT mice (Table 1) (Kumari et al., 2022).
The most common form of syndromic IRD is Usher syndrome, causing sensorineural hearing loss and RP (Castiglione and Möller, 2022).RNA-Seq from retinal organoids from Usher type 1B (myosin 7A (MYO7A)) patient-derived iPSC showed an increase in TSPO expression in 7-week-old organoids while in 28-week-old organoids its expression was decreased (LogFC<1) (Leong et al., 2022).However, the lack of retinal vasculature and resident microglia in this model also limits modeling of mutation-induced inflammatory responses and thus the precise role of Tspo expression.

Glaucoma
Glaucoma, a worldwide leading cause of irreversible vision loss, is a heterogenous group of diseases characterized by loss of retinal ganglion cells mostly due to increased intraocular pressure (IOP).The aqueous humor is drained either by trabecular meshwork and Schlemm's canal or via uveoscleral outflow through the iris (Weinreb et al., 2014).Impairment or decreased outflow facility of aqueous humor results in increased IOP followed by thinning of the retinal nerve fiber layer and optic disc cupping (Stein et al., 2021;Weinreb et al., 2014).So far, glaucoma is treated by lowering IOP using hypotensive eye drops, laser trabeculoplasty or surgery (Weinreb et al., 2014).Although the etiology is not fully known, a growing body of evidence suggests a crucial role of inflammation mediated by resident microglia and complement system (Wang et al., 2016).Interestingly, several studies highlighted a potential role for Tspo during glaucomatous disease progression.In an ex vivo glaucoma rat model, Tspo expression increased in the proximity of the ganglion cell layer (GCL) in a pressure-dependent manner (Table 1).Additionally, treatment with Tspo ligand PK11195 resulted in increased allopregnanolone synthesis and reduced pressure-mediated ganglion cell death (Ishikawa et al., 2016).In a pathologically high intraocular pressure rat model, increased Tspo expression was also detected in the retina.In addition, silencing Tspo resulted in decreased numbers of Iba1 + cells and reduced retinal damage (Zeng et al., 2023).Interestingly, increased levels of Tspo were also detected in human PBMCs isolated from glaucoma patients with high IOP (Table 2) (Zeng et al., 2023).

Other ocular diseases
Uveitis is the collective term for various infectious and noninfectious diseases of the uvea composed of ciliary body, iris and choroid (Ghadiri et al., 2023).In both forms, the immune system plays a major role and current treatment approaches rely on corticosteroids or disease-modifying antirheumatic drugs such as methotrexate (Mayhew et al., 2022).Recently, a study using single cell RNA-Seq of retinas from autoimmune regulator (Aire) KO mice, a mouse model for spontaneous, chronic, and progressive uveoretinitis, revealed increased Tspo expression in microglia and Müller cells (Table 1) (Heng et al., 2019).Hence, targeting Tspo in uveitis might have beneficial effects on disease progression.
Retinal ischemia is a sight threatening complication in various eye diseases such as glaucoma, DR and retinal vein occlusions.Interestingly, increased levels of Tspo were found in both microglia and Müller cells after transient retinal ischemia (Table 1) (Mages et al., 2019).Treatment with Tspo ligand XBD173 resulted in reduced gliosis and improved survival of inner retinal neurons (Mages et al., 2019).

Conclusion and future perspectives
There is convincing evidence that Tspo upregulation in glial cells represents a common hallmark of various retinal diseases with different underlying etiologies.Thus, increased Tspo expression has been found in different preclinical model systems of retinal degenerative diseases and human samples including AMD, DR and glaucoma.Therefore, Tspo can be regarded as critical a disease modifier and consequently represents an attractive target for therapeutic interventions.
In the eye, expression of Tspo is not only confined to microglia and RPE but is also found in other retinal cells such as Müller cells and ECs.So far, no data are available for Tspo expression in further retinal cells including astrocytes, pericytes, choroidal and perivascular macrophages.Because Tspo expression was identified in these specific cells in the brain, it is very likely that the corresponding retinal cells may also express Tspo, but this aspect clearly needs further cell-specific investigations including single cell transcriptomics or in situ analyses.Furthermore, deeper mechanistic insights into transcriptional regulation of Tspo have so far only been gained for retinal microglia and RPE cells.Knowledge about Tspo gene regulation in other retinal cells could definitely unravel additional signaling pathways that are involved in Tspo dysregulation in pathological conditions.
To date, several in vitro and in vivo studies have been carried out to decipher the precise role of Tspo in retinal cells under both normal and pathological conditions.Especially, Tspo-dependent effects on microglia functions have been extensively studied and some are well established.In this context, it has been clearly demonstrated that Tspo modulates the secretion of cytokines and chemokines as well as ROS production.In addition to microglia, significant research has been done to define the exact role of Tspo in RPE cells that show constitutive expression of this protein.Here, the involvement of Tspo in ROS and lipid transport and accumulation is also well documented.
In addition, Tspo has been associated with a variety of functions, including oxidative stress, Ca2+ signaling, neurosteroid synthesis, energy metabolism and cholesterol efflux, which are still much debated.Some of the functions attributed to Tspo have only been demonstrated by Tspo ligands, which have been shown to have off-target effects.Future studies with these ligands should therefore aim to characterize their effects in depth in order to decipher their precise functions in conjunction with cell-specific Tspo targeting to demonstrate specificity.
However, data on the role of Tspo in other retinal cells, such as Müller cells and ECs, remain limited and need to be further investigated by comprehensive cell-specific studies.
In addition, the subcellular localization of Tspo was mainly located to the mitochondrial network in retinal microglia, RPE cells and ECs.However, other subcellular localizations such as the plasma membrane, cytoplasm and nucleus were also discussed, which need to be further characterized and investigated for possible differences in function when studying the role of Tspo in retinal cells.These differences could also be important for evaluating the potential therapeutic options of Tspo ligands and their side effects.
Several ligands used in preclinical studies have been shown to influence Tspo functions and to have immunomodulatory and neuroprotective properties in retinal disease models.However, beside the specificity of Tspo ligands another important point to consider is their solubility.Tspo ligands such as XBD173, PK11195 or Ro5-4864 have a poor water solubility which lead to low bioavailability after oral administration and thus require a higher dose to achieve the minimum pharmacologically effective concentration.Additionally, systemic treatment approaches with Tspo ligands could also affect other Tspoexpressing cells or tissues in the body, which could lead to adverse effects.
In this case, intravitreal injection would be the preferred route of drug delivery, but this is still a challenge due to poor water solubility.Therefore, future studies with these ligands should aim not only to thoroughly characterize their effects to determine their precise functions on Tspo, but also to develop improved and more cell-selective Tspo ligands that could be used to enhance or suppress specific functions of Tspo for future use in ocular inflammatory diseases.In this context, cellmediated drug delivery systems could also help to improve the therapeutic specificity and efficacy of Tspo ligands while reducing their side effects.To make further progress in this field, we also need to understand the different mechanisms that lead to Tspo induction in these cells.

Fig. 2 .
Fig. 2. Transcriptional regulation of Tspo in ARPE-19 and BV-2 microglia cells.Schematic representation of cis-elements and transcription factors regulating TSPO gene expression in ARPE19 cells (A) and BV-2 microglia during normal (B) and LPS-induced conditions (C).The first 150 bp of the Tspo promoter contain all core promoter elements needed to drive constitute TSPO expression in ARPE-19 cells, while the minimal promoter in BV-2 cells extends − 845 bp upstream.The promoter region − 593 to − 520 is crucial for inducible Tspo expression in BV-2 cells.Here Pu.1, Ap1 (cJun/cFos), Stat3, Sp1, Sp3 and Sp4 factors bind to proximal and distal elements on the Tspo promoter to regulate Tspo transcription.LPS induces an enhanced recruitment of these transcription factors to the promoter to augment transcription, resulting in upregulated Tspo levels in BV-2 microglia.Tlr4, Toll-like receptor 4; LPS, Lipopolysaccharide.

Fig. 3 .
Fig. 3. Expression of Tspo in the developing and mature murine retina and RPE.A-B Expression of Tspo in the C57BL/6J retina (A) and RPE (B).A significant correlation with age using two-tailed Pearson correlation is only detected in the retina (R 2 = 0.8147 and p < 0.0001) but not in the RPE (R 2 = 0.03456 and p < 0.5071).Mouse eyes were analyzed from postnatal day (p) 0 up to 105 weeks of age.Data are shown as mean ± SEM. n = 6-12 mice.

Fig. 4 .
Fig. 4. Expression of Tspo in the healthy and diseased retina.A Representative image of TSPO-stained human cryosection of a 72-year-old donor.Nuclei were counterstained with DAPI.B-C Representative images of Tspo-stained cryosections from healthy mouse eye (B) and acute retinal degeneration after light exposure for 15,000 lux for 1 h (C).Nuclei were counterstained with DAPI.Scale bar: 100 μm.D Representative images of Tspo-stained laser lesions in mouse retina and RPE/ choroid of Cx3cr1 GFP reporter mice.Quantel Medical Vitra, 532 nm green laser; power 100 mW; duration 100 m and spot size 100 μm.Scale bar: 100 μm.E Representative images of Tspo-and MitoTracker Red-stained mitochondria from isolated primary microglia.Nuclei were counterstained with DAPI.Scale bar: 6 μm.

Fig. 5 .
Fig. 5. Functional role of Tspo in retinal cells.A Schematic overview of the retinal cross-section and the organization of the retinal cells.BM, Bruch's membrane; RPE, retinal pigment epithelium; OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.B Tspo has been shown to modulate cholesterol efflux in endothelial cells.C In RPE cells, Tspo affects cholesterol efflux, the production of reactive oxygen species (ROS) and mitophagy.D In microglia, Tspo is modulating cytokine and chemokine signaling, Ca 2+ -dependent ROS production via Nox1, and cholesterol transport into the mitochondrial intermembrane space, representing the rate limiting step for pregnenolone synthesis.Additionally, Tspo is involved in the regulation of the mitochondrial membrane potential (ΔΨm).E Müller cells express the endogenous Tspo ligand diazepam-binding-inhibitor protein (DBI).Moreover, Tspo is influencing the glutamate-glutamine cycle.Red arrows indicate pathways and mechanisms that are affected or modulated by Tspo.

Table 1
Tspo in animal models for ocular diseases.

Table 2
TSPO in human ocular diseases.