Retinoic Acid Receptor-Related Orphan Receptors
There are three retinoic acid receptor – related orphan receptors (RORs), RORα (NR1F1), RORβ (NR1F2) and RORγ (NR1F3). RORγ exists as two isoforms RORγ1, or simply RORγ, and RORγ2 also called RORγt. RORγt has a shorter N- terminus compared to RORγ but with otherwise has identical domains. RORγt is highly expressed in thymus and is an essential transcription factor for Th17 cell development. Th17 cells are pro-inflammatory T-helper cells (CD4+) that express interleukin 17 (IL-17). Th17 cells are mediators of EAE, the mouse model of multiple sclerosis (Duc, Vigne & Pot, 2019). RORγ is expressed in multiple organs, but not normally in the CNS. However, RORγt-immunoreactive cells have been found in the meninges, the three membranes that envelops the brain and spinal cord, of multiple sclerosis patients, presumably as infiltrating immune cells (Serafini, Rosicarelli, Veroni, Zhou, Reali & Aloisi, 2016). RORα and RORβ are both expressed in brain. RORα is abundant in cerebellum and thalamus, and plays a key role in development, particularly in the regulation of the maturation and survival of Purkinje cells (Jetten & Joo, 2006). RORβ is highly expressed in brain and also the retina (Jetten & Joo, 2006). Oxysterols have been found to bind to the ligand binding domains of both RORα and RORγ (including RORγt), but not as yet RORβ (Duc, Vigne & Pot, 2019).
In 2010 a number of papers were published identifying sterols and oxysterols as ligands to RORα and RORγ (Jin, Martynowski, Zheng, Wada, Xie & Li, 2010; Wang, Kumar, Crumbley, Griffin & Burris, 2010; Wang et al., 2010), these included the archetypical neuro-oxysterol 24S-HC, 24S,25-EC, 20S-hydroxycholesterol (20S-HC), 22R-hydroxycholesterol (22R-HC), 25-HC, 7-oxocholesterol (7-OC), 7α-hydroxycholesterol (7α-HC) and 7β-hydroxycholesterol (7β-HC), all of which have been identified in mouse or human brain (Figure 1) (Yutuc et al., 2020). It is noteworthy that the side-chain oxysterols 20S-HC, 22R-HC 24S-HC, 24S,25-EC and 25-HC are also ligands to the LXRs (Janowski et al., 1999; Janowski, Willy, Devi, Falck & Mangelsdorf, 1996; Lehmann et al., 1997) and with the exception of 20S-HC, which has not been tested, to INSIG (Radhakrishnan, Ikeda, Kwon, Brown & Goldstein, 2007). Cholesterol sulphate, the dominating neuro-sterol sulphate in rat brain (Liu, Sjovall & Griffiths, 2003), has also been shown to bind to the ligand binding domain (LBD) of RORα (Kallen, Schlaeppi, Bitsch, Delhon & Fournier, 2004). RORα is an unusual nuclear receptor in that it is constitutively active in the absence of ligand and despite binding to the LBD of RORα cholesterol sulphate does not appear to affect the transcriptional activity of RORα or RORγ (Wang et al., 2010). In contrast, Wang et al reported that 7α-HC, 7β-HC and also 7-OC to be ligands to RORα and RORγ, and supress the transcriptional activity of the receptors as inverse agonists (Wang et al., 2010). Although CYP7A1 the enzyme required to synthesise 7α-HC and 7-OC is not expressed in brain, 7α-HC, 7-OC and 7β-HC could cross the BBB and be imported from the periphery (Griffiths et al., 2019a; Meaney, Bodin, Diczfalusy & Bjorkhem, 2002), or alternatively, be formed via non-enzymatic reactions initiated by reactive oxygen species (Griffiths & Wang, 2020; Murphy & Johnson, 2008). Interestingly, HSD11B1 is expressed in brain and will convert 7-OC to 7β-HC (Cobice et al., 2013; Mitic et al., 2013) (Figure 1).
24S-HC has also been found to be an inverse agonist of RORα and RORγ supressing the constitutive activity of these receptors (Wang, Kumar, Crumbley, Griffin & Burris, 2010). Interestingly, 24S,25-EC selectively supresses the activity of RORγ (Wang, Kumar, Crumbley, Griffin & Burris, 2010). Conversely, 20S-HC, 22R-HC and 25-HC have been shown to be agonists towards RORγ, promoting the recruitment of co-activators (Jin, Martynowski, Zheng, Wada, Xie & Li, 2010). The crystal structure of the LBD of RORγ with 20S-HC, 22R-HC or 25-HC bound showed the AF-2 helix in a conformation that is permissive for interactions with coactivator proteins. Importantly, mutations that disrupt the binding of these hydroxycholesterols abolish RORγ transcriptional activity, suggesting a critical role for hydroxycholesterols in activating RORγ (Jin, Martynowski, Zheng, Wada, Xie & Li, 2010). With respect to brain it is interesting to note that CYP46A1, the enzyme that generates 24S-HC, is expressed in Purkinje cells (Lund, Guileyardo & Russell, 1999) and that RORα plays a role in the regulation of the maturation and survival of these cells (Jetten & Joo, 2006). The cerebellum of RORα-deficient mice contains significantly fewer Purkinje cells and exhibits a loss of cerebellar granule cells (Jetten & Joo, 2006). It is tempting to speculate that 24S-HC plays a role in modulating maturation and survival of Purkinje cells via its inverse agonist activity towards RORα. Interestingly, Cyp7b1 has been reported as a target gene of RORα (Wada et al., 2008). CYP7B1 was first reported in brain and functions as an oxysterol 7α-hydroxylase to side-chain oxysterols including 25-HC and (25R)26-HC (Rose et al., 1997), but not 24S-HC, where CYP39A1 is the 7α-hydroxylase (Li-Hawkins, Lund, Bronson & Russell, 2000).
There is good evidence for the production of 7α,(25R)26-diHC in brain from either imported or in situ synthesised (25R)26-HC (Iuliano et al., 2015; Meaney, Lutjohann, Diczfalusy & Bjorkhem, 2000; Yutuc et al., 2020). Based on the publications of Jin et al which indicates that (25R)26-HC activates RORγ and Wang et al that 7α-HC supresses the activity of RORγ it is difficult to predict whether 7α,(25R)26-diHC should be an agonist or inverse agonist towards RORγ (Jin, Martynowski, Zheng, Wada, Xie & Li, 2010; Wang et al., 2010). Soroosh et al have answered this conundrum and shown that 7α,(25R)26-diHC and also 7β,26-dihydroxycholesterol (7β,26-diHC also called 7α,27-diHC) presumably 7β,(25R)26-diHC, are RORγt agonists, reversing the inhibitory effects of the RORγt antagonist, ursolic acid, in cell-based reporter assays (Soroosh et al., 2014). In primary cells both oxysterols were found to enhance the differentiation of IL-17-producing cells in a RORγt dependent manner and Th17 cells were found to produce both oxysterols (Soroosh et al., 2014). Importantly, the pro-inflammatory cytokine IL17-producing CD4+ Th17 cells play a key pathogenic role in multiple sclerosis, and as stated above, RORγt-immunoreactive cells have been found in the meninges of multiple sclerosis patients, presumably from infiltrating immune cells (Serafini, Rosicarelli, Veroni, Zhou, Reali & Aloisi, 2016). While reaction mechanisms for the formation of 7α,(25R)26-diHC are well established, mostly reactions catalysed by CYP27A1 and CYP7B1 or CYP7A1 and CYP27A1 (Griffiths & Wang, 2020), the formation of 7β,(25R)26-diHC is less clear cut, however, it has been identified in plasma from patients suffering from Niemann Pick (NP) disease where 7β-HC and 7-OC are abundant (Griffiths et al., 2019b) and SLOS patients where 7-dehydrocholesterol (7-DHC) is abundant, so mechanisms are available for its formation (Shinkyo, Xu, Tallman, Cheng, Porter & Guengerich, 2011), at least extra-cerebrally (Figures 1 & 2).