Liver X Receptors
Perhaps the most well studied activity of oxysterols is as ligands to nuclear receptors (Evans & Mangelsdorf, 2014; Warner & Gustafsson, 2015). Oxysterols are ligands to the liver X receptors α (LXRα, NR1H3) and β (LXRβ, NR1H2) (Janowski et al., 1999; Janowski, Willy, Devi, Falck & Mangelsdorf, 1996; Lehmann et al., 1997), the latter of which is highly expressed in brain (Wang, Schuster, Hultenby, Zhang, Andersson & Gustafsson, 2002). The most potent oxysterols are ones in which the side-chain of cholesterol has been modified by hydroxylation to give monohydroxycholesterols (HC), epoxidation to give 24S,25-epoxycholesterol (24S,25-EC) (Janowski et al., 1999; Janowski, Willy, Devi, Falck & Mangelsdorf, 1996; Lehmann et al., 1997) or carboxylation to give a carboxylic acid (Ogundare et al., 2010; Song & Liao, 2000; Theofilopoulos et al., 2014), although other oxysterols also activate these receptors (Segala et al., 2017).
Following birth, the dominant neuro-oxysterol in brain is 24S-HC (Bjorkhem, 2007; Dietschy & Turley, 2004; Ercoli & Ruggieri, 1953). This is a ligand to both the LXRs. Surprisingly, the cholesterol 24-hydroxylase knock-out (Cyp46a1-/- ) mouse has a mild phenotype but does show defective motor learning and in vitro studies indicate impaired hippocampal long-term potentiation (LTP) (Kotti, Ramirez, Pfeiffer, Huber & Russell, 2006; Lund, Xie, Kotti, Turley, Dietschy & Russell, 2003). Thanks to the tight regulation of cellular cholesterol levels by cholesterol itself (Goldstein, DeBose-Boyd & Brown, 2006), the cholesterol levels in brain of the Cyp46a1-/- mouse are not distorted (Lund, Xie, Kotti, Turley, Dietschy & Russell, 2003; Meljon, Wang & Griffiths, 2014). However, Kotti et al found that a reduced rate of production of cholesterol precursors, specifically geranylgeraniol, as a consequence of an absence of CYP46A1, was the explanation for impaired hippocampal LTP (Kotti, Ramirez, Pfeiffer, Huber & Russell, 2006).
Before birth, 24S,25-EC is the major neuro-oxysterol (Theofilopoulos et al., 2013; Wang et al., 2009), and can be formed by at least two pathways. In the cholesterol biosynthesis pathway, squalene epoxidase (SQLE) introduces a 2,3-epoxy group into squalene, the resultant 3S-squalene-2,3-epoxide is then cyclised to lanosterol by the enzyme lanosterol synthase (LSS, Figure 2). If levels of 3S-squalene-2,3-epoxide are elevated squalene epoxidase can introduce a second epoxy group to give 3S-squalene-2,3;22,23-dioxide which is metabolised in parallel to 3S-squalene-2,3-epoxide by the same enzymes (except 24-dehydrocholesterol reductase, DHCR24) to give 24S,25-EC rather than cholesterol (Gill, Chow & Brown, 2008; Nelson, Steckbeck & Spencer, 1981). The second pathway involves oxidation of desmosterol to give 24S,25-EC. In brain the likely catalyst is CYP46A1 expressed in neurons (Goyal, Xiao, Porter, Xu & Guengerich, 2014), however, the reaction can also proceed in fibroblasts raising the possibility of the involvement of an alternative catalyst, or CYP46A1 expression in these cells (Saucier, Kandutsch, Gayen, Nelson & Spencer, 1990).
Theofilopoulos and colleagues have shown the importance of 24S,25-EC in the neurogenesis of midbrain dopaminergic neurons through activation of the LXRs (Theofilopoulos et al., 2013). They showed that midbrain progenitor cells derived from Lxr double knockout (Lxr α-/-Lxr β-/-) mouse embryos have reduced neurogenic capacity (Sacchetti et al., 2009), while exogenous 24S,25-EC promoted dopaminergic neurogenesis in midbrain progenitor cells derived from wild type embryos (Theofilopoulos et al., 2013). 24S,25-EC also promoted dopaminergic differentiation of embryonic stem cells, suggesting that 24S,25-EC, or perhaps more chemically stable LXR ligands, may contribute to the development of cell replacement and regenerative therapies for Parkinson’s disease, a disease characterised by the loss of dopaminergic neurons (Theofilopoulos et al., 2013). In the CYP46A1  transgenic mouse (CYP46A1 tg) overexpressing human CYP46A1, the concentration of 24S,25-EC is elevated in the developing ventral midbrain (Theofilopoulos et al., 2019). Theofilopoulos et al exploited this mouse to show an increase in midbrain dopaminergic neurons in vitro and in vivo.Importantly, 24S-HC which is also elevated in CYP46A1 tg mouse developing midbrain does not affect in vitro neurogenesis of midbrain dopaminergic neurons (Theofilopoulos et al., 2019; Theofilopoulos et al., 2013). Intracerebroventricular injection of 24S,25-EC to WT mouse brain embryos in utero increased the number of midbrain dopaminergic neurons in vivo adding further weight to the hypothesis that the neuro-oxysterol 24S,25-EC promotes dopaminergic neurogenesis (Theofilopoulos et al., 2019). This led to the suggestion that increasing the levels of 24S,25-EC in vivo may be a useful strategy to combat the loss of midbrain dopaminergic neurons in Parkinson’s disease. Interestingly, the concept of adeno-associated virus (AAV) gene transfer of CYP46A1 for the treatment of Alzheimer’s disease (Burlot et al., 2015), Huntigton’s disease (HD) (Boussicault et al., 2016; Kacher et al., 2019) and spinocerebellar ataxias (Nobrega et al., 2019) has been tested in mouse models of these diseases with success, with the aim of enhancing cholesterol metabolism via oxidation to 24S-HC. Such administration of CYP46A1 no doubt enhances 24S,25-EC biosynthesis as well, although this was not measured in these studies, and could potentially be used to treat Parkinson’s disease also. In the study by Kacher et al using the zQ175 mouse model of HD, CYP46A1 gene transfer to striatum was found to rectify defects in cholesterol metabolism and alleviate the HD phenotype (Kacher et al., 2019). The expression of the LXR target gene Apoe was upregulated and the transport of cholesterol from astrocytes to neurons partially explained the improved phenotype (Kacher et al., 2019). It should be noted that the anti-HIV drug Efavirenz activates CYP46A1 and could provide an alternative route to enhancing 24S-HC and 24S,25-EC formation in brain (Mast et al., 2017).
Studies on theLxr α-/-Lxr β-/- adult mouse reveal progressive accumulation of lipids in the brain and loss of spinal cord motor neurons and ventral midbrain dopaminergic neurons, indicating further that these receptors are important in brain in both the developing and adult mouse (Wang, Schuster, Hultenby, Zhang, Andersson & Gustafsson, 2002). Surprisingly, studies of theCYP46A1 tg adult mouse found little change in the mRNA of LXR target genes in brain (Shafaati et al., 2011), this is despite elevation in 24S-HC and 24S,25-EC levels (Shafaati et al., 2011; Theofilopoulos et al., 2019). This may be a consequence of making global measurements where changes in specific brain regions are lost by averaging signal levels in bulk tissue.
Cholestenoic acids are also ligands to LXRs, these include 3β-hydroxycholest-5-en-(25R)26-oic (3β-HCA), 3β-hydroxy-7-oxocholest-5-en-(25R)26-oic (3βH,7O-CA) and 3β,7α-dihydroxycholest-5-en-(25R)26-oic (3β,7α-diHCA) acids, but not its down-stream metabolite 7α-hydroxy-3-oxocholest-4-en-(25R)26-oic (7αH,3O-CA) acids (Figure 3) (Ogundare et al., 2010; Song & Liao, 2000; Theofilopoulos et al., 2014). All of acids are identified in human CSF, with the exception of 3βH,7O-CA which has been found in plasma (Griffiths et al., 2019b; Ogundare et al., 2010; Theofilopoulos et al., 2014), and the two 7α-hydroxy acids have been identified in mouse brain making them neuro-oxysterol-acids (Yutuc et al., 2020). 3β-HCA, 3β,7α-diHCA and 7αH,3O-CA have also been identified in human brain. Remarkably, 3β-HCA is neurotoxic towards motor neurons while 3β,7α-diHCA is protective and 3βH,7O-CA promotes maturation of precursor cells into motor neurons, with each activity mediated by LXRs (Theofilopoulos et al., 2014). Theofilopoulos et al suggested that the loss of motor function in two diseases resulting from inborn errors of metabolism, cerebrotendinous xanthomatosis (CTX, deficiency in CYP27A1) and hereditary spastic paresis type 5 (SPG5, deficiency in CYP7B1), is a consequence of a reduced production of 3β,7α-diHCA in CTX with the additional over production of neurotoxic 3β-HCA in SPG5 providing a double-hit mechanism in the latter disease (see Figure 3 for CYP catalysed reactions) (Theofilopoulos et al., 2014). Interestingly, 3β,7α-diHCA is found to be most abundant in mouse cerebellum but is absent in cerebellum from CTX patients (Yutuc et al., 2020). Cerebellar ataxia, impaired co-ordination of voluntary movements due to underdevelopment of the cerebellum, is a common characteristic of CTX, and may occur in SPG5 also (Bjorkhem, 2013; Clayton, 2011; Salen & Steiner, 2017), linking cholestenoic acids and LXRs to brain development. Besides the cholestenoic acids, (25R)26-hydroxycholesterol ((25R)26-HC, also called by the non-systematic name 27-hydroxycholesterol, 27-HC, note if stereochemistry at C25 is not defined it is assumed to be 25R), a weak LXR agonist (Fu et al., 2001), also accumulates in cerebrospinal fluid of SPG5 patients (Schols et al., 2017; Theofilopoulos et al., 2014) and Hauser et al have suggested that neurotoxic effects of (25R)26-HC are major contributors to the SPG5 phenotype (Hauser et al., 2019). It is noteworthy that neitherCyp27a1-/- or Cyp7b1-/- mice shows a motor neuron phenotype. A possible explanation for these differences between human and mouse is the prevalence of alternative pathways to produce neuroprotective 3β,7α-diHCA in mouse (Griffiths et al., 2019a; Meljon et al., 2019), or alternatively the presence of lower levels of neurotoxic neuro-oxysterols in mouse than human (Hauser et al., 2019).
Neuro-oxysyerols in the form of 24S-HC and its activation of LXRs has been suggested to be protective against glioblastoma, the most common primary malignant brain tumour in adults (Han et al., 2020). Efavirenz, an antiretroviral medication that crosses the BBB and activates CYP46A1 (Mast et al., 2017), was shown to inhibit glioblastoma growth, the effect being explained by enhanced synthesis of 24S-HC, activation of LXR and inhibition of the cholesterol synthesis pathway by inhibition of SREBP processing (Han et al., 2020). The results reported by Han et al are interesting in a number of regards (Han et al., 2020):- (i) despite the name, the cellular origin of glioblastoma is unknown, but if glia are the origin of the cancer the effect of 24S-HC must be via a paracrine mechanism as CYP46A1 is expressed in neurons not glia (Lund, Guileyardo & Russell, 1999); (ii) the SREBP studied by Han et al was SREBP-1, not SREBP-2 the dominating transcription factor regulating cholesterol synthesis (Horton, Goldstein & Brown, 2002), 24S-HC was found to reduce nuclear SREBP-1, which is expected (Wang, Muneton, Sjovall, Jovanovic & Griffiths, 2008), but SREBP-1, primarily activates fatty acid not cholesterol synthesis (Horton, Goldstein & Brown, 2002), hence the link to reduced cholesterol synthesis and uptake via LDLR is less clear. Never-the-less the beneficial effects of 24S-HC suggest CYP46A1 as a potential therapeutic target.