Introduction
It has been known for decades that C18-C21 steroids are ligands to nuclear receptors and since the 1990’s that oxysterols are ligands to “orphan”, now “adopted”, nuclear receptors (Evans & Mangelsdorf, 2014). Importantly, nuclear receptors are expressed in brain where oxysterols are also abundant (Warner & Gustafsson, 2015). These receptors work via regulating gene and hence protein expression.N -methyl-D-aspartate receptors (NMDARs) are expressed in nerve cells and work on a much shorter time scale. They are ligand-gated ion channels activated by the neurotransmitter glutamate, critical to the regulation of excitatory synaptic function. NMDARs are modulated by excitatory neurosteroids and by the neuro-oxysterol 24S-hydroxycholesterol (24S-HC, Figure 1, see Supporting Information Table S1 for systematic and common names and their abbreviations) (Paul et al., 2013).
For simplicity, in this article we use the term oxysterol to cover oxidised forms of cholesterol and its precursors (Javitt, 2008; Schroepfer, 2000), and use the term sterol to specifically embrace cholesterol and its cyclic precursors. By extension neuro-oxysterols and neuro-sterols are the respective terms to define oxysterols and sterols found in the central nervous system (CNS). The sterol definition is at variance with the formal definition of sterols by Lipid Maps to includeall molecules based on the cyclopentanoperhydrophenanthrene skeleton and ring-opened versions thereof (Fahy et al., 2005).
G protein-coupled receptors (GPCR) are cell membrane receptors that are activated by molecules outside the cell and activate signal transduction pathways within the cell. Cell membranes are rich in cholesterol and membrane cholesterol plays an important role in GPCR structure and function (Sengupta & Chattopadhyay, 2015). Several cholesterol interaction sites have been identified in GPCRs whose occupancy may modulate GPCR activity. Smoothened (SMO), a member of the Frizzled-class of GPCRs and a critical component of the hedgehog (Hh) signalling pathway, has a cholesterol binding site within its extracellular cysteine rich domain (CRD), mutational modification of which impairs the ability of SMO to transmit Hh signals (Byrne et al., 2016). The Hh pathway is critical for tissue patterning during development and abnormal function is associated with birth defects and cancer (McCabe & Leahy, 2015). Defective Hh signalling is implicated in Smith-Lemli-Opitz syndrome (SLOS) which presents, not only with dysmorphology, but also learning and behavioural problems, highlighting the importance of Hh signalling in brain (Cooper et al., 2003; Kelley et al., 1996). Numerous oxysterols have also been found to activate the Hh signalling pathway by binding to the CRD of SMO (Nedelcu, Liu, Xu, Jao & Salic, 2013; Qi, Liu, Thompson, McDonald, Zhang & Li, 2019; Raleigh et al., 2018), many of these are found in brain and are hence neuro-oxysterols (Yutuc et al., 2020). GPCR183, also known as Epstein Barr Virus Induced GPCR or EBI2, is involved in the trafficking of immune cells towards their EBI2 ligands i.e. oxysterols with a dihydroxycholesterol (diHC) structure, where one additional hydroxy group is at C-7α and the second on the side-chain (Hannedouche et al., 2011; Liu et al., 2011). As T-cell trafficking plays a major role in the neurodegenerative autoimmune disease multiple sclerosis and in its animal model, experimental autoimmune encephalitis (EAE), GPCR183 and neuro-oxysterols have been studied in this regard (Duc, Vigne & Pot, 2019). 7α,25-Dihydroxycholesterol (7α,25-diHC) and 7α,(25R)26-dihydroxycholesterol (7α,(25R)26-diHC, also known as 7α,27-dihdroxycholesterol, note, if stereochemistry at C25 is not indicated it is assumed to be 25R) are both GPCR183 agonists present in brain (Griffiths et al., 2019a) and 7α,25-diHC has been found to be increased in spinal cord during EAE development (Wanke et al., 2017).
All cells in vertebrates express the enzymatic machinery to synthesise cholesterol. The cholesterol biosynthesis pathway, also known as the mevalonate pathway, (see http://www.lipidmaps.org/pathways/pathway_lipids_list.php for exact details) is regulated by the master transcription factor SREBP-2 (sterol regulatory element-binding protein 2) which also regulates the expression of the low density lipoprotein receptor (LDLR) (Horton, Goldstein & Brown, 2002). When cholesterol levels are low, SREBP-2 is transported by SCAP (SREBP cleavage activating protein) from the endoplasmic reticulum to the Golgi where it is processed to its active form which translocates to the nucleus and activates target gene transcription, hence up-regulating cholesterol biosynthesis and import via LDLRs and restoring cholesterol levels in the cell (Goldstein, DeBose-Boyd & Brown, 2006). When cholesterol levels are elevated, cholesterol in the endoplasmic reticulum binds to SCAP which becomes tethered to the resident endoplasmic reticulum protein INSIG (insulin induced gene) and prevents transport of SREBP-2 to the Golgi for activation (Goldstein, DeBose-Boyd & Brown, 2006; Sun, Seemann, Goldstein & Brown, 2007). In this way cholesterol regulates its own biosynthesis and import via the LDLR. Oxysterols also inhibit cholesterol biosynthesis, but in this case, by binding to INSIG and tethering the SCAP - SREBP-2 complex in the Golgi (Radhakrishnan, Ikeda, Kwon, Brown & Goldstein, 2007). Although the turnover of cholesterol in the adult brain is slow (0.4% per day in mouse, 0.03% in human) the rate of synthesis is much higher during development (Dietschy & Turley, 2004). In the foetal mouse, the blood-brain barrier (BBB) is formed at E10 - E11 and after E11 - 12 the brain is the source of essentially all new sterols (Tint, Yu, Shang, Xu & Patel, 2006). Importantly, cholesterol 24-hydroxylase (cytochrome P450 46A1, CYP46A1) expression is low until E18, conserving cholesterol and restricting the biosynthesis of the neuro-oxysterol 24S-HC (Tint, Yu, Shang, Xu & Patel, 2006). CYP46A1 is the dominant cholesterol hydroxylase in brain and hydroxylation of cholesterol to 24S-HC is responsible for 50 - 60% of all cholesterol metabolism in adult brain (Russell, Halford, Ramirez, Shah & Kotti, 2009).
Other receptors for oxysterols include cytoplasmic oxysterol-binding protein (OSBP) and a family of proteins showing sequence homology to OSBP called OSBP-related (ORP) or OSBP-like (OSBPL) (Olkkonen & Hynynen, 2009). OSBP regulates lipid transport between the endoplasmic reticulum and Golgi and also can act as a sterol-dependent scaffold for protein phosphatases that dephosphorylate ERK (Olkkonen & Hynynen, 2009).