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.