Localization of sterols and oxysterols in mouse brain reveals distinct spatial cholesterol metabolism

Abstract

Dysregulated cholesterol metabolism is implicated in a number of neurological disorders. Many sterols, including cholesterol and its precursors and metabolites, are biologically active and important for proper brain function. However, spatial cholesterol metabolism in brain and the resulting sterol distributions are poorly defined. To better understand cholesterol metabolism in situ across the complex functional regions of brain, we have developed on-tissue enzyme-assisted derivatization in combination with microliquid extraction for surface analysis and liquid chromatography-mass spectrometry to locate sterols in tissue slices (10 µm) of mouse brain. The method provides sterolomic analysis at 400-µm spot diameter with a limit of quantification of 0.01 ng/mm2. It overcomes the limitations of previous mass spectrometry imaging techniques in analysis of low-abundance and difficult-to-ionize sterol molecules, allowing isomer differentiation and structure identification. Here we demonstrate the spatial distribution and quantification of multiple sterols involved in cholesterol metabolic pathways in wild-type andcholesterol 24S-hydroxylaseknockout mouse brain. The technology described provides a powerful tool for future studies of spatial cholesterol metabolism in healthy and diseased tissues.

Soticlestat, a novel cholesterol 24-hydroxylase inhibitor shows a therapeutic potential for neural hyperexcitation in mice

“…S2B). These observations also agree with a recent study indicating a low level of 24S-hydroxycholesterol in the cerebellum 33 , while an earlier immunohistochemistry study detected a relatively high level of CH24H expression in Purkinje cells 34 . To further characterize the CH24H binding specificity of soticlestat, biodistribution experiments were conducted in vivo, comparing CH24H-KO and WT mice following intravenous injection (Fig.…”

Neuro‐oxysterols and neuro‐sterols as ligands to nuclear receptors, GPCRs, ligand‐gated ion channels and other protein receptors

“…Theofilopoulos et al suggested that the loss of motor function in two diseases resulting from inborn errors of metabolism, cerebrotendinous xanthomatosis (deficiency in CYP27A1) and hereditary spastic paresis type 5 (deficiency in CYP7B1) is a consequence of a reduced production of 3β,7α-diHCA in cerebrotendinous xanthomatosis, with the additional over production of neurotoxic 3β-HCA in spastic paresis type 5 providing a double-hit mechanism in the latter disease (see Figure 4 for CYP catalysed reactions) (Theofilopoulos et al, 2014). Interestingly, 3β,7α-diHCA is found to be most abundant in mouse cerebellum (Yutuc et al, 2020) Table S1 cholestenoic acids, (25R)26-hydroxycholesterol ((25R)26-HC, also called by the non-systematic name 27-hydroxycholesterol, 27-HC, note if stereochemistry at C-25 is not defined it is assumed to be 25R), a weak liver X receptor agonist (Fu et al, 2001), also accumulates in CSF of spastic paresis type 5 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 spastic paresis type 5 phenotype (Hauser et al, 2019). It is noteworthy that neither Cyp27a1 −/− or Cyp7b1 −/− mice shows a motor neuron phenotype.…”

“…There is good evidence for the production of 7α,(25R)26-diHC in the brain from either imported or in situ synthesised (25R)26-HC (Heverin et al, 2005;Iuliano et al, 2015;Yutuc et al, 2020 (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).…”

“…Conversely, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO, Figure 3), an oxysterol derived from 7-DHC and identified in the brain of a mouse model of Smith-Lemli-Opitz syndrome where 7-DHC is abundant (Xu et al, 2012), binds to SMO and blocks hedgehog signalling (Sever et al, 2016 (Theofilopoulos et al, 2013;Wang et al, 2009). In the new-born mouse, 24S,25-EC is still the most abundant oxysterol (Meljon et al, 2012), but in the adult mouse, 24S-HC is by far the most abundant oxysterol and 20S-HC is also present but at a low level (Meljon et al, 2012;Yutuc et al, 2020). Smith-Lemli-Opitz syndrome phenocopies dysregulated hedgehog signalling (Cooper et al, 2003), however, at least in the new-born mouse the pattern of SMOactivating oxysterols in the brain is similar to the WT (Meljon, Watson, Wang, Shackleton, & Griffiths, 2013).…”

“…oxysterols as ligands to RORα and RORγ(Jin et al, 2010;Wang, Kumar, Crumbley, Griffin, & Burris, 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…”