Oxysterols: From cholesterol metabolites to key mediators

    Oxysterols: From cholesterol metabolites to key mediators Valentin Mutemberezi, Owein Guillemot-Legris, Giulio G. Muccioli PII: DOI: Reference:

S0163-7827(16)30032-7 doi: 10.1016/j.plipres.2016.09.002 JPLR 926

To appear in: Received date: Revised date: Accepted date:

14 June 2016 13 September 2016 23 September 2016

Please cite this article as: Mutemberezi Valentin, Guillemot-Legris Owein, Muccioli Giulio G., Oxysterols: From cholesterol metabolites to key mediators, (2016), doi: 10.1016/j.plipres.2016.09.002

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ACCEPTED MANUSCRIPT Oxysterols: from cholesterol metabolites to key mediators

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Valentin Mutemberezi1, Owein Guillemot-Legris1 and Giulio G. Muccioli1*

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Bioanalysis and Pharmacology of Bioactive Lipids Research Group Louvain Drug Research Institute Université catholique de Louvain

Corresponding author

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Prof Giulio G. Muccioli Bioanalysis and Pharmacology of Bioactive Lipids Research Group Louvain Drug Research Institute Université catholique de Louvain Av. E.Mounier, 72 (B1.72.01) 1200 Bruxelles Belgium

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Tel : +32 2 764 7231 Fax : +32 2 764 7293 [email protected]

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ACCEPTED MANUSCRIPT Abstract: Oxysterols are cholesterol metabolites that can be produced through enzymatic or radical processes. They constitute a large family of lipids (i.e. the oxysterome) involved in a plethora of physiological

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processes. They can act through GPCR (e.g. EBI2, SMO, CXCR2), nuclear receptors (LXR, ROR, ERα)

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and through transporters or regulatory proteins. Their physiological effects encompass cholesterol, lipid and glucose homeostasis. Additionally, they were shown to be involved in other processes such

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as immune regulatory functions and brain homeostasis. First studied as precursors of bile acids, they quickly emerged as interesting lipid mediators. Their levels are greatly altered in several pathologies and some oxysterols (e.g. 4β-hydroxycholesterol or 7α-hydroxycholestenone) are used as biomarkers

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of specific pathologies. In this review, we discuss the complex metabolism and molecular targets (including their binding properties) of these bioactive lipids in human and mice. We also discuss the

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genetic mouse models currently available to interrogate their effects in pathophysiological settings. We also summarize the levels of oxysterols reported in two key organs in oxysterol metabolism (liver and brain), plasma and cerebrospinal fluid. Finally, we consider future opportunities and directions in

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the oxysterol field in order to gain a better insight and understanding of the complex oxysterol

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system.

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Key words: bioactive lipids, cytochrome, hydroxycholesterol, INSIG, squalene epoxidase, oxysterome

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ACCEPTED MANUSCRIPT Abbreviations: ABC: ATP-binding cassette; ACAT: acyl-coenzyme A cholesterol acyltransferase; C4: 7hydroxycholestenone; CA: cholestanoic acid; CH25H: cholesterol-25-hydroxylase; ChEH: cholesterol

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epoxide hydrolase; CNS: central nervous system; CSF: cerebrospinal fluid; CXCR2: chemokine (C-X-C

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motif) receptor 2; CYP: cytochrome P450; D8D7I: 3β-hydroxysterol-delta8-delta7-isomerase; DHC:

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dehydrocholesterol; DHCR7: 3β-hydroxysterol-delta7-reductase; EAE: experimental autoimmune encephalomyelitis; EBI2: Epstein-Barr virus-induced gene 2; Epoxychol: epoxycholesterol; ER: estrogen receptor; GPCR: G protein-coupled receptor; HMG-CoAR: 3-hydroxy-3-methylglutaryl coenzyme A reductase; HSD: hydroxysteroid dehydrogenase; Insig: insulin-induced gene; Ketochol:

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ketocholesterol; LCAT: lecithin–cholesterol acyltransferase; LDLR: low-density lipoprotein receptor; LXR: liver X receptor; NMDAR: N-methyl-D-aspartate receptor; NPC: Niemann-Pick protein type C;

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OHC: hydroxycholesterol; OHCnone: hydroxycholestenone; ORP: oxysterol-binding protein related protein; OSBP: oxysterol-binding protein; RNS: reactive nitrogen species; ROR: retinoic acid receptorrelated orphan receptor; ROS: reactive oxygen species; RXR: retinoid X receptor; SCAP: SREBP

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cleavage-activating protein; SERM: selective estrogen receptor modulator; Smo: smoothened; SOAT:

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sterol O-acyltransferase; SQLE: squalene epoxidase; SREBP: sterol regulatory element-binding protein; StAR: steroidogenic acute regulatory protein; STARD: StAR-related lipid transfer protein;

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SULT: sulfotransferase

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ACCEPTED MANUSCRIPT 1.

Introduction Oxysterols are 27-carbons molecules which, similarly to cholesterol, are made of a steroid

backbone and a 6-methylheptan-2-yl side chain. Carbon 3 of the steroid moiety can either be

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substituted by a hydroxyl moiety, similarly to cholesterol, or by a ketone function (Figure 1 and Table 1). Furthermore, the 3-hydroxyl group allows for the esterification or sulfation of

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both cholesterol and oxysterols. Chemically, the distinction between cholesterol and oxysterols is based on the additional oxygenated function (one or several) present on oxysterols. Importantly, although in many publications the term hydroxycholesterol (OHC) is

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frequently used to describe oxysterols with reference to an additional hydroxylation, chemically speaking oxysterols are not limited to the addition of a single hydroxyl group.

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Indeed, as it is illustrated in the Figure 1, in addition to the hydroxylated compounds (e.g. 4β-OHC, 7α-OHC, 7α,25-dihydroxycholesterol (7α,25-di-OHC),…), there are also oxysterols with a ketone group (e.g 7-ketocholesterol, 7α-hydroxycholestenone (7α-OHCnone)),

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epoxides (e.g. 5,6-epoxycholesterol (5,6-epoxychol), 24(S),25-epoxycholesterol (24(S),25-

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epoxychol),…) and molecules with two different functional groups (e.g. 7-keto-27hydroxycholesterol). Recently, molecules with a carboxyl group (the very early intermediates

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of bile acids synthesis) have been suggested to enter the oxysterol family, hence including the 27-cholestanoic acids (27-CA)[1;2]. Historically, the metabolism of oxysterols was perceived as a simple step of

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biotransformation of an endobiotic – i.e. cholesterol – into more polar compounds to favor its elimination. It was later found that oxysterols are also intermediates of pregnenolone and steroid hormones synthesis [3] and that they were also able to interact with specific receptors (see below). This clearly increased the interest in studying their metabolism and mechanisms of action. As a group, the endogenous oxysterols can be formed either by free radical oxidation or by enzyme-mediated mechanisms. The chemical oxidation, that will not be further developed here, is mainly initiated when a hydrogen atom is abstracted at the C-7 position by reactive oxygen species (ROS) such as OH• or reactive nitrogen species (RNS) such as ONOO- [4;5]. In addition, the carbons C-20 and C-25 were shown in vitro to be prone to free radical oxidation via a bi-radical oxygen attack [5]. Nevertheless, in contrast to position 7, in 4

ACCEPTED MANUSCRIPT vivo, this radical reaction does not seem to be responsible for a large proportion of the endogenous 25-OHC [6]. Moreover, to date there are no reports on the in vivo quantification of 20-OHC.

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We will discuss in the next paragraph the different metabolic pathways leading to the

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large variety of oxysterols. We will also summarize the molecular targets mediating

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oxysterols’ actions, before describing the different models available to study these

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interesting bioactive lipids.

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Table 1: Trivial and systematic names, as well as Lipid Maps and CAS numbers of the main oxysterols.

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Systematic name cholest-5-en-3β,4β-diol 5α,6α-epoxy-5α-cholestan-3β-ol cholestane-3β,5α,6β-triol 5β,6β-epoxy-cholestan-3β-ol cholestan-3β,6α-diol 7α-hydroxycholest-4-en-3-one cholest-5-en-3β,7α-diol cholest-5-en-3β,7α,24-triol cholest-5-en-3β,7α,25-triol 5-cholestene-3β,7α,26-triol 5-cholestene-3β,7β-diol (25R)-cholest-5-en-3β,7β,27-triol cholesta-5,7-dien-3β-ol 7-oxo-cholest-5-en-3β-ol (3β)-3,25-dihydroxycholest-5-en-7-one (3β)-3,26-dihydroxycholest-5-en-7-one cholest-5-en-3β,22R-diol cholest-5-en-3β,24S-diol 24S,25-epoxy-cholest-5-en-3β-ol cholest-5-en-3β,24S,26-triol cholest-5-en-3β,25-diol cholest-(25R)-5-en-3β,26-diol 3β-hydroxycholest-5-en-25R-26-oic acid cholest-5,24-dien-3β-ol

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Common name 4β-hydroxycholesterol 5α,6α-epoxycholesterol 5α,6β-dihydroxycholesterol 5β,6β-epoxycholesterol 6α-hydroxycholesterol 7α-hydroxycholestenone 7α-hydroxycholesterol 7α,24(S)-dihydroxycholesterol 7α,25-dihydroxycholesterol 7α,27-dihydroxycholesterol 7β-hydroxycholesterol 7β,27-dihydroxycholesterol 7-dehydrocholesterol 7-ketocholesterol 7-keto-25-hydroxycholesterol 7-keto-27-hydroxycholesterol 22(R)-hydroxycholesterol 24(S)-hydroxycholesterol 24(S),25-epoxycholesterol 24(S),27-dihydroxycholesterol 25-hydroxycholesterol 27-hydroxycholesterol Cholestenoic acid Desmosterol

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General formula

Abbreviation 4β-OHC 5α,6α-epoxychol 5α,6β-di-OHC 5β,6β-epoxychol 6α-OHC 7α-OHCnone 7α-OHC 7α,24(S)-di-OHC 7α,25-di-OHC 7α,27-di-OHC 7β-OHC 7β,27-di-OHC 7-DHC 7-ketochol 7-keto,25-OHC 7-keto,27-OHC 22(R)-OHC 24(S)-OHC 24(S),25-epoxychol 24(S),27-di-OHC 25-OHC 27-OHC 27-CA Desm

CAS number 17320-10-4 1250-95-9 1253-84-5 4025-59-6 41083-73-2 3862-25-7 566-26-7 245523-67-5 64907-22-8 4725-24-0 566-27-8 240129-43-5 434-16-2 566-28-9 64907-23-9 240129-30-0 17954-98-2 474-73-7 77058-74-3 642093-75-2 2140-46-7 20380-11-4 56845-87-5 313-04-2

Lipid Map ID LMST01010014 LMST01010011 LMST01010052 LMST01010010 LMST01010135 LMST04030123 LMST01010013 LMST04030174 LMST04030166 LMST04030081 LMST01010047 LMST04030178 LMST01010069 LMST01010049 LMST01010086 LMST01010019 LMST01010012 LMST01010129 LMST01010018 LMST01010088 LMST04030072 LMST01010016

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ACCEPTED MANUSCRIPT 2.

Metabolism of oxysterols in mice and human In this section, we will discuss the complex metabolism leading from cholesterol, and its

precursors, to the numerous endogenous oxysterols.

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As illustrated in Figure 2, the main enzymes involved in oxysterol metabolism can be

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classified into three groups: oxidoreductases (i.e. cytochromes P450, cholesterol

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hydroxylase, hydroxysteroid dehydrogenases and squalene epoxidase), hydrolases (cholesterol epoxide hydrolase, cholesterol esterase) and transferases (hydroxysteroid sulfotransferases, acyl-CoA cholesterol transferase, lecithin-cholesterol acyltransferase).

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Table 2 summarizes the enzymes (human and mouse orthologues) involved in oxysterol

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metabolism, their subcellular localization as well as their catalytic mechanisms.

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Table 2. Enzymes involved in oxysterol metabolism. For each enzyme, the table shows the subcellular localization, the catalyzed reaction(s) as well as the

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main oxysterol(s) produced by the enzyme. When available, a description of murine models and human pathologies related to the enzyme are also

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mentioned.

Oxidoreductases +

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Mouse orthologue

Subcellular localization

Oxysterol formed

CYP3A4

Cyp3a11

Endoplasmic reticulum

Cholesterol  4β-OHC Cholesterol  25-OHC

Cyp3a13

Endoplasmic reticulum

Cyp7a1

Endoplasmic reticulum

Cyp7b1

CYP11A1

Cyp11a1

Mitochondrial (inner membrane)

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CYP7B1

Endoplasmic reticulum

Cholesterol  4β-OHC

Cholesterol  7α-OHC 7-DHC  7-ketochol (a)

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CYP7A1

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Human gene

CYP3A5

+

Cholesterol + NADPH+ H + O2  Oxysterol + NADP + H2O

Cytochrome P450

27-OHC  7α,27-OHC [17] 25-OHC  7α,25-OHC [17]

Cholesterol  22(R)-OHC [23] 22(R)-OHC  20(S),22(R)-diOHC [23]

Model description

* Cyp3a

−/−

Related human pathology

mice [7] * Essential hypertension [10]

* Humanized model [8;9] -/-

* Cyp7a1 [11-13] * Cyp7a1 overexpression [14] * Cyp7b1

-/-

[18;19]

-/-

* Cyp11a1 [24]

* Hypercholesterolemia [15] * Neonatal cholestasis [16] * Bile acid synthesis defect [20] * Congenital spastic paraplegia [21;22] * Adrenal and gonadal insufficiencies [25]

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* Single ko: Cyp27a1

Sqle

Endoplasmic reticulum

-/-

Ch25h [33] * Humanized models [34;35]

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-/-

* Cerebrotendinous xanthomatosis [36;37] * Neonatal hepatitis [36]

-

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* knock-down: Cyp46a1 WT [41;42] -/* Single ko: Cyp46a1 [43] -/-

Cholesterol  24(S)-OHC [39] Desmosterol  24(S),25-epoxychol [40]

* Double ko: Cyp46a1 Cyp27a1 [32] -/-

* Triple ko: Cyp46a1 Ch25h -/Cyp27a1 [33]

-/-

-

-/-

* Humanized models [44] +

Squalene  (3S)-2,3-epoxy-2,3dihydrosqualene [45]

-

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7-hydroxycholesterols + NAD  oxocholesterol + NADH +

Dehydrogenase

HSD3B7

-/-

-/-

Squalene + [NADPH--hemoprotein reductase] + O2  (3S)-2,3-epoxy-2,3-dihydrosqualene + [NADP --hemoprotein reductase] + H2O

Epoxidase

SQLE

Endoplasmic reticulum

Cyp46a1

* Triple ko: Cyp27a1 Cyp46a1

24(S)-OHC  7α,24(S)-OHC [38]

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Cyp46a1

Endoplasmic reticulum

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CYP46A1

Cyp39a1

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CYP39A1

* Double ko: Cyp27a1 [32]

[29-31]

-/-

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Cholesterol  27-OHC [26;27] Cholesterol  25-OHC [28]

Mitochondria

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Cyp27a1

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CYP27A1

-/-

Hsd3b7

Endoplasmic reticulum

7α-OHC  7α-OHCnone [46]

-/-

* Hsd3b7 [47]

* Cholestasis with symptoms of congenital bile acid synthesis defect type 1 [46;48]

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ACCEPTED MANUSCRIPT

Hsd11b1

Endoplasmic reticulum

7β-OHC  7-ketocholesterol [49;50]

Cholesterol + AH2+ O2  25-OHC + A + H2O (b) Endoplasmic reticulum and cytosol

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Cholesterol  25-OHC [55]

* Single ko: Ch25h

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[56]

-/-

* Triple ko: Ch25h Cyp46a1

-/-

-

-/-

Cyp27a1 [33]

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Ch25h

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Hydroxylase

CH25H

* Cortisone reductase deficiency [54]

-/-

* Hsd11b1 [51-53]

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HSD11B1

Hydrolases Epoxycholesterol + H2O  dihydroxycholesterol

Hydrolase

5α,6α-epoxychol  5α,6β-di-OHC [57] 5β,6β-epoxychol  5α,6β-di-OHC [57]

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Endoplasmic reticulum

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ChEH

Sulfo-transferase SULT2B1b

Sult2b1b

Cytosol

SULT2A1

Sult2a1

Cytosol

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ChEH

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Transferases Oxysterol + PAPS  3-sulfated oxysterol + PAP (c) -/-

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Sult2b1b [58]

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-

-

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(a) 7-DHC : 7-dehydrocholesterol; (b) AH2 : reduced di-iron cofactor, A : oxidized di-iron cofactor; (c) PAPS : 3′-phosphoadenosine 5′-phosphosulfate

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2.1. Oxidoreductases

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the better described enzymes in oxysterols’ metabolism (Figure 2).

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Among the oxidoreductases, cytochromes P450 (CYP in human or Cyp in mouse [59]) are by far

Cholesterol 7alpha-hydroxylase (CYP7A1) and cholesterol 27-hydroxylase (CYP27A1) were of

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the first CYP described, certainly owing to their key role in bile acid synthesis [27;60]. Indeed, CYP7A1 initiates the classical or neutral pathway of bile acid synthesis, while CYP27A1 initiates the alternative or acidic pathway [61]. Actually, the formation of 7α-OHC by CYP7A1 constitutes one of

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the rate-limiting steps in bile acid synthesis. However, in spite of its liver-specific expression, we and other groups could not find any correlation between 7α-OHC levels in plasma or liver and

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hepatic expression or activities of CYP7A1[62-64]. This may be due to the dual origin (enzymatic and free radical oxidation) of 7α-OHC. Interestingly, one of the metabolites of 7α-OHC, namely 7αOHCnone (also known as C4 in the literature), which is formed by the action of the 3β-

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hydroxysteroid dehydrogenase type 7 (HSD3B7), was shown to correlate with CYP7A1 expression

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and activity in the liver [62;64;65]. Thus, although not a direct product of its activity, 7α-OHCnone is suggested as a biomarker of the bile acid diarrhea, a disease in which CYP7A1 activity is involved

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in the pathogenesis [66-68]. Finally, similarly to other CYPs, cholesterol is not the only substrate of CYP7A1 as it can also catalyze the oxidation of 7-dehydrocholesterol (DHC) into 7-ketocholesterol [69;70] (Figure 2).

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The CYP27A1 is one of the most promiscuous CYPs. Indeed, it is involved in both the formation and catabolism of 27-OHC, but it is also involved in the metabolism of multiple other oxysterols (Figure 2). However, to date, no direct comparison of the different substrates is available for this specific enzyme. The deficiency in this cytochrome was shown to cause a genetic metabolic disorder, known as cerebrotendinous xanthomatosis, characterized by increased 7α-OHCnone and cholestanol plasma and tissue levels [37;71]. These patients present a wide range of symptoms such as dementia, ataxia, xanthomas (in the nervous system and tendons), diarrhea, or peripheral neuropathy [37]. The cytochromes CYP3A4 and CY3A5 (the mouse orthologues being Cyp3a11 and Cyp3a13, respectively) are responsible for the synthesis of 4β-OHC which is among the most abundant and stable (half-life of about 60h [72]) oxysterols in human plasma [73]. Both cytochromes are well 11

ACCEPTED MANUSCRIPT characterized, in human and mouse, in terms of xenobiotic metabolism but unfortunately only little is known about their respective contribution to 4β-OHC synthesis. Nevertheless, 4β-OHC levels are clinically used as markers for CYP3A isoforms activities [74-78]. Furthermore, it was

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shown in vitro that 4β-OHC can be metabolized into a 27-hydroxylated metabolite (4β,27-diOHC)

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by CYP27A1, a 7-hydroxylated derivative (4β,7-diOHC) by CYP7A1, or into a 24-hydroxylated

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metabolite (4β,24-diOHC) by the CYP46A1 [72]. However, to date, these metabolites are not commonly detected in biological matrices.

CYP46A1, also known as cholesterol 24-hydroxylase, is another key enzyme in oxysterol metabolism as it is responsible for the synthesis of 24(S)-OHC, the key intermediate in brain

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cholesterol metabolism [39]. Brain cholesterol metabolism essentially takes place in the neurons where CYP46A1 is highly expressed [43;79]. Indeed, cholesterol from glial cells is transported to

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the neurons via lipoproteins, ATP binding cassette transporters (e.g. ABCA1) and LDL receptors were it is transformed into oxysterols [80]. Although most of the 24(S)-OHC crosses the blood

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brain barrier to be further metabolized in the liver (thus explaining the high level of this oxysterol

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in human plasma [81;82]), a fraction of it is directly converted, in the brain, into cholestanoic acids (i.e. bile acid precursors) through the successive intervention of CYP39A1 (i.e. 24(S)-

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hydroxycholesterol 7α-hydroxylase), CYP27A1 and HSD3B7 [38;81]. Recently, Stiles et al. identified 5 polymorphisms in the CYP39A1 sequence ((rs12192544, R23P); (rs2277119, R103H); (rs17856332, Y288H); (rs7761731, N324K); (rs41273654, K329Q)) which result in increased 24(S)-

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OHC serum levels[83]. They showed that these mutations do not affect the mRNA and protein expression of the enzyme, but reduced its activity from 15% to 100 %, depending on the variant. In humans, these CYP39A1 alleles explained about 10.8% of the interindividual variation observed in the 24(S)-OHC serum levels [83]. In the periphery, CYP46A1 is also involved in the synthesis of other oxysterols, including in the production of 24(S),25-epoxycholesterol (24(S),25-epoxychol) from desmosterol [39;40] (Figure 2). 24(S),25-epoxychol is one of the few oxysterols that can be produced from a substrate other than cholesterol. Indeed, another anabolic pathway for 24(S),25-epoxychol originates in a shunt of the cholesterol synthesis pathway in which the squalene epoxidase (SQLE) transforms the squalene2,3-epoxide into 2,3,22,23-dioxidosqualene which, through several reactions, results in the synthesis of 24(S),25-epoxychol [84;85].

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ACCEPTED MANUSCRIPT Another cytochrome playing a role in the synthesis of oxysterols is the cholesterol side-chain cleavage enzyme known as CYP11A1. This key enzyme in the pathway leading to C21-steroid hormones is also responsible for the production of 22(R)-OHC and 20(S),22(R)-di-OHC, two

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intermediates in the pathway leading from cholesterol to pregnelone [86-88].

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Finally, the corticosteroid 11-beta-dehydrogenase isozyme 1 (HSD11B1) and the cholesterol 25-

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hydroxylase (CH25H), are two important oxidoreductases that are not member of the cytochrome P450 superfamily. HSD11B1 is responsible for the reversible interconversion of 7-ketocholesterol and 7β-OHC [89]. Of note, this enzyme is also responsible for the interconversion of cortisol and cortisone [90].

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CH25H is a member of a small family of enzymes that use oxygen and a di-iron cofactor to catalyze hydroxylation reactions [55]. CH25H catalyzes the synthesis of 25-OHC from cholesterol.

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However, the exact origin of this oxysterol in the tissues is quite complex. Indeed, beside the activity of CH25H, other cytochromes (CYP3A4, CYP27A1, CYP46A1) and even ROS-induced

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reactions can form this specific oxysterol [6;91;92] (Figure 2). Interestingly, among all these

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enzymes able to hydroxylate the C-25 position, CH25H has been shown to be the most dynamically regulated enzyme and especially so in inflammatory conditions [93;94]. Hydrolases and transferases

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2.2

As mentioned above, the main hydrolases involved in the metabolism of oxysterols are the cholesterol epoxide hydrolase (CHEH) and the cholesterol esterases.

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CHEH is a bifunctional enzyme having both a steroid-isomerase (D8D7I) and a steroid-reductase (DHCR7) function [57]. This enzyme catalyzes the hydration of 5α,6α-epoxychol and 5β,6βepoxychol, to give 5α,6β-di-OHC (or cholestane-3β,5α,6β-triol) with a clear preference for the αcompared with the β-substrate [57;95] (Figure 2). 5α,6β-di-OHC was put forth as one of the potential biomarker of inherited disorders with disturbance in cholesterol metabolism such as Niemann-Pick type C disease, cerebrotendinous xanthomatosis or lysosomal acid lipase deficiency [96-98]. It was also suggested that the accumulation of epoxycholesterols could have beneficial effects on cancer, thus explaining the anti-tumor properties described for CHEH inhibitors [99]. Similarly to cholesterol, oxysterols are subject to an esterification in position 3 of the sterol moiety via the enzymes acyl-CoA cholesterol transferase (ACAT, also known as SOAT) or lecithincholesterol acyltransferase (LCAT). The esterified forms of oxysterols can in turn be hydrolyzed by 13

ACCEPTED MANUSCRIPT cholesterol esterases into free-form of oxysterols [100-102]. Although the role of these esterified forms still need to be clarified, several authors already take into consideration the presence of this fraction of oxysterols when quantifying oxysterols in biological matrices (see Table 5 and below).

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Finally, three hydroxysteroid sulfotransferases, SULT2B1b, SULT2B1a and SULT2A1, are involved

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in the introduction of a sulfate group in position 3 of the steroid backbone of several oxysterols

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(e.g. 7-ketocholesterol, 24(S)-OHC or 25-OHC) [103-105]. In vitro experiments demonstrated that SULT2B1b has a more pronounced affinity towards 7-ketocholesterol (Km=7.7 µM) compared with SULT2B1a or SULT2A1 (Km=24 and 17 µM respectively) [104]. Of the different compounds tested, cholesterol displays a higher affinity for SULT2B1b (Km=1.1 µM) when compared with oxysterols

Of

note, the

sulfated

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while 7α-OHC (Km=2.3 µM) has a higher affinity than 7β-OHC (Km=5.9 µM) for this enzyme [104]. hydroxycholesterol species,

25-OHC-3S, was

studied

in

the

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pathophysiological context of obesity and metabolic syndrome where it displayed beneficial effects [106;107].

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Although it is tempting to point to a given enzyme as being responsible for the synthesis of a

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specific oxysterol, it is clear that this would often be speculative. Indeed, many enzymes can catalyze the formation of a given oxysterol, the same oxysterol can be formed from different

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substrates (e.g. 7-ketocholesterol can be formed from cholesterol, dehydrocholesterol, and 7βOHC), and for some oxysterols (e.g. 7α-OHC) part of their origin can be explained by both enzymatic and non-enzymatic (ROS) mechanisms (see Figure 2). While animal models have clearly

humans. 3.

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helped (see Table 2), further efforts are needed to fully unravel oxysterol metabolism, especially in

Molecular targets of oxysterols The complexity of studying oxysterols not only arises from the large number of molecules

within this family and their complex metabolism but also from the large number of molecular targets identified for these bioactive lipids. Moreover, some targets such as nuclear receptors (see below) act as permissive heterodimers, adding more complexity to the interpretation of the targeted genes and the implication of oxysterols in the studied physiopathological conditions. In this section we will briefly describe the different molecular targets binding the oxysterols as well as the effect of each oxysterol on these targets (Table 3 and Table 4). From a functional point

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ACCEPTED MANUSCRIPT of view, the proteins that bind the oxysterols can be classified into receptors (nuclear and GPCRs) and regulatory or transport proteins (e.g. Oxysterol binding protein). 3.1. Nuclear receptors

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Liver X receptors (LXR) were the first receptors described for oxysterols. LXR form a heterodimer with the retinoic X receptor (RXR). Following activation by oxysterols (LXR) or by

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retinoic acid (RXR), the heterodimer recruits co-activators proteins (nuclear receptor co-activator 1 and activating signal co-integrator 2) and can initiate transcriptional activity [108]. The genes targeted by the heterodimer LXR/RXR are mainly those involved in the reverse

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cholesterol transport, such as ATP-binding cassette transporters (ABCA1, ABCG5, ABCG8), or apolipoprotein E [109]. However, LXR also control the expression of genes involved in many

MA

process such as macrophage recruitment and activation, apoptosis and CNS myelination [110;111]. Importantly, although oxysterols (e.g. 24(S),25-epoxychol) are generally perceived as the endogenous ligands of LXR, not all the oxysterols behave as full agonists, and several of them

D

actually behave as antagonists (Table 3). Some authors found a quite complex pharmacological

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behavior for the oxysterols. For instance, Berrodin and colleagues reported that 5α,6α-epoxychol behaves as antagonist or (partial) agonist depending on the LXRα downstream gene studied [112].

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RAR-related orphan receptors (RORα, RORβ and RORγ) are another family of nuclear receptors binding oxysterols. Upon activation, ROR recognize specific ROR response elements and activate gene transcription (via recruitment of co-activators). Thus, ROR function as constitutive activators

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of transcription. Over the years, several oxysterols (e.g. 24(S)-OHC and 7-ketocholesterol) were described as inverse agonists of both RORα and RORγ (Table 3). To date however no oxysterol was reported to bind to RORβ. The binding of oxysterols such as 24(S)-OHC and 7-ketocholesterol to ROR results in decreased gene transcription due to their inverse agonist functionality [113-115]. More recently, other oxysterols (e.g. 7β,27-di-OHC and 7α,27-di-OHC) were described as agonists of RORγt (a specific RORγ variant found in immune cells) which is a key driver of Th17 cell differentiation. This finding further implicated ROR in the context of oxysterol-mediated immune effects [116;117]. However, some apparently conflicting results exist regarding oxysterols’ function at RORγ. For instance, 24(S),25-epoxychol was described either as an agonist or as an inverse agonist of RORγ [113;116]. This discrepancy, that may originate from the different models used to characterize their functionality, calls for additional studies. 15

ACCEPTED MANUSCRIPT The estrogen receptor α (ERα) is another nuclear receptor activated by oxysterols. Indeed, 27OHC and 25-OHC were shown to bind to ERα (but not to ERβ) [118]. 25-OHC is characterized as an ERα agonist, while 27-OHC is described as selective estrogen receptor modulator (SERM)

T

[118;119]. Indeed, 27-OHC modulates ERα activity in a tissue-specific manner. For instance, 27-

IP

OHC was shown to impair the beneficial effects of estrogen on vascular function, but to stimulate

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ER-positive breast tumor growth and progression in mice. The binding of oxysterols to ERα also

AC

CE P

TE

D

MA

NU

explains part of the effects of oxysterols observed in bone homeostasis (e.g. osteoporosis) [120].

16

ACCEPTED MANUSCRIPT Table 3. Receptors binding oxysterols Targets

Oxysterols ligands

Functionality

pharmacological parameters

LXRα agonist

ND

(a)

LXRβ agonist

ND

(a)

[121]

(b)

5α,6α-epoxychol-3sulfate

D

7-ketochol-3-sulfate

Liver X receptors (LXRs α and /or β)

no binding

LXRα antagonist

EC50=2.0 µM

LXRα

ki>10 µM ki>10 µM

LXRα

ki>5 µM

LXRβ

ki>5 µM

LXRα antagonist

EC50=5 µM

LXRα agonist

(d)

TE CE P

LXRβ agonist LXRα antagonist LXRβ antagonist LXRα agonist

AC

24(S)-OHC LXRβ agonist LXRα agonist

24(S),25-epoxychol

27-OHC Retinoic-related orphan receptors (RORα, RORβ, RORγ)

7α-OHC

ki= 0.38 µM EC50=2.3-5 µM ki= 0.13 µM EC50=0.5-3 µM ki=0.15 µM ki=0.16 µM ki=0.11 µM EC50=4 µM ki=0.10 µM EC50=3 µM ki=0.20-0.37 µM EC50=0.87-4 µM

LXRβ agonist

ki=0.20-0.39 µM EC50=0.81-3 µM

LXRα agonist

ki=0.18 µM EC50=7-22 µM

LXRβ agonist

ki=0.30 µM (e) EC50=3 µM

25-OHC

25-OHC-3-sulfate

[122]

[123]

22(R)-OHC

22(S)-OHC

[112]

[123]

LXRβ

MA

7-ketochol

LXRβ

NU

7α-OHC

(c)

EC50=1.7 µM

SC R

5α,6α-epoxychol

IC50= 0.08 µM

IP

LXRα antagonist

T

4β-OHC

References

LXRα antagonist LXRβ antagonist

ND

LXRα partial agonist LXRβ partial agonist RORα inverse agonist

EC50=0.09 µM EC50=0.07 µM Ki ~ 0.020 µM EC50=1.3 µM

RORγ inverse agonist

Ki ~ 0.025 µM EC50=1.7 µM

[122]

[112;123]

[123]

[123]

[112;123]

[112;123]

[124] [125]

[114;126]

17

ACCEPTED MANUSCRIPT

RORα inverse agonist

ki=0.027 µM EC50=0.6 µM

RORγ inverse agonist

ki=0.025 µM EC50=1.3 µM

RORγ inverse agonist

ki=0.02 µM EC50=0.28 µM

[113]

RORγ agonist

EC50=1.7 µM

[116]

RORγ agonist

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7β,27-di-OHC

RORγ agonist

MA

D

TE

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[114]

RORγ agonist

RORγ agonist

25-OHC

Ki ~ 0.025 µM EC50=0.7 µM

[114]

ki >5,0 µM EC50>6 µM ki=1.0 µM EC50=3.2 µM ki=0.12 µM EC50=0.52 µM ki=0.18 µM EC50=0.48 µM EC50=0.02-0.04 µM

7α,27-di-OHC

24(S),25-epoxychol

AC

Ki ~ 0.020 µM EC50=1.4 µM

RORγ

24(S)-OHC

GPR183 (or EBI 2 receptor)

RORα inverse agonist

7α,25-di-OHC

22(R)-OHC

Smoothened

Ki ~ 0.025 µM EC50=2.2 µM

RORγ inverse agonist

7-keto,27-OHC

Estrogen receptor α ERα

RORγ inverse agonist

T

7-ketochol

Ki ~ 0.020 µM EC50=0.7 µM

IP

7β-OHC

RORα inverse agonist

RORα ligand

Kd = 3nM

(f)

[116] [116] [116] [116] [127]

[113]

[128]

RORγ agonist

ki=0.28 µM EC50=0.02 - 1.0 µM

[116;127]

27-OHC

RORγ agonist

ki=0.47 µM EC50=0.65 µM

[116]

25-OHC

agonist

IC50 ~0.15µM

[118]

27-OHC

SERM

ki=1.3 µM

[119]

20(S)-OHC

allosteric modulator

EC50=3 µM

[129;130]

25-OHC

allosteric modulator

7α-OHC

agonist

ND IC50=1.3 µM

[130] [131]

IC50=3.5 µM

[132]

7α,25-di-OHC

agonist

IC50= 0.24 nM

[131]

IC50=70 nM

[132]

7β,25-di-OHC

agonist

IC50=0.004 µM

[131]

IC50=2.63 µM

[132]

7α,27-di-OHC

agonist

IC50= 0.78 nM

[131]

IC50= 362 nM

[132]

7β,27-di-OHC

agonist

IC50= 0,34 µM

[131]

IC50 > 10 µM

[132]

18

ACCEPTED MANUSCRIPT [132]

agonist

IC50=0.9 nM EC50= 0.7 nM

[133]

22(R)-OHC

agonist

IC50= 0.9 nM EC50= 0.2 nM

[133]

27-OHC

agonist

IC50=0.6 nM EC50= 5 nM

[133]

22(R)-OHC

agonist

EC50= 1.32 µM

[134]

7α-OHC

T

IC50 > 10 µM

IP

CXCR2

[131]

agonist

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GPR17

IC50=1,26 µM

25-OHC

AC

CE P

TE

D

MA

NU

ND: not determined (a) no IC50 or EC50 were reported by the authors, 4β-OHC significantly activated LXRs when tested at 7.8 and 31.2 µM [121] (b) 5α,6α-epoxychol has a complex pharmacology, as it was able to antagonize LXR-mediated gene expression but also to induce LXR-mediated gene expression in some cellular systems [112]. (c) when tested in the same affinity assay, 5α,6α-epoxychol was found to have an higher affinity for LXRα than the “reference” ligand 24(S),25-epoxychol [112]. (d) ref [123] discusses the relative efficacy of oxysterols at LXRα and LXRβ. They found 24(S),25epoxychol to be the agonist with the highest efficacy among the two dozen oxysterol tested. (e) found to be an antagonist by [123] but an agonist by [112;116] (f) Kumar et al determined the Kd values for [3H]-25-OHC on RORα (3nM) and RORγ (5nM).

19

ACCEPTED MANUSCRIPT 3.2. Cell membrane receptors GPR183 (also known as EBI2 for Epstein Barr virus-induced G protein–coupled receptor 2) is one of the membrane receptors known to bind oxysterols. Upon activation, GPR183

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couples to Gi/o proteins and inhibits adenylate cyclase activity. Among the oxysterols that

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have been tested, 7α,25-di-OHC is the most potent GPR183 endogenous agonist

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[131;132;135]. GPR183 is thought to play important immunomodulatory roles. Indeed, it is required for normal migration of B cells to appropriate intra- and extra-follicular sites of the secondary lymphoid tissues [93;132]. Furthermore, 7α,25-di-OHC has been shown to

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promote the migration of activated CD44+CD4+ T cells in a GPR183-dependent manner and thereby to play a pro-inflammatory role in a murine experimental autoimmune

MA

encephalomyelitis model of multiple sclerosis [136]. GRP183 is therefore suggested as an interesting therapeutic target to investigate in the context of autoimmune diseases [136;137].

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CXCR2 (C-X-C motif chemokine receptor 2), a Gi/o protein-coupled receptor, is another

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receptor involved in immunity and was shown to bind at least one oxysterol. Indeed, Raccosta et al. have shown that 22(R)-OHC (but not 22(S)-OHC and 4β-OHC) was able to bind

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and activate CXCR2. They also showed that 22(R)-OHC, plays a role in neutrophils’ migration towards tumors in a CXCR2-dependent (and LXR-independent) way, hence promoting neoangiogenesis and tumor growth [134].

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Sensi and co-workers have shown that GPR17 is another Gi/o protein-coupled receptor that is able to bind oxysterols. Indeed, they have reported affinities in the nanomolar range for 7α-OHC, 22(R)-OHC and 27-OHC [133]. The affinities of other oxysterols have not been described yet. As other endogenous compounds also bind this receptor (e.g. cysteinylleukotrienes and uracil nucleotides [138]) further studies will have to investigate the involvement of this receptor in potentially mediating oxysterols’ specific effects. This GPCR remains to be further characterized but has been found to be mostly expressed in the nervous system, heart, and kidney [138]. Oxysterols have also been characterized as allosteric modulators of two receptors. Nachtergaele et al. showed that 20(S)-OHC allosterically activates (EC50=3µM) the GPCR smoothened (SMO) [130]. It was subsequently found that 7-keto-25-hydroxycholesterol and 7-keto-27-hydroxycholesterol, two metabolic intermediate in the elimination of 720

ACCEPTED MANUSCRIPT ketocholesterol, bind and activate the SMO receptor via the same pocket than 20(S)-OHC [139]. Other oxysterols, including 22(R)-OHC, 22(S)-OHC and 7β-OHC, were found to be inactive on SMO receptor [130]. The allosteric binding site was further characterized as

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extracellular and clearly distinct form the transmembrane orthosteric site [129;140].

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Besides, 24(S)-OHC has been characterized as an allosteric modulator (EC50=1.2 µM) of N-

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methyl-D-aspartate (NMDA) receptors [141]. Here also, a quite stringent regioselectivity was observed as several other oxysterols failed to enhance NMDA receptor signaling. Interestingly, in a subsequent study, the same group reported that 25-OHC non-

AC

CE P

TE

D

MA

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competitively antagonizes the effects of 24(S)-OHC on NMDA receptors [142].

21

ACCEPTED MANUSCRIPT Table 4. Non-receptor proteins binding oxysterols Protein (and gene) name

Subcellular localization

Oxysterols ligand

Affinity & efficacy parameters ND

(a)

ND

(a)

[143]

ND

(a)

[143]

27-OHC

ND

(a)

[143]

24(S),25-epoxychol

ND

(a)

[143]

25-OHC

Oxysterol binding proteins (OSBP related, OSBPL or

* nucleus membrane * late endosome

* late endosome membrane

CE P

AC

(NPC1)

TE

D

* cytosol

Niemann-Pick protein C1

[144]

22(R)-OHC

kd= 14 nM

[144]

25-OHC

kd= 10-20nM

[144;145]

5α,6β-di-OHC

EC50=1.7µM

[146]

7-ketochol

EC50=8.7µM

[146]

22(R)-OHC

EC50=2.9µM

[146]

24(S)-OHC

-

[147]

EC50=3.3µM

[146]

kd= 10 nM

[147]

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ORPs)

membrane

[143]

kd= 160 nM

7-ketochol

NU

* endoplasmic reticulum

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Insulin induced gene (Insig) membrane

IP

24(S)-OHC

* endoplasmic reticulum

T

22(R)-OHC

(b)

References

* lysosome membrane 25-OHC 27-OHC

ND

(a)

[147]

25-OHC

ND

(a)

[148-150]

Steroidogenic acute

regulatory-related lipid

* cytosol

transfer (StarD4/5)

(a) Binding was shown, but no actual values characterizing the binding were given (ND : not determined) (b) OSBP and ORP constitute a large family of cytoplasmic lipid transporters, the kd values reported refer to multiple proteins.

22

ACCEPTED MANUSCRIPT

3.3. Transport proteins as oxysterols targets In addition to bind to nuclear or cell membrane receptors, oxysterols are also able to bind

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regulation of cholesterol and lipids homeostasis (Table 4).

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and regulate the function of several other proteins which play a direct or indirect role in the

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The insulin induced gene protein (Insig) is a regulatory protein for the sterol regulatory element-binding protein (SREBP), a transcription factor which upregulates the expression of enzymes involved in cholesterol biosynthesis [143]. It was shown that 25-OHC is able to bind

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Insig, thereby inducing a close interaction between Insig and scap, thus preventing SREBP export to the Golgi and subsequent activation. A similar binding was observed for 24(S)-OHC,

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22(R)-OHC and 27-OHC [143]. This interaction is central in the control exerted by some oxysterols on cholesterol metabolism.

Niemann-Pick protein (NPC) 1 is a membrane glycoprotein which resides primarily in the

D

late endosomes and transiently in lysosomes [151]. This protein shares the sequence

TE

homology with other regulatory protein involved in the cholesterol homeostasis such as SREBP and SCAP [152]. The NPC2 is a soluble lysosomal protein. NPC proteins bind not only

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cholesterol but also oxysterols (e.g. 25-OHC, IC50: 5 nM for NPC1) [147;153]. Even if the exact role of oxysterol binding to NPCs is not well understood, in vitro studies showed that NPC1 and NPC2 regulate cellular cholesterol homeostasis through generation of oxysterols (i.e. 25-

AC

OHC, 27-OHC) [154]. Indeed, failure to transport cholesterol from lysosomes to mitochondria and ER prevents the synthesis of 27-OHC (by mitochondrial CYP27A1) and 25OHC (by endoplasmic reticulum/golgi CH25H). This results in reduced LXR activation and increased SREBP-dependent gene expression thus altering cholesterol synthesis [154]. The oxysterol binding proteins family (OSBP related, OSBPL or ORPs) constitute a family of sterol and phosphoinositide binding/transfer proteins [149]. More than 16 human members divided in 6 main groups and 12 members in mice have been identified [155;156]. They are characterized by a carboxy-terminal OSBP-related ligand-binding domain which binds a variety of lipids such as phosphatidylinositol-4-phosphate, cholesterol, and oxysterols [149]. Only the affinity of a few oxysterols for ORPs was studied. For instance, 25-OHC was shown to bind human OSBP with a Kd of 10 nM (compared to 173 nM for cholesterol). 22(R)-OHC, 23

ACCEPTED MANUSCRIPT 7-ketocholesterol and 25-OHC were shown to bind ORP2 (Kd of 14, 140 and 3900 nM, respectively) [157]. OSBP and its related proteins (ORPs) are involved in vesicle trafficking, lipid metabolism

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and signal transduction [149;155;158]. The diversity of these proteins and the large number

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of their ligands explain the few studies that specifically address the interactions between

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these proteins and oxysterols and their pathophysiological relevance. However, although oxysterols are not the sole ligands of these proteins, it seems that upon binding they can modulate their functionality [159]. For instance, Wang and co-workers showed that OSBP, when bound to cholesterol, oligomerizes with a serine/threonine phosphatase (PP2A). This

NU

newly formed oligomer can dephosphorylate phosphorylated ERK. The binding of an oxysterol to OSBP results in the dissociation of this OSBP-PP2A complex suggesting a

MA

mechanism through which oxysterols can control this crucial cell signaling pathway [159]. ORP8, another OSBP-related protein, is able to bind 25-OHC but not 24(S)-OHC nor 7-

D

ketocholesterol [160]. This ORP was suggested to act as a sterol sensor as it affects the

TE

reverse cholesterol transport process via modulation of ABCA1 expression and cholesterol efflux in macrophages in vitro [160].

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Steroidogenic acute regulatory–related lipid transfer (START) domain proteins are transport proteins able to control cholesterol metabolism. StarD1 binds cholesterol but not oxysterols, while StardD4 and 5 bind cholesterol as well as 7α-OHC and 25-OHC, respectively

AC

[161;162]. The overexpression of StarD1 in macrophages results in increased cholesterol efflux through apoA1 in a LXR- and cyp27A1-dependent manner [163;164]. StarD1 shuttles cholesterol to the mitochondria where cyp27A1 is expressed, favoring 27-OHC formation and subsequent modulation of LXR-dependent genes. While the role of StarD proteins is being unraveled, the implication of oxysterol binding to some of these proteins remains to be clarified.

4.

Murine models available to study some of the role of oxysterols Different murine models have been developed to study the metabolism and roles of

oxysterols. Most of them consist in knockout animals for one of the enzymes involved in oxysterol metabolism. Mice expressing the human orthologue of the gene of interest have also been developed for some CYP. Furthermore, several knockout mice have been 24

ACCEPTED MANUSCRIPT generated in order to study the receptors and proteins mediating oxysterols’ effects. Here we chose to focus our discussions on the models in which oxysterols’ metabolism was directly affected (see Table 2).

T

4.1 Cytochrome 3a knockout (Cyp3a -/-)

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The enzymes of this subfamily are among the most studied CYP in human because of their

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role in the metabolism of xenobiotics (e.g. they metabolize over 50% of drugs). As discussed, these enzymes are involved in the synthesis of 4β-OHC (CYP3A4 and 5 in human and Cyp3a11 and 13 in mice) and 25-OHC (CYP3A4 in human) from cholesterol (see Table 2 &

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Figure 2). Recently, Hashimoto et al. studied the impact on cholesterol metabolism of the genetic invalidation of the entire Cyp3a subfamily in mice (Cyp3a-/-) [7]. They found an

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increased expression of enzymes involved in cholesterol synthesis (e.g. HMG-CoA synthase 1, SQLE) and in oxysterol metabolism in the liver (e.g. Cyp7a1, Cyp8b1, Cyp7b1) as well as in the intestine (e.g. HMG-CoA reductase). Moreover, they found that hepatic 25-OHC levels

D

were lower in Cyp3a-/- mice compared with wild-type mice. Although this was not shown in

TE

vitro, this could suggest that Cyp3a, similarly to human CYP3A4, is able to form 25-OHC. This could also be due to an increased catabolism by Cyp7b1 (the expression of which is

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increased in this model). Moreover, 7α-OHCnone (C4) levels were increased which is in line with the increased Cyp7a1 expression. Unfortunately, in this interesting model, the levels of 4β-OHC, an oxysterol which synthesis depends directly on Cyp3a11 and/or 13 activities in

AC

mice were not reported.

4.2 Cytochrome 7a1 knockout (Cyp7a1 -/-) Historically, this model was set up to study the metabolism of bile acids [13;165;166]. Indeed, Cyp7a1 is responsible for catalyzing the rate-limiting step of the classical (or neutral) pathway of bile acid metabolism in both human and rodents. To date, the effect of Cyp7a1 invalidation on oxysterols levels has not been reported. However, in one study, it was shown that Cyp7a1-/- mice have an increased expression of the oxysterol 7α-hydroxylase (Cyp7b1) which could constitute a rescuing or compensatory mechanism for the bile acid deficiency which is typical of this genetic model [11]. In these Cyp7a1-/- mice, the in vitro synthesis of 7α,25-di-OHC from 25-OHC was maintained in line with the role of Cyp7b1 as catabolic enzyme of 25-OHC [11]. 25

ACCEPTED MANUSCRIPT 4.3 Cytochrome 7b1 knockout (Cyp7b1 -/-) The role of Cyp7b1 in the oxysterols’ metabolism and more generally the metabolism of steroids was further studied using Cyp7b1-/- mice. The levels of 25-OHC and 27-OHC, two

T

oxysterols substrate of Cyp7b1, are elevated in the liver, kidney and plasma of Cyp7b1-/-

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mice compared with wild type mice [18;167]. However, the levels of 24(S)-OHC were not, or

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only slightly, affected [18;167]. This model should help understanding two human genetic disorders characterized by a CYP7B1 deficiency, namely neonatal liver disease [20] and spastic paraplegia type 5 [21]. These two pathologies have in common the same mutation but the evolution toward one specific pathology seems to depend on the accumulation of

NU

substrates (i.e. oxysterols, steroids or other lipids) [168]. Finally, Cyp7b1-/- mice represent an interesting tool in the context of pathologies with an inflammatory component. Indeed, the

MA

products generated by this enzymes, i.e. 7α,25-di-OHC and 7α,27-di-OHC, have been shown to modulate the immune response via GPR183 receptor (chemoattractant properties) and

D

RORγ receptor (Th17 polarization), respectively [116;132;169].

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4.4 Cholesterol 25-hydroxylase knockout (Ch25h -/-) Ch25h-/- mice are largely used to study the role of 25-OHC in immunity, inflammatory and

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antiviral processes [6;170-173]. However, the exact role and the mechanisms mediating the effects of this oxysterol remain a subject of controversy. For instance, the deletion of Ch25h in an experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis has

AC

been shown to significantly attenuate the symptoms of the disease by limiting the trafficking of pathogenic CD4(+) T lymphocytes to the central nervous system [136]. However, in another study, a faster development of the disease was observed in Ch25h-/- EAE mice compared with wild type mice [173]. The authors also reported an increased sensitivity to septic shock [173]. In the latter study, the Ch25h-/- mice have also been studied in the context of viral infections. The antiviral effects of interferons (e.g. growth inhibition of several enveloped viruses) have been shown to be mediated by the production of 25-OHC [93;94]. Finally, Ch25h-/- mice were used to confirm the key role of Ch25h in the synthesis of the GPR183 ligand 7α,25-di-OHC and confirmed this oxysterol as an endogenous ligand of GPR183 receptor [132].

26

ACCEPTED MANUSCRIPT 4.5 Cytochrome 27a1 knockout (Cyp27a1 -/-) and overexpression The cholesterol 27-hydroxylase is also a well-studied enzyme in oxysterol metabolism. Its crucial role in bile acid synthesis (alternative pathway) and the fact that its deficiency is the

T

cause of cerebrotendinous xanthomatosis explain this interest [37]. For this ubiquitous

IP

enzyme, two types of models have been developed, a model in which Cyp27a1 expression is abolished [29] and a mouse model overexpressing the human Cyp27a1 (Cyp27a1overexp) [34].

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Consistent with their genotype, Cyp27a1overexp mice have higher circulating and tissue levels of 27-OHC [30;34] while Cyp27a1-/- have drastically reduced levels of this oxysterol [29]. Bile acid metabolism is clearly affected in these mice pointing to the important role of Cyp27a1

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[29;174]. As for cholesterol metabolism, although its hepatic and brain synthesis seems to be increased [30;174], plasma cholesterol levels remain unaltered by the deletion of Cyp27a1

MA

[29;174]. Shiri-Sverdlov’s group used these models to study the role of Cyp27a1 and 27-OHC in hepatic inflammation. The transfer of bone marrow from Cyp27a1 -/- mice to irradiated

D

Ldlr-/- mice fed a high fat and cholesterol diet resulted in increased inflammation and liver damage compared with mice that were transplanted with wild type bone marrow [175].

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When the same mice were transplanted with bone marrow from Cyp27a1overexp mice hepatic

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inflammation was reduced [35]. However, neither mice transplanted with Cyp27a1-/- bone marrow nor those transplanted with Cyp27aoverexp bone marrow displayed altered levels of 27-OHC in the liver or plasma [35;175]. Cyp27a1-/- mice were also used to study the link between hypercholesterolemia and breast cancer development [176]. Nelson and

AC

collaborators found that Cyp27a1-/- mice were less susceptible to develop ER-positive breast cancer than wild type mice, and that, consistent with its role as SERM [119], the effects of 27-OHC were mediated by ERα [177]. 4.6 Cytochrome 46a1 knockout (Cyp46a1 -/-) and overexpression The pivotal role of Cyp46a1 in regulating cholesterol metabolism in the central nervous system and the suggested link between oxysterols and neurodegenerative diseases and neuroinflammation [178] explain the interest in developing Cyp46a1-/- mice. This model has been used to directly explore cholesterol metabolism and pathways of cholesterol efflux from the brain. As expected, Cyp46a1-/- mice display decreased brain and serum levels of 24(S)-OHC [43;179]. Although cholesterol levels are not affected, brain cholesterol synthesis 27

ACCEPTED MANUSCRIPT is decreased in the Cyp46a1-/- mice compared with the wild type mice [43]. This suggests the implementation of compensatory mechanisms in order to cope with the decreased cholesterol efflux.

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Cyp46a1 knock-out and knock-down models allowed to study the role of 24(S)-OHC in several CNS diseases where cholesterol metabolism is known to play a key role. For instance,

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knock-down of Cyp46a1 (ShCyp46a1) in the striatum of wild-type mice resulted in neuron degeneration and motor deficits reproducing Huntington's disease phenotype [41]. Moreover, Cyp46a1 overexpression in the striatum of the R6/2 mice model of Huntington's

NU

disease led to a reduction of the neurodegeneration and motor deficits observed [41]. Finally, in a model of Alzheimer’s disease (APP23 mice), overexpression of Cyp46a1 using a

MA

viral vector increased 24(S)-OHC levels but did not change cholesterol levels. The APP23 mice overexpressing Cyp46a1 had a decreased astrogliosis and microgliosis and showed

D

reduced amyloid deposits and improved spatial memory [180].

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4.7 Cytochrome double or triple knockout models in oxysterols studies In addition to the single knockout models described above, some double or triple

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knockout models have been described. For instance Pikuleva’s group used Cyp46a1−/− Cyp27a1−/− mice to study cholesterol’s metabolism in the retina [32]. Another example is the triple knockout mice - Cyp46a1−/− Cyp27a1−/− Ch25h−/− - model developed by Russell’s group

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to study LXR signaling by decreasing the levels of three important LXR ligands (24(S)-OHC, 27-OHC and 25-OHC) [33]. 4.8 Humanized models Mouse and human cytochromes may differ in terms of expression, regulation, and activity [59]. Thus, using humanized mice models might represent an interesting tool to better understand oxysterol metabolism in humans. Furthermore, such model allows studying selected CYP isoforms. As an example, this strategy was used to study drug metabolism for instance using Cyp3a /-

mice expressing human CYP3A4 [181]. This model could be interesting in the study of 4β-

OHC which is one of the most abundant and stable oxysterol in human plasma.

28

ACCEPTED MANUSCRIPT A model overexpressing the human CYP27A1 in mice was used to study the role of this enzyme in the metabolism of cholesterol. The authors found that CYP27A1 was not crucial in cholesterol metabolism and that overexpression of human CYP27A1 in mice resulted in

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T

decreased 24(S)-OHC plasma levels [34]. 5. Oxysterols levels in human and mouse

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Based on the different sections discussed above, it is undeniable that several oxysterols are important mediators in many pathophysiological conditions [182;183]. However, some challenges remain in order to gain a better understanding of their roles. Indeed, the

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interconnections in the metabolic pathways, as well as the possibility for many species to activate the same molecular targets, contribute to a great complexity. One potential way to

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solve this problem would be to quantify the whole “oxysterome” when studying an animal model or human disease.

Mass spectrometry has clearly facilitated the development of quantification methods

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based on liquid (HPLC or UPLC) or gas (GC) separation of the oxysterols. Nowadays, validated

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HPLC-MS and GC-MS methods allow for the quantification of a large number of oxysterol species in human and animal models [184-186]. However, both techniques often require

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time consuming sample pre-treatment in order to efficiently eliminate cholesterol which can interfere during the analysis by reacting with free radicals and create artefactual oxygenated species of cholesterol (i.e. 7β-OHC, 7-ketocholesterol, 5α,6α-epoxycholesterol, 5β,6β-

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epoxycholesterol). Furthermore, numerous methods are characterized by a time-consuming derivatization step used to favor the detection and identification of the oxysterols [187]. Another element to take into consideration when discussing oxysterol levels is the fact that they are present in both free and esterified forms due to transferase activity (e.g. ACAT and LCAT). Thus, in literature, both free and total oxysterol levels can be reported (see Table 5). Actually, some oxysterols (e.g. 7α-OHC or 27-OHC) can be found in the esterified form at more than 90% [185;188;189]. Table 5 summarizes the oxysterol levels found in two key tissues – brain and liver – as well as in two important biofluids, i.e. plasma/serum and cerebrospinal fluid. Indeed many oxysterols have been investigated, and some suggested, as biomarkers. This is the case for

29

ACCEPTED MANUSCRIPT instance of 24(S)-OHC that was put forth as a biomarker for neurodegenerative diseases [190;191].

---

Plasma or serum (ng/mL)

107.1 [64]

---

brain (ng/g) CSF (ng/mL)

-----

-----

Liver (ng/g)

413.6 [186]

Liver (ng/g) 5α,6β-di- Plasma or serum (ng/mL) OHC brain (ng/g) CSF (ng/mL)

-----

56.7 [186]

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5β,6β- Plasma or serum (ng/mL) epoxychol brain (ng/g) CSF (ng/mL) Liver (ng/g) Plasma or serum (ng/mL) 6-OHC brain (ng/g) CSF (ng/mL)

7α-

12.4 – 77.0 [81;82;185;192]

-----

-----

---

---

---

---

1.3 [193]

6.0 – 9.0 [81;192]

-----

-----

-----

---

---

--4.2 – 27.0 [81;192] -------

-----

-----

1.4 – 9.0 [97;98;193] -----

741.2 [186]

---

---

194.4 [64]

---

6.0 [193]

-------

-------

-------

18.7 – 30.0 [81;192] -------

1.5 [1]

---

0.3 – 2.0 [71;192]

---

---

---

---

0.7 [1]

---

--0,03 - 0.2 [71;187]

---

---

---

1.2 [194]

0.3 – 3.5 [71;192;193]

9.0 – 145.0 [82;184;185;192]

---

---

---

413.9 (b) [186] Plasma or serum 25.0 – 52.2 (b) (ng/mL) [1;64] 116.0 (b) brain (ng/g) [195] Liver (ng/g)

7α-OHC

---

---

52.8 [64]

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Liver (ng/g)

81.3 [64]

---

21.9 – 30.0 [74]

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5α,6α- Plasma or serum (ng/mL) epoxychol brain (ng/g) CSF (ng/mL)

Total OHCs

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2119.2 [186]

human

Free OHCs

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Liver (ng/g)

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Mouse Free OHCs Total OHCs

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4β-OHC

Tissue

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OHCs

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Table 5. Oxysterol levels (a) in the liver, plasma, brain and cerebrospinal fluid (CSF).

---

CSF (ng/mL)

0.4 [1]

---

0.02 – 0.03 [71;187]

---

Liver (ng/g)

125.1 [186]

---

---

---

30

ACCEPTED MANUSCRIPT

0.1 [1] 327.2 [186] 2.4 – 44.2 [1;50;64] 60.0 -840.0 [195;197] ------

brain (ng/g)

----

3.0 – 43.0 [81;184;185;192]

---

---

0.03 [71] --3.6 – 27.0 [71;98;192;193]

----10.7 – 98.0 [81;184;185;192]

---

---

---

------

0.03 [71;187] ----

--10.0 [198]

----

----

----

---------7.0 – 64.0 [37;71;192]

---------33.2 – 227.0 [81;82;185;192]

----

----

0.08 [71] ----

-------

----

----

0.1 - 2.0 [198] ----

240.0 – 350.0 [197] ----1.2 [194]

------------------5.8 – 6.8 14.0 – 40.0 [81] [1;64] 1841.0 26600.0 27910.0 51000.0 [84;111;179] [30;197] 0.09 [1] ----------

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CSF (ng/mL) Liver (ng/g) Plasma or serum (ng/mL) 22(R)-OHC brain (ng/g) CSF (ng/mL) Liver (ng/g) Plasma or serum (ng/mL) 24(S)-OHC brain (ng/g)

0.3 – 1.02 [71;192]

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7-ketochol

1.0 [194]

AC

24(S),25epoxychol

CSF (ng/mL) Liver (ng/g) Plasma or serum (ng/mL) brain (ng/g) CSF (ng/mL) Liver (ng/g)

25-OHC

Plasma or serum (ng/mL)

brain (ng/g) CSF (ng/mL) Liver (ng/g) 7α,25-di- Plasma or serum OHC (ng/mL) brain (ng/g)

----

T

CSF (ng/mL) Liver (ng/g) Plasma or serum (ng/mL)

---

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116.0 [195]

---

TE

7β-OHC

---

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brain (ng/g)

Liver (ng/g)

-----

0.88 – 22.0 [37;71;192;196] -----

---

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Plasma or serum (ng/mL)

10.0 – 14.7 [7;64] ----413.9 (b) [186] 1.4 – 9.1 [1;50]

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OHCnone Plasma or serum (ng/mL) brain (ng/g) CSF (ng/mL)

27.5 [82] -----

400.0 – 1400.0 [84;179] ---21.4 [186]

----

----

---20.0 [7]

------

1.22 [1]

0.6 [194]

138.0 [111] 0.005 [1] 4.7 [186]

-------

0.5 [1]

---

0.4 [192]

0.1 [199]

---

---

---

---

-----2.0 – 31.0 1.3 – 4.0 [71;192] [81;82;184;192;1 98] ----0.03 [71] -------

31

ACCEPTED MANUSCRIPT

---

--0.01 [1] ---

-------

28.1 [1] --2.5 [1]

-------

---

---

T

----43.6 – 196.0 [82;184;185;192]

0.2 [71] ---

-----

0.6 – 0.9 [71;192]

1.4 [199]

--0.03 [71] --18.6 - 39.4 [71;192] -----

-------

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0.4 [1]

0.03 [71] --11.6 – 154.0 [37;71;192]

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CSF (ng/mL) Liver (ng/g) Plasma or serum 7α,27-di(ng/mL) OHC brain (ng/g) CSF (ng/mL) Liver (ng/g) Plasma or serum 7α,27-di(ng/mL) CA brain (ng/g) CSF (ng/mL)

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brain (ng/g)

0.04 [1] --6.2 [186] --7.3 - 8.4 --[1;64] 150.0 – 580.0 4000.0 – 6000.0 [111;179;195] [81] 0.03 [1] --7.8 [186] ---

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27-OHC

CSF (ng/mL) Liver (ng/g) Plasma or serum (ng/mL)

-------

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(a) Several techniques are used to quantify oxysterol in tissues. In the references [64;182;189;191;195] oxysterols were analyzed using an HPLC-MS method without derivatization. Charge-tagging (with Girard’s reagent P, picolinic acid or a quaternary aminooxy reagent) before HPLC-MS analysis is also frequently used [1;7;71;81-83;175;183;188;192;194]. In the references [94;95;180] oxysterols were transformed in the dimethylglycine derivatives and analyzed by HPLCESI-MS. Finally GC-MS can also be used, as in references [30;37;50;74;108;181;188;190;193] where oxysterols were analyzed as trimethylsilyl derivatives. (b) Measured as a pool of both 7α-OHC & 7β-OHC

Another issue in the oxysterol quantification is the inter-laboratory variability. This was

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clearly evidenced by a study in which the same serum samples were analyzed by 21 laboratories [187]. Such variability makes it hard to find a global consensus on reference values for oxysterol levels. At this point, the operational working groups on the topic (such as those suggested by the European network for oxysterol research) could be helpful in order to standardize protocols and perhaps to set up a common quality control system. Another point of interest is the fact that the levels of 7α-OHCnone were shown to have diurnal variations [200;201]. To date little is known about the potential circadian variations, as well as the effect of the feeding state, on most oxysterol levels. This interesting topic clearly deserves to be studied.

32

ACCEPTED MANUSCRIPT 6.

Biochemical considerations: oxysterols, more than simple metabolic intermediates? Based on the elements discussed, one question could be “are oxysterols to be considered

as intermediates in the catabolism of cholesterol or as full fledge bioactive lipids?”. We will

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discuss here arguments supporting both definitions, and show that these lipids actually meet

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the requirements of both.

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Oxysterols are clearly metabolites issued from cholesterol metabolism. Indeed, a definition of a metabolite (as proposed by the FDA) could include the following concepts: a structural similarity of the metabolite to its precursors, it has to be present inside cells,

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processed by enzymes, and its products can enter subsequent reactions. Metabolites have a finite half-life and do not accumulate in cells. Numerous metabolites have the ability to

MA

regulate metabolism and exert biological functions inside cells. Regardless of their origin (from cholesterol or from cholesterol’s precursors) all oxysterols are closely structurally related to cholesterol, retaining cholesterol’s sterol ring and

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methylheptyl side chain. Oxysterols are intracellularly produced, with most of them

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originating from enzymes localized in the endoplasmic reticulum, with the notable exception of CYP27A1 and CYP11A1 which are mitochondrial. Of note, oxysterols have been quantified

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in whole cells but also different organelles (e.g. nuclei, mitochondria, plasmalemma or endoplasmic reticulum) [202]. Oxysterols are products of enzymatic reactions having cholesterol as substrate but they are also substrate of subsequent enzymatic reactions,

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often leading to bile acids (as illustrated in Figure 2). Oxysterols have a finite half-life as they do not accumulate (in physiological conditions) in cells, they can even be considered as an important cellular efflux system for cholesterol. The plasma half-life of different oxysterols was studied by several research groups and found to be highly variable depending on the oxysterol [72;203;204]. For instance, Bodin et al. showed that 4β-OHC has a very long half-life in human plasma (60 hours) while oxysterols such as 7α-OHC and 27-OHC have a half-life of less than 1 hour [72]. This short half-life could be explained by the fact that these two oxysterols are also the first principal intermediates in bile acids synthesis pathways. Oxysterols exert regulatory functions as it was clearly demonstrated that they control cholesterol, glucose, and lipid metabolism as well as immune functions [182;205-208]. However, whether oxysterols regulate their own metabolism is still unclear. Indeed, it is not 33

ACCEPTED MANUSCRIPT known whether oxysterols exert a regulatory feedback on the expression or activity of the enzyme responsible for their formation. In mice at least our group could not find any correlation between oxysterol levels in tissue and the expression of the corresponding

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enzyme [64]. Different and not mutually exclusive hypothesis could explain this discrepancy.

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The first one is that the enzymes involved in the oxysterol system are redundant: the same

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enzyme can metabolize different oxysterols and different enzymes can metabolize the same oxysterol. Another explanation could be the presence of a cross-regulation in the system where a given oxysterol could modulate the expression of an enzyme that is not involved in its own metabolic pathway.

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Besides these characteristics defining oxysterols as metabolites, several elements point to oxysterols as bioactive lipids. Bioactive lipids are endogenous lipid mediators whose changes

MA

in levels have functional consequences. These physiological repercussions are often mediated by specific molecular targets even if changes in cell membrane properties (e.g.

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changes in micro-domain composition) cannot be excluded. Although not the main focus of

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this review, several pathophysiological situations (e.g. obesity, atherosclerosis, Alzheimer’s disease) are characterized by altered oxysterol levels [182;183]. We also described the large

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number of proteins, including several receptors, which are able to interact with one or more oxysterols. Consequently, in addition to their role in controlling cholesterol metabolism, oxysterols have been shown to be involved in many processes such as immunity

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[116;132;171;209], bone mass homeostasis (e.g. osteoclast precursors migration and differentiation) [120;210;211], nervous system development and homeostasis (e.g. promotion

of neurogenesis, promotion of myelination and remyelination) [71;111;212]. In conclusion, it is clear that oxysterols cannot be reduced to simple metabolic intermediates. Indeed, in addition of being intermediates in bile acid synthesis, they constitute a form of cholesterol transport and directly participate in its homeostasis. Furthermore, through the activation of an increasing number of receptors, they are directly involved in several processes implicated in metabolism, immunity, brain homeostasis and many others. Concluding remarks

34

ACCEPTED MANUSCRIPT In this review we provided an updated overview and discussion about oxysterol metabolism and on evidence pointing to oxysterols as bioactive lipid mediators. However, as in numerous fields, the more we learn the more questions arise and need to be explored.

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One of the many challenges to tackle in the complex oxysterol system is their metabolism.

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The complexity in the metabolic pathways leading to the formation of oxysterols is largely

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due to the enzymes’ redundancy and the coexistence of both enzymatic and non-enzymatic synthesis (Figure 2). This clearly does not facilitate the interpretation of results. In many studies where oxysterol levels are altered by genetic modulations of synthesis and/or

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degradation enzymes, quite often only a few oxysterols are quantified. Thus the potential presence of compensatory mechanisms is often not directly studied. Hence, the

MA

quantification of the global “oxysterome” in such models may contribute to a better understanding of the intricate and complex oxysterol metabolism. Attempts have been made to link specific oxysterol levels with the activity of one specific metabolic enzyme.

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Although not always successful, this allowed for some oxysterols to be considered as proper

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biomarkers of enzyme activity (e.g. 4β-OHC levels and CYP3A4 activity). However, these associations remain the exception as enzyme expression, or activity, most of the time, do

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not correlate with oxysterol levels. Another potential explanation for this lack of direct correlation between oxysterol levels and enzyme expression/activity could be the fact that oxysterols also exist in esterified form. Actually, any oxysterol species can be esterified by

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ACAT or LCAT giving rise to a pool of esterified oxysterols. This esterified pool can be very important when compared with the oxysterol free form (e.g. 90% of 27-OHC has been found to be in the esterified form). So far, the esterified oxysterols are still considered to be physiologically inactive unlike the free form. One of the main challenges in the future will be to better understand how that stored pool can be mobilized in a given condition and if the esterified forms can have any effects per se in physiopathological conditions [213]. 3-Sulfate conjugated oxysterols are another conjugated form of oxysterols that should attract more attention in the future. Indeed, the most studied sulphated oxysterol, 25hydroxycholesterol-3-sulfate, has biological effects that are distinct from those of its free form [124]. However, beside 25-hydroxycholesterol-3-sulfate little is known about the levels

35

ACCEPTED MANUSCRIPT and roles of these oxysterol conjugates. Dedicated quantification methods would facilitate the study of sulfated oxysterol levels in physiopathological situations. One major difficulty when studying the effect of oxysterols in pathophysiological settings is

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that modulating the levels of a specific oxysterol is quite challenging. The administration of

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the oxysterol of interest is one option. However the majority of oxysterols have a short half-

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life due to efficient metabolism processes. Therefore, even when effects are observed, the question remains on the actual molecule responsible. Another strategy quite often used to test the effect of an oxysterol is to use knockout models of the enzyme responsible for its

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production (e.g. Ch25h-/- mice to study the involvement of 25-OHC). However, knockout of an enzyme of the oxysterol system can lead to profound alteration in oxysterols (and

MA

sometimes bile acids) levels well beyond those of the oxysterol of interest. From a translational standpoint, it is important to bear in mind that there are

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dissimilarities in cytochromes P450 between mouse and human [59;214]. For instance, some

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enzymes such as CYP3A4 are not present as such in mouse (its orthologue being Cyp3a11). In this context, mice models such as the humanized mice models (e.g. mice expressing the

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CYP3A4 [9] or CYP27A1 [35]) could be useful. Another element is that, in humans, the activity of several cytochromes P450 is affected by genetic variations (polymorphisms) [215]. So far, only a limited number of studies evaluated the effect of these polymorphisms on

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oxysterol levels [77;216].

Oxysterols are a large family of lipids exerting pleiotropic functions. As discussed in this review, their metabolic pathways are quite complex and they have multiple molecular targets. In this context, a good understanding of oxysterols’ effects will be favored by both the quantification of the largest number of oxysterols possible and the assessment of the expression of the corresponding enzymes.

Acknowledgments VM and OGL are research fellows of the “Fonds pour la recherche dans l’industrie et l’agriculture” (FRIA, Belgium). GGM is the recipient of subsidies from the Fonds Spéciaux de Recherches (FSR, Université catholique de Louvain) and from the FRS-FNRS, Belgium (grant J.0160.13). 36

ACCEPTED MANUSCRIPT Figure Legends

Figure 1: Structures of the main oxysterols detected in biological samples. The blue frame

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(A) contains the enzymatically-formed oxysterols, while the oxysterols formed by free radical

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oxidation are contained in the red frame (B). Several oxysterols (e.g. 7-OHC, 25-OHC and 7-

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ketochol) can be formed by both enzymatic reaction and free radical oxidation (C). Figure 2: Metabolic pathways involved in oxysterol synthesis (see the text for more details). The enzymes names correspond to the human nomenclature. Most of the human and

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mouse orthologues have the same name, except for the mouse orthologues of CYP3A4 and CYP3A5 which are Cyp3a11 and Cyp3a13, respectively.

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CH25H: cholesterol-25-hydroxylase; ChEH: cholesterol epoxide hydrolase; DOS: 2,3dioxidosqualene; HSD: hydroxysteroid dehydrogenase; MOS: 2,3(S)-monooxidosqualene; RNS: reactive nitrogen species; ROS: reactive oxygen species; SQLE: squalene epoxidase;

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SULT: sulfotransferase.

37

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