Advances in the physiological and pathological implications of cholesterol

Biol. Rev. (2013), pp. 000–000. 1doi: 10.1111/brv.12025

Advances in the physiological and pathologicalimplications of cholesterol

Victor A. Cortes1,∗, Dolores Busso1, Pablo Mardones1, Alberto Maiz1, AntonioArteaga1, Flavio Nervi2 and Attilio Rigotti1

1Department of Nutrition Diabetes and Metabolism, School of Medicine, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Marcoleta

367 Edifico de Gastroenterologia 4 piso, Santiago, Chile2Department of Gastroenterology, School of Medicine, Faculty of Medicine, Pontificia Universidad Catolica de Chile, Santiago, Chile

ABSTRACT

Cholesterol has evolved to fulfill sophisticated biophysical, cell signalling, and endocrine functions in animal systems.At the cellular level, cholesterol is found in membranes where it increases both bilayer stiffness and impermeabilityto water and ions. Furthermore, cholesterol is integrated into specialized lipid-protein membrane microdomains withcritical topographical and signalling functions. At the organismal level, cholesterol is the precursor of all steroidhormones, including gluco- and mineralo-corticoids, sex hormones, and vitamin D, which regulate carbohydrate,sodium, reproductive, and bone homeostasis, respectively. This sterol is also the immediate precursor of bile acids,which are important for intestinal absorption of dietary lipids as well as energy homeostasis and glucose regulation.Complex mechanisms maintain cholesterol within physiological ranges and the dysregulation of these mechanismsresults in embryonic or adult diseases, caused by either excessive or reduced tissue cholesterol levels. The causativerole of cholesterol in these conditions has been demonstrated by genetic and pharmacological manipulations in animalmodels of human disease that are discussed herein. Importantly, the understanding of basic aspects of cholesterol biologyhas led to the development of high-impact pharmaceutical therapies during the past century. The continuing effortto offer successful treatments for prevalent cholesterol-related diseases, such as atherosclerosis and neurodegenerativedisorders, warrants further interdisciplinary research in the coming decades.

Key words: cholesterol, mevalonate pathway, atherosclerosis, nuclear receptors, Alzheimer’s disease.

CONTENTS

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2II. General aspects of cellular cholesterol biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

(1) Cholesterol and membrane structure and physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2(2) Cholesterol biosynthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5(3) Cellular cholesterol uptake . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5(4) Cellular cholesterol efflux . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5(5) Intracellular cholesterol regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

III. Cholesterol physiological regulation and pathological implications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7(1) Whole-body cholesterol homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

(a) Nuclear receptors LXR and FXR coordinate body cholesterol and bile acids homeostasis . . . . . . . . . 8(b) LXR transcriptionally regulates cholesterol transport from extrahepatic tissues to the liver for its

catabolism and body elimination . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9(c) Hepatic de novo lipogenesis is regulated by LXR and is metabolically coupled to ACAT-dependent

cholesterol esterification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 9(2) Cholesterol in the central nervous system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

(a) The central nervous system (CNS) is segregated from body cholesterol metabolism . . . . . . . . . . . . . . . . 10(b) CNS cholesterol efflux requires its conversion to 24(S)-hydroxycholesterol and inhibition of this

pathway results in learning disability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10

* Author for correspondence (Tel: ++56 23 546389; E-mail: [email protected]).

Biological Reviews (2013) 000–000 © 2013 The Authors. Biological Reviews © 2013 Cambridge Philosophical Society

2 Victor A. Cortes and others

(c) Defects in cholesterol synthesis or intracellular trafficking determine neurodegeneration . . . . . . . . . . . 11(d ) Relationship between CNS apolipoprotein E4, cholesterol trafficking and Alzheimer’s disease . . . . . 11

(3) Cholesterol in embryonic and fetal development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12(4) Origins of embryonic/fetal cholesterol . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13(5) Cholesterol cytotoxicity: implications for atherosclerosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13

IV. Is cholesterol essential for life? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13V. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14

VI. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 14VII. Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

VIII. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

I. INTRODUCTION

In what could be considered a fateful connection topathology, cholesterol was first identified in 1769 as themain component of human gallstones (Olson, 1998). Sincethen, it has been implicated in a variety of humandiseases, such as atherosclerosis, Alzheimer’s disease anddiverse malformation syndromes. However, this versatilelipid molecule has also been linked directly or indirectly to astunning diversity of biological roles, such as membranestructure and function, cell signalling, morphogenesis,intestinal lipid digestion and absorption, reproduction, stressresponse, sodium and water balance, and calcium andphosphorous metabolism. Due in part to this wide rangeof functions, research on cholesterol has been instrumentalin the progress of several biomedical fields, reflected inthirteen Nobel laureates for their discoveries on differentaspects of this molecule (Brown & Goldstein, 1986).

In contrast to most other naturally occurring lipidscharacterized by long aliphatic chains, cholesterol is a27-carbon polycyclic molecule, comprised of 4 fused ringsin a planar conformation. It contains one double bond(C5-C6), one β-hydroxyl substitution (C3), and a simpleeight-carbon aliphatic tail (Fig. 1A). Notably, the β-hydroxylgroup not only confers a slight polarity to the molecule, whichdetermines its orientation within phospholipid bilayers,but it can also be esterified with fatty acyl chains byacyl-CoA:cholesterol acyl transferases (ACATs). Cholesterylesters are important for storage and transport of cholesterolin the hydrophobic core of intracellular lipid droplets andplasma lipoproteins, respectively. Unlike other commonlipids, cholesterol cannot be used as an energy source inanimal cells since they lack enzymes that can break the sterolnucleus down to its original acetyl-CoA units. Therefore, the‘catabolic’ metabolism of cholesterol implies its conversioninto other biologically active molecules.

Plant sterols, also called phytosterols, share the samesteroid ring structure with cholesterol but have a bulkierside chain, substituted with methyl and/or ethyl groups(Fig. 1B, C). Despite their structural similarities, animals haveevolved very efficient mechanisms to prevent phytosterolaccumulation, suggesting that plant sterols cannot fulfillcholesterol functions and/or that they exert toxic actions onanimal systems. Indeed, the large number of genes involvedin cholesterol synthesis, catabolism, trafficking, and excretion

further support the hypothesis that this lipid is critical foranimal cells.

II. GENERAL ASPECTS OF CELLULARCHOLESTEROL BIOLOGY

(1) Cholesterol and membrane structure andphysiology

Cholesterol and its fatty acyl esters are virtually insolublein water. This determines that they are exclusively found inmembranes and other lipid and/or lipid-protein complexes,such as lipid droplets and lipoproteins. Within membranes,cholesterol interacts with phospholipids and sphingolipidfatty acyl chains resulting in increased membrane bilayerrigidity and reduced permeability to water and ions(Bloch, 1983).

At a subcellular level, cholesterol is heterogeneouslydistributed, with only 0.5–1% of total cholesterol presentin the endoplasmic reticulum (ER) (Lange et al., 1999) and60–80% in the plasma membrane (Lange & Ramos, 1983;Liscum & Munn, 1999; van Meer, Voelker & Feigenson,2008). In this latter organelle, cholesterol accounts for 20%of total lipid mass (Haines, 2001) and appears to be criticalfor its organization and function. In fact, the formation ofspecialized membrane microdomains, called ‘lipid rafts’,can only be achieved when cholesterol concentration in theplasma membrane reaches a 10% threshold (Simons & Ehe-halt, 2002). These cholesterol- and sphingolipid-enrichedmicrodomains are dynamic protein/lipid assemblies thatdrift interspersed in the liquid-disordered membrane bilayerrecruiting a number of receptors and accessory proteins thatparticipate in extracellular ligand binding and intracellularsignal transduction (Simons & Toomre, 2000).

Paradoxically, in spite of its crucial cellular functions inmetazoan cells, cholesterol can only be synthesized from basicacetyl-CoA building blocks by vertebrates. Arthropods andnematodes lack the critical cholesterol biosynthetic enzymessqualene synthase and lanosterol synthase (see Section II.2),and must obtain cholesterol, or other steroid precursors, fromdietary sources (Svoboda, 1999; Kurzchalia & Ward, 2003).

Perhaps reflecting drastic disparities in environmentalavailability and biosynthetic capacity, cellular cholesterolrequirements are markedly different among species. For


Cholesterol in health and disease 3

(A)

(B)

(C)

(D)

Fig. 1. Structure of cholesterol, phytosterols and hopanoids.Cholesterol (A) is a 27-carbon, planar 3-β-hydroxylated sterolmolecule. Minor structural modifications preclude normalbiological functioning, indicating very selective molecularrequirements. Campesterol (B) and β-sitosterol (C) are the twomore abundant phytosterols in the human diet (95% of totalplant sterols in a normal diet). They are structurally related tocholesterol, but bulky substitutions in the side chain make themmore rigid and hydrophobic, enabling them to prevent protonleaks in plant cell membranes which are normally exposed tomore intense proton concentration gradients than animal cells.Bacteriohopane-32,33,34,35-tetrol (D) is the most abundanthopanoid in nature. Hopanoids are anaerobically derived fromsqualene and, like sterols, are multi-ring molecules and areembedded in membrane monolayers. Hopanoids possibly playimportant roles in limiting proton membrane permeability. Thecarbon numbering system for sterol molecules is shown in A–C.

example, biochemical determinations have shown that,on average, Caenorhabditis elegans cells have 20-fold lowertotal cholesterol content than mammalian cells (normalizedto phosphatidylcholine). More strikingly, morphologicalstudies with filipin, a fluorescent macrolide that binds andstains cholesterol, have shown that these nematodes haveentire groups of cells that are virtually free of cholesterol(Matyash et al., 2001; Merris et al., 2003). Interestingly, evenat this very limited level, cholesterol is still essential for thisinvertebrate species since a synthetic cholesterol enantiomer,with biophysical properties identical to the natural molecule,is incapable of supporting their growth and reproduction(Crowder et al., 2001).

Prokaryotic organisms, as a general rule, do not havecholesterol in their cellular structures but instead have devel-oped alternative molecular strategies to carry out someof the membrane-organising properties of sterols. In fact,either complex linear carotenoids or squalene-derived cyclichopanoids (Fig. 1D) are found across bacterial species exert-ing a cholesterol-like effect over membrane rigidity and per-meability. Considering that both squalene cyclization to formlanosterol and lanosterol demethylation to generate choles-terol require molecular oxygen (see Section II.2 and Fig. 2),it has been speculated that prokaryotes evolved hopanoidsbefore the appearance of molecular oxygen in the atmo-sphere, hinting at a convergent adaptation of cellular mem-branes to increasingly complex environments (Haines, 2001).

There are, however, a few exceptions to this ‘nocholesterol’ rule in prokaryotes. Notable examples areMethylococcus capsulatus, which can synthesize non-cholesterol4,4-dimethyl- and 4-monomethyl sterols (Bloch, 1979), andintracellular pathogens from Mycoplasma spp. and Coxiella

burnetti, the causative agents of some forms of atypicalpneumonia and the zoonosis fever Q, respectively (Howe &Heinzen, 2006). C. burnetti, like other intracellular microbes,thrives in pathological organelles called parasitophorousvacuoles (PVs), which resemble phagolysosomes in theirluminal acidity, the presence of hydrolytic enzymes, and theexpression of lysosomal markers. Notably, PVs are isolatedfrom the host’s vesicular traffic while their membranesremain highly enriched in cholesterol and contain lipid raftproteins such as flotillin. The source and the mechanismsby which cholesterol is incorporated into PV membranesremain unknown (Gilk, Beare & Heinzen, 2010) but mightconstitute new pharmacological targets against relevantinfectious diseases.

Indeed, C. burnetti as well as Toxoplasma gondii – an obligateintracellular protozoan – require cholesterol for growthin human cells (Coppens, Sinai & Joiner, 2000; Howe &Heinzen, 2006). Similarly, the persistence of Mycobacterium

tuberculosis in chronically infected, interferon-γ stimulatedmacrophages requires the incorporation of cholesterol fromthe host through active mechanisms that involve membersof the ATP binding cassette (ABC) transporter family(Pandey & Sassetti, 2008). This observation has tremendousimplications for human health since modern medicine lackseffective therapeutic tools against the globally expanding



Fig. 2. Cholesterol is the end product of the mevalonate pathway. The first step in cholesterol biosynthesis is the generation ofa 3-hydroxy-3-methyl-glutaryl CoA (HMG-COA) from acetyl-CoA units. Then, HMG-CoA is converted to mevalonate by theaction of HMG-CoA reductase on endoplasmic reticulum membrane. This is the only limiting rate reaction in cholesterol synthesisand is the target of statins (cholesterol-lowering drugs). Mevalonate is next converted to the activated isoprenoids isopentenylpyrophosphate and dimethyallyl pyrophosphate (not shown), from which originate farnesyl pyrophosphate (PP) and squalene. Thefollowing cyclization and oxygenation of squalene to lanosterol involves the action of squalene monooxygenase and lanosterolsynthase (also known as oxidosqualene cyclase, not shown) and requires molecular oxygen and nicotinamide adenine dinucleotidephosphate. This conversion only happens in vertebrates. Finally, the generation of cholesterol involves the following lanosterolmodifications: (i) demethylation at C4α, C4β, and C14; (ii) isomerization of the �8(9) double bond to a �7 double bond;(iii) desaturation to form a �5 double bond; and (iv) reduction of �14, �24, and �7 double bonds. Importantly, the reduction ofthe �24 double bond, catalyzed by 3β-hydroxysterol �24-reductase (DHCR24) can happen in any post-lanosterol intermediate,generating essentially two parallel pathways, which result in either 7-dehydrocholesterol or desmosterol, which are reduced tocholesterol by 7-dehydrocholesterol reductase (DHCR7) or DHCR24, respectively.



strains of multi-drug-resistant M. tuberculosis. Intriguingly,mycobacteria do not seem to require cholesterol forstructural or signalling functions but they rather use it asan energy source. In fact, M. tuberculosis can cleave ringA from the rest of the cholesterol molecule to generatepyruvate, which is later oxidized to CO2 in the tricarboxylicacid cycle. This species can also derive propionyl-CoAfrom cholesterol’s side chain for metabolic fuel production(Pandey & Sassetti, 2008). No other pathogens are knownto catabolize cholesterol for energy purposes, therefore thiscan be considered a unique metabolic adaptation, possiblytransmitted to M. tuberculosis by its saprophytic ancestors.

(2) Cholesterol biosynthesis

The complex structure and biosynthesis of cholesterol wereelucidated by the combined efforts of Nobel laureates H.O.Wieland (1927), L. Ruzicka (1939), R. Robinson (1947), K.Bloch and F. Lynen (1964), and J. W. Cornforth (1975),through almost four decades of painstaking work (Bloch,1965; Goldstein & Brown, 1990).

Cholesterol is synthesized in the endoplasmic reticulum(ER) by the concerted action of over 30 enzymes organizedin the mevalonate pathway (Fig. 2), whose first partinvolves four fundamental steps: (i) condensation of threeacetyl-CoA units to form 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA); (ii) HMG-CoA reduction mediated bynicotinamide adenine dinucleotide phosphate (NADPH)to generate mevalonate; (iii) conversion of mevalonate intothe activated isoprenoids isopentenyl pyrophosphate anddimethylallyl pyrophosphate; and (iv) polymerization ofsix isoprenoid units into squalene. Next, linear squaleneundergoes a series of oxygenation and cyclization reactionsto form lanosterol and, finally, lanosterol is converted tocholesterol by sequential oxidative demethylations anddouble-bond isomerizations and reductions.

The rate-limiting reaction in cholesterol biosynthesisis the conversion of HMG-CoA into mevalonate cat-alyzed by HMG-CoA reductase (HMG-CoA-R). Thisenzyme is tightly regulated at both transcriptional andpost-translational levels and is the pharmacological target ofstatins, the most potent cholesterol-lowering drug family incurrent use (see Section II.5).

Importantly, isoprene intermediates in the mevalonatepathway generate a variety of other bioactive molecules,including ubiquinone and heme A, involved in mitochon-drial electron transport; dolichols, utilized in the synthesisof glycoproteins; isopentyl adenine, present in some transferRNAs; and farnesyl and geranyl groups, required formembrane protein prenylation (Goldstein & Brown, 1990).

(3) Cellular cholesterol uptake

In addition to being synthesized intracellularly, cholesterolcan be incorporated into mammalian cells from plasmalipoproteins through cell surface receptor-mediated endo-cytosis (Fig. 3). Michael Brown and Joseph Goldstein firstdissected the now prototypical low density lipoprotein (LDL)

receptor (LDLR) pathway (Goldstein & Brown, 1974),demonstrating that specific cell surface receptors are requiredfor extracellular ligand endocytosis (Brown & Goldstein,1986). In this model, circulating LDL particles bind tothe LDLR on the cell surface and are incorporated, asa lipoprotein-receptor complex via clathrin-coated vesicles,into endolysosomal compartments for further processing. Atthis level, the LDLR is recycled back to the cell surface,whereas LDL particles are fully degraded into their individ-ual components. More specifically, LDL-derived cholesterylesters are hydrolyzed to form free cholesterol and fatty acidsby the action of lysosomal acid lipase. Deficiency of thisenzyme leads to the build-up of cholesteryl esters and otherlipids in tissues, causing a lysosomal storage disorder knownas Wolman’s disease (Wolman, 1995).

The importance of lysosomal processing of lipoproteinsfor cellular cholesterol homeostasis is further underscored byNiemann Pick type-C (NPC) disease, a lethal conditioncharacterized by intracellular unesterified cholesterolaccumulation. NPC is caused by mutations in the NPC1 orNPC2 genes that preclude endolysosomal cholesterol export(Klein et al., 2006). NPC1 is a large multispan lysosomalintegral membrane protein that binds cholesterol at the 3β

hydroxyl position (Kwon et al., 2009). NPC2, on the otherhand, is a soluble protein located within the lysosomal lumenthat binds to cholesterol’s aliphatic side chain (Wang et al.,2010). Brown and Goldstein’s group recently demonstratedthat lysosomal cholesterol export requires the concertedaction of both NPC proteins, possibly involving the transferof cholesterol from NPC2 to NPC1 and the subsequentinsertion of its aliphatic side chain into the lysosomalmembrane. Endolysosomal cholesterol is then exported toother intracellular sites and incorporated in the metabolicallyactive intracellular pool, by mechanisms that have yet tobe elucidated. In fact, currently there is no evidence ofdirect cholesterol transfer from the endolysosome to theER membrane, the primary site of intracellular cholesterolsensing and regulation (Section II.5).

As opposed to LDL cholesterol uptake, high-densitylipoprotein (HDL) cholesterol is incorporated into cells bynon-endocytic mechanisms (Fig. 3). The scavenger receptorclass B type I (SR-BI), an integral membrane protein foundmostly in the liver and steroidogenic tissues, directly bindsHDL particles and facilitates the selective transfer of HDLcholesterol into the plasma membrane with no internaliza-tion of the lipoprotein holoparticle (Rigotti, Miettinen &Krieger, 2003). Cholesterol-depleted HDL particles are thenreleased back into the extracellular space whereas transferredcholesteryl esters are hydrolyzed by extralysosomal neutralcholesteryl ester hydrolases and trafficked towards the ERby unknown mechanisms (Leiva et al., 2011).

(4) Cellular cholesterol efflux

Several ABC transporter superfamily members can mediatethe efflux of cholesterol towards extracellular acceptors(Fig. 3). ABC subfamily A, member 1 (ABCA1) translocatesplasma membrane cholesterol to lipid-poor discoidal HDL



Fig. 3. Lipoprotein cholesterol uptake and cholesterol efflux are mediated by specific cell surface proteins. Low-density lipoprotein(LDL) binds to LDL receptor (LDLR) on the surface of numerous cell types, including hepatocytes, corticoadrenal epithelium,macrophages and skin fibroblasts. After binding, the LDL-LDLR complexes are internalized into endosomes, where, afteracidification of the organelle lumen, they dissociate into free LDL and LDLR. The LDLR is recycled back to the surface, for newbinding/internalization rounds, whereas the LDL particles are trafficked towards lysosomal compartments for their full degredation.The released free cholesterol is transported to other membranous organelles such as plasma membrane and the endoplasmicreticulum for structural or metabolic roles. Alternatively, this cholesterol can be esterified by acyl-coA:cholesterol acyltransferase(ACAT, not shown) for storage in lipid droplets. In contrast to LDL, high-density lipoprotein (HDL) binds to the scavengerreceptor class B member I (SR-BI) and is not internalized to the cells. In fact, HDL cholesterol is selectively transferred fromHDLs particles to intracellular regulatory pools, with no endocytosis or degradation of the lipoprotein particles. As a result, matureHDLs are converted into smaller, cholesterol-depleted lipoproteins, which are released back to the circulation for new rounds ofcholesterol transportation. The cellular cholesterol efflux is mediated by ATP binding cassette (ABC) family members. ABCA1,a multiple membrane-spanning protein with two nucleotide-binding domains, interacts with discoid lipid-poor apolipoprotein (apo)A-I-containing HDL precursors, transferring phospholipids and cholesterol to it. These lipidated apo A-I-containing precursors arethen transformed into HDL in the blood by the action of lecithin:cholesterol acyl transferase (LCAT, not shown). ABCG1 transfercholesterol to more mature HDL particles, contributing to their full lipid loading.

precursors. Tangier’s disease, a condition caused by ABCA1deficiency, is characterized by very low plasma HDL levelsand cholesterol accumulation in macrophages, possiblyas a result of defective cholesterol exportation (Francis,Knopp & Oram, 1995). ABCG1, which is also expressedin macrophages, mediates cholesterol efflux to lipid-loadedspherical HDL particles (Wang et al., 2004), perhapscontributing to the full maturation of this lipoproteinclass in vivo. SR-BI has also been reported to play

a role in cholesterol efflux into mature HDL particles(Rigotti et al., 2003).

ABCG5 and ABCG8 transporters are expressed on thecanalicular (apical) surface of hepatocytes as well as on theluminal membrane of enterocytes. In these locations, bothtransporters actively pump cholesterol and phytosterols outof cells, contributing to the net elimination of body sterols (Yuet al., 2002). Concordantly, sitosterolemia, a genetic diseasecaused by mutations in ABCG5 and ABCG8, is characterized



by phytosterol and cholesterol accumulation in tissues, highplasma sterol concentrations, and accelerated atherosclerosis,indicating that the main function of these transporters is theelimination of phytosterols and excess cholesterol from theorganism (Berge et al., 2000).

(5) Intracellular cholesterol regulation

As exemplified by the lysosomal storage disorders describedabove, cholesterol accumulation can be deleterious or evenlethal for cells. Consequently, many protective mechanismshave evolved to prevent cholesterol’s toxic effects. Theunderlying causes of this cytotoxicity are not completelyclear, but may include: (i) abnormalities in membraneprotein conformational plasticity; (ii) increased productionof oxysterols by oxidative stress; (iii) intracellular cholesterolcrystallization leading to mechanical membrane disruptionand (iv) increased apoptosis (Tabas, 2002).

Cellular cholesterol is kept within homeostatic levelsby the concerted action of both transcriptional and post-transcriptional mechanisms (Brown & Goldstein, 1997;Horton, Goldstein & Brown, 2002). At the transcriptionallevel, sterol regulatory element binding protein (SREBP), amembrane-bound member of the basic helix-loop-helix fam-ily of transcription factors, controls the expression of LDLR,HMG-CoA reductase and other genes encoding enzymes ofthe mevalonate pathway (Fig. 4). SREBP-cleavage activatingprotein (SCAP), a multi-spanning ER membrane protein,directly binds cholesterol and interacts with SREBP andother ER proteins to regulate SREBP activation. Specifically,when ER cholesterol levels are high (Fig. 4A), SCAP phys-ically interacts with insulin-induced genes 1 and 2 (INSIG1and INSIG2), two ER membrane-resident proteins (Engelk-ing et al., 2004, 2005) that sequester the SCAP/SREBPcomplex in the ER preventing its translocation to theGolgi apparatus. By contrast, when cholesterol levels arelow (Fig. 4B), the SCAP-INSIGs interaction is abolished,and SCAP/SREBP complexes are transported to the Golgi,where the active form of SREBP is released from the mem-brane by the sequential action of site-1 and site-2 proteases(S1P and S2P, respectively). This mature membrane-freeSREBP form translocates into the nucleus where it binds tosterol responsive element (SRE) sequences within the regu-latory region of target genes, activating their transcription.

HMG-CoA reductase (HMG-CoA-R) is additionallyregulated at the post-translational level. When ER cholesterolconcentration rises, HMG-CoA-R – a multi-spanning ERmembrane protein itself –binds directly to cholesterol and toa membrane-bound ubiquitin ligase, gp78. This interactionresults in HMG-CoA-R polyubiquitination and subsequent26S proteasome-mediated degradation (Goldstein, DeBose-Boyd & Brown, 2006; DeBose-Boyd, 2008). Conversely,when ER cholesterol is low, the protein turnover rateof the reductase is slowed down as a result of decreasedpolyubiquitination and proteosomal degradation.

Statins, currently the most important family ofhypocholesterolemic drugs, are competitive inhibitors ofHMG-CoA-R and thus work through an indirect activation

of the SREBP system. By lowering cholesterol synthesis andconcentration in the ER, statins lead to increased SCAP-modulated SREBP activation and, subsequently, to elevatedLDLR levels on the cell surface, accelerated LDL clearancerates, and lower plasma LDL cholesterol levels.

The regulatory systems described above are based on thedetection of small fluctuations in ER cholesterol concentra-tions. Indeed, it has been determined that changes as smallas 5% in the content of this sterol in the ER are sufficientto trigger functional activation/inactivation of the SREBPsystem (Radhakrishnan et al., 2008). Mechanistically, itseems logical to sense cholesterol in a compartment where itsconcentrations are low; however, it has to be emphasized thatcholesterol is actively kept low in the ER by mechanisms thatcontinuously vectorize it to the plasma membrane and othercellular organelles. This is achieved by cholesterol bindingto proteins that are sorted through the secretory pathway, aswell as cholesterol transport proteins present in the cytosoland the preferential interaction of cholesterol with othermembrane lipids destined to plasma membrane biogenesis(e.g. sphingolipids) (van Meer et al., 2008). Even though theentire process is only partially understood, it is clear thatexquisitely fine-tuned mechanisms are required to maintaincholesterol balance among different subcellular structuresand fulfill the varying sterol needs of different organelles(Ikonen, 2008).

III. CHOLESTEROL PHYSIOLOGICALREGULATION AND PATHOLOGICALIMPLICATIONS

(1) Whole-body cholesterol homeostasis

Cholesterol balance is a major challenge for entire organisms,where the presence of diverse specialized cell types andtissue compartments adds additional levels of complexity toits regulation. Even though the discovery of the SREBPsystem provided a solid and elegant explanation for cellularcholesterol homeostasis, the question of how different tissues,organs and systems coordinate and balance their cholesterolcontent to ensure adequate supply and avoid noxiousaccumulation remained an enigma until very recently.

In mammals, the liver is at the centre of cholesterolmetabolism for it is, quantitatively, the main site of cholesterolsynthesis, lipoprotein production, and cholesterol excretionfrom the body into the bile (Dietschy, Turley & Spady, 1993;Turley, Spady & Dietschy, 1995). It is therefore not surprisingthat the master molecular regulators of body cholesterolbalance were originally cloned from liver cDNA libraries(Apfel et al., 1994; Forman et al., 1995). In particular, twomembers of the heterodimeric nuclear receptors superfamilyof transcription factors, liver X receptor (LXR, isoformsα and β) and farnesoid X receptor (FXR), are largelyresponsible for the activation of transcriptional programsacross different cell types that maintain body cholesterolhomeostasis.



(A)

(B)

Fig. 4. Cell cholesterol homeostasis is regulated at transcriptional level by the sterol regulatory element binding protein (SREBP)system. SREBP-cleavage activating protein (SCAP), a multi-spanning endoplasmic reticulum (ER) membrane protein, bindscholesterol through its luminal loop 1. This stabilizes SCAP interaction with an ER resident protein, insulin-induced genes (INSIG)(A), anchoring the SCAP-SREBP complex to the ER membrane and preventing SREB activation. When ER cholesterol is low (B),cholesterol-free SCAP detaches from INSIG, possibly as a consequence of conformational changes, and exposes COPII bindingsites in its cytosolic loop 6. This enables SCAP-SREBP complex migration to the Golgi. In this organelle, precursor (p)SREBPis sequentially cleaved by site 1 protease (S1P), and site 2 protease (S2P), releasing a mature membrane-free SREBP form. Thisbasic helix-loop-helix (bHLH)-containing fragment translocates to the nucleus (nSREBP) and binds to the sterol responsive elements(SREs) in the regulatory region of SREBP target genes, such as low-density lipoprotein receptor (LDLR), 3-hydroxy-3-methyl-glutarylCoA reductase (HMG-CoA-R) and others, elevating their transcriptional rates.

(a) Nuclear receptors LXR and FXR coordinate body cholesterol andbile acids homeostasis

Like other nuclear receptors, LXRs and FXR are activatedby specific ligands that induce their heterodimerization withthe retinoid X receptor (RXR). In their active form, theybind to specific regulatory motifs in the promoter regionof target genes and recruit complex sets of transcriptionalco-activators and chromatin remodelling enzymes toincrease transcription rates.

Historically, a major clue to ascribe to LXR and FXR afunction in cholesterol homeostasis was the identification oftheir physiological ligands: LXRs bind products of cholesteroloxidation, namely oxysterols (Janowski et al., 1996; Lehmannet al., 1997), whereas FXR binds the final products of

cholesterol catabolism in the liver, the bile acids (BAs)(Makishima et al., 1999; Parks et al., 1999).

In fact, it seems that hepatic LXRα and LXRβ senseoxysterols as an early sign of excessive cholesterol build-up in the body. In rodents, LXRs accelerate cholesterolcatabolism and elimination by transcriptionally activatingthe rate-limiting enzyme of BA synthesis, cholesterol-7-α-hydroxylase (Cyp7a) (Lehmann et al., 1997; Peet et al., 1998).Simultaneously, LXRs activation results in elevated levels ofcanalicular cholesterol transporters, Abcg5 and Abcg8 (Repaet al., 2002), that actively move cholesterol to the biliary treeand then to the intestinal lumen.

LXRs also induce Abcg5/Abcg8 transcription in enterocytes(Repa et al., 2002), resulting in elevated levels of Abcg5/g8 on



the luminal surface of the intestinal epithelium. As mentionedabove, the lack of these transporters results in cholesterol andphytosterol accumulation in the blood and tissues of bothsitosterolemia patients and Abcg5/g8-deficient mice (Yuet al., 2002).

In contrast to LXR, FXR primary function seemsto be protecting hepatocytes from excessive exposure toBAs, which are cytotoxic molecules in themselves, ratherthan preventing body cholesterol accumulation. In theliver, BA-activated FXR increases the expression levels ofsmall heterodimeric partner-1 (SHP-1), an atypical nuclearreceptor that lacks a DNA-binding domain but insteadinteracts with liver receptor homolog 1 (LRH-1) and thusprevents its action as a constitutive activator of Cyp7atranscription (Goodwin et al., 2000). Consequently, Cyp7aprotein levels are lowered, which reduces BA synthesis andconcentration in hepatocytes.

FXR also induces the transcription of the bile salt exportpump (BSEP/ABCB11), an ABC transporter located on thecanalicular membrane of hepatocytes that extrudes BAstowards the biliary tree (Plass et al., 2002).

Finally, FXR inhibits the expression of proteins thatenhance the uptake and transport of BAs back to the liver forreutilization (Grober et al., 1999; Chen et al., 2003), furtherfavouring the removal of these cholesterol metabolites fromthe body.

(b) LXR transcriptionally regulates cholesterol transport fromextrahepatic tissues to the liver for its catabolism and body elimination

The enterohepatic circuit is not only important to handlecholesterol and BAs derived from the liver or food sourcesbut is also key for disposing of cholesterol exported from‘peripheral’ tissues. Macrophages participate in the so-called ‘reverse-cholesterol transport’ pathway that essentiallyconsists in the net transfer of excess cholesterol from extra-hepatic tissues to the liver for its catabolic processing. Inmacrophages, LXRs transcriptionally activate the cholesteroltransporters ABCA1 and ABCG1, as well as apolipoprotein E(APOE) (Venkateswaran et al., 2000a, b; Laffitte et al., 2001),leading to an increased efflux of cellular cholesterol towardscirculating HDL, which can then be cleared by hepaticSR-BI-mediated selective uptake, secreted into the bile byABCG5/G8, and ultimately eliminated in the faeces for itsfinal deposition (Naik et al., 2006).

The ability of LXRs to promote cholesterol removal fromcells makes this nuclear receptor a prime therapeutic targetfor atherosclerotic artery diseases (Im & Osborne, 2011). Inthese coronary and cerebrovascular conditions, lipoprotein-derived cholesterol accumulates inside subendothelialmacrophages, disrupting their function and structure, andtriggering local inflammation. This leads to atheroscleroticplaque formation, artery occlusion and ischemic cell death ofdownstream tissues. Therefore, LXR-dependent promotionof reverse-cholesterol transport might, theoretically, decreasecholesterol loading of macrophages and prevent furthervascular complications. Additionally, it has been shownthat LXRs also possess potent anti-inflamatory effects, in

both cultured macrophages and living mice (Joseph et al.,2003), that should further boost LXR anti-atherogenicpotential. Different lines of evidence strongly suggest thatLXR activation might indeed be an excellent therapy foratherosclerosis: first, LXR activation with the nonsteroidalLXR agonist GW3965 significantly decreases plaque lesionsurface in atherosclerosis-prone mouse models (Josephet al., 2002); second, macrophage-specific deficiency of bothLXR isoforms in mice (LXRαβ−/−) significantly aggravatesatherosclerotic aortic lesions in hypercholesterolemic mice(Tangirala et al., 2002); and third, LXR activation promotesreverse-cholesterol transport and faecal net elimination ofcholesterol in dyslipidemic hamsters (Briand et al., 2010) andrabbits (Honzumi et al., 2011), two models of lipid metabolismand atherosclerosis more similar to human physiology thanmice.

(c) Hepatic de novo lipogenesis is regulated by LXR and ismetabolically coupled to ACAT-dependent cholesterol esterification

It has to be noted that LXRs potently induce fattyacid and triglyceride synthesis in hepatocytes, elevatingboth liver and plasma triglyceride levels in murinemodels. This undesirable effect has largely prevented theimplementation of clinical trials of non-selective LXRactivators as antiatherogenic agents. Mechanistically, LXR-induced hyperlipidemia depends on its ability to directlyactivate the transcription of the SREBP-1c variant, which, inturn, regulates the expression of lipogenic enzymes suchas hepatic fatty acid synthase, acetyl-CoA carboxylase-1, stearoyl-CoA desaturase, as well as enzymes involvedin the generation of NADPH, an essential cofactor inde novo lipogenesis (Horton et al., 2002). In fact, it hasbeen speculated that LXR-dependent activation of lipogenicpathways in the liver evolved to provide extra fatty acidsubstrates for ACAT-mediated cholesterol esterification andthus prevent the hepatotoxic effects of cellular cholesteroloverload (Horton et al., 2002). Consequently, before LXRactivators can be introduced to clinical practice as partof a viable antiatherogenic therapy, new strategies thatpreserve their effects over reverse cholesterol transport andanti-inflamatory responses, while avoiding the activation ofhepatic lipogenesis, must be developed (Miao et al., 2004;van der Hoorn et al., 2011).

Recent studies have unveiled unexpected metabolic rolesof both LXRs and FXR that go beyond their directinfluence on body cholesterol or BA regulation. Primeexamples are the insulin-sensitizing action of LXR inanimal models of severe insulin resistance, possibly bydirect transcriptional inhibition of the gluconeogenic enzymephosphoenolpyruvate carboxykinase (PEPCK) (Baranowski,2008), and FXR effects on BA conjugation and mucosalimmunity in the intestine (Song et al., 2001; Inagaki et al.,2006). FXR has also been linked to carbohydrate metabolismin hepatocytes (Ory, 2004; Duran-Sandoval et al., 2005).Collectively, these observations extend the therapeuticimplications of LXRs and FXR to other prevalent conditions,such as type 2 diabetes mellitus and inflammatory intestinal



diseases (Cortes et al., 2005; Schulman, 2010; Jakobssonet al., 2012).

(2) Cholesterol in the central nervous system

(a) The central nervous system (CNS) is segregated from bodycholesterol metabolism

The most distinctive feature of CNS cholesterol metabolismstems from the blood–brain barrier (BBB) blockage ofplasma lipoprotein transit to the cerebrospinal fluid (CSF),resulting in negligible access to cholesterol from dietaryor hepatic origin and leaving neural tissue completelydependent on its own cholesterol synthesis and trafficking(Turley et al., 1996; Quan et al., 2003; Dietschy & Turley,2004). Somewhat paradoxically, the CNS has the highestcontent of cholesterol per gram of tissue, mainly as aconsequence of the large surface area of neurons andastrocytes, combined with the presence of cholesterol-richSchwann cells on myelinated axons (Dietschy & Turley,2004; Simons & Trotter, 2007; Pfenninger, 2009). In fact,pioneering tracer studies by H. Waelsch, later extended andrefined by John Dietschy and colleagues, demonstrated thatthe CNS basically relies on local cholesterol synthesis as itsonly sterol source and that the rate of sterol synthesis peaksin periods of increased cholesterol demand, especially duringpostnatal axon myelination (Waelsch, Sperry & Stoyanoff,1940; Dietschy & Turley, 2004).

This virtual isolation of the CNS from exogenouscholesterol sources required the evolution of similarly self-sufficient intra-CNS cholesterol trafficking systems, in whichglial cells, namely astrocytes, act as cholesterol suppliersto meet the relatively high neural sterol demand (Pfrieger,2003; Pfrieger & Ungerer, 2011). Supporting this claim,current estimations derived from primary cultured CNS cellshave shown that astrocytes synthesize cholesterol significantlymore efficiently than neurons, which seem to be particularlymeagre in the processing of lanosterol (Nieweg, Schaller &Pfrieger, 2009). Unlike neurons, astrocytes express APOE,the major lipid carrier protein in the CNS (Amaratungaet al., 1996; DeMattos et al., 2001; Vance & Hayashi,2010), and secrete lipoprotein-like particles towards the CSF(Roheim et al., 1979; Pitas et al., 1987; LaDu et al., 1998).Other apolipoproteins found in the CNS are apolipoproteinA-I (APOAI), produced by endothelial and choroid plexusepithelial cells, apolipoprotein J/clusterin (APOJ/CLU), andapolipoprotein D (APOD), expressed by astrocytes (Haddadet al., 1986; Mockel et al., 1994; Rassart et al., 2000). Eventhough normal neurons do not express significant amounts ofapolipoproteins, some studies have shown that, upon variousinsults, neurons may induce APOE and APOD expression(Ong et al., 1997; Franz et al., 1999; Aoki et al., 2003; Xu et al.,2006), probably as a component of a more complex cellularstress response evolved to enhance lipid dynamics in timesof acute need. Concordantly, glia-derived APOE-cholesterolpromotes synaptogenesis (Mauch et al., 2001), suggestingthat astrocytes provide neurons with substrates for synapticmembrane remodeling.

In contrast to apolipoprotein expression patterns, everycell type in the CNS harbours lipoprotein receptors ontheir plasma membrane. These include: LDLR, very lowdensity lipoprotein (VLDL) receptor, APOE receptor 2and the LDLR-related proteins 1 and 4 (LRP1 and LRP4,respectively), all members of the LDLR family. Importantly,besides lipoprotein binding and uptake, these receptorsare implicated in various signalling pathways that cruciallydetermine cell survival, migration and morphogenesis in theCNS (reviewed in Herz & Bock, 2002; Herz, 2009; Herzet al., 2009).

Neurons and glial cells also express all main ABCtransporters involved in cholesterol efflux, namely, ABCA1,ABCG1 and ABCG4. It is thought that these transportersare implicated in offloading excessive cholesterol intoAPOE-containing acceptors along with contributing toCSF lipoprotein maturation (Koldamova et al., 2003; Kim,Weickert & Garner, 2008; Tarr & Edwards, 2008).

(b) CNS cholesterol efflux requires its conversion to24(S)-hydroxycholesterol and inhibition of this pathway results inlearning disability

The impermeability of BBB endothelia to cholesterol andlipoproteins not only impedes inward transport of thismolecule to the CSF but also determines that, in order to beremoved from the CNS, cholesterol must first be convertedinto 24(S)-hydroxycholesterol (24-HC). This oxysterol candiffuse freely through the BBB and gain access to the generalcirculation (Bjorkhem et al., 2001). Soon after its departurefrom the CNS, 24-HC reaches the liver, where it is terminallyconverted into BAs and secreted into the bile. Importantly,the formation of 24-HC is catalyzed by cholesterol-24(S)-hydroxylase (CYP46A1), a highly conserved cytochromeP450 that is almost exclusively expressed by neurons (Lund,Guileyardo & Russell, 1999; Russell et al., 2009).

Mice lacking Cyp46a1 (Cyp46a1−/−) have decreasedCNS cholesterol turnover rates and proportionally lowerCNS cholesterol synthesis rates that closely match theamount of 24-HC eliminated per day (Lund et al., 2003).This compensatory effect on cholesterol synthesis results innormal CNS cholesterol content (Dietschy & Turley, 2004).Interestingly, although Cyp46a1−/− mice have no grossmetabolic, morphological or reproductive abnormalities,different functional tests demonstrated that they suffer fromsevere learning disabilities, most likely due to diminishedmemory function. These memory abnormalities closelycorrelate with reduced long-term potentiation (LTP) in invitro stimulated Cyp46a1−/− hippocampal slices, indicatingdecreased synaptic strengthening (Kotti et al., 2006; Russellet al., 2009). Since these mice display no anatomical ordevelopmental defects in their CNS, it is probable that subtleneuron-specific metabolic deficiencies are the primary causeof impaired synaptic function.

Given that mevalonate can rescue statin-induced loss ofLTP in wild-type hippocampal slices (Russell et al., 2009)and that Cyp46a1−/− mice have reduced brain cholesterolbiosynthesis, that is, a slower substrate flow through the



mevalonate pathway, David Russell and colleagues aimedto rescue LTP abnormalities by systematically addingback mevalonate pathway intermediates into electricallystimulated Cyp46a1−/− hippocampal slices. Surprisingly,they found that an isoprenoid related to geranylgeranyldiphosphate was sufficient to recover normal LTP (see Fig. 2)(Kotti et al., 2008).

Taken together, these results demonstrate, for thefirst time, that normal cholesterol homeostasis in theCNS is required for proper synaptic plasticity andmemory. Additionally, they raise the question of whethersubtle defects in CNS sterol metabolism, caused byunidentified genetic variations in enzymes of the mevalonate-pathway, could precipitate memory, learning, or behaviouraldisorders in predisposed individuals subjected to differentstressors. This hypothesis might explain, for instance, thecognitive impairment reported in some statin-treated patients(Muldoon et al., 2000, 2004), within whom HMG-CoAreductase inhibition could unbalance mevalonate-derivedmetabolites for proper synapse formation and/or function.In fact, when cellular mevalonate concentrations are limiting,for example, during statin treatment, this metabolite ispreferentially shunted towards the synthesis of non-sterolproducts due to the higher substrate affinity of the enzymesin the non-sterol branches of the mevalonate pathway (Brown& Goldstein, 1980; Goldstein & Brown, 1990).

(c) Defects in cholesterol synthesis or intracellular trafficking determineneurodegeneration

As discussed above, cholesterol directly influences thebiophysical properties of cellular membranes, somethingof paramount importance for neural function. Actually,cholesterol content determines the electrical properties of theplasma membrane as well as neurotransmitter receptor andion channel activities, axon myelination, synapse formationand pruning, and synaptic vesicle trafficking. Consequently,in the few instances where defects of cholesterol metabolismdo not result in embryonic lethality (see Section III.3),both intracellular cholesterol accumulation or deficiency areinvariably associated with severe CNS pathology (Carsteaet al., 1997; Nwokoro, Wassif & Porter, 2001; Waterham,2006). This is the case for NPC disease (Section II.3and below), Smith-Lemli-Opitz syndrome (Section III.3),desmosterolosis (Section IV), and Pelizaeus-Merzbacherdisease (Saher et al., 2012).

NPC disease evolves with intralysosomal accumulation offree cholesterol in neurons, progressive neurodegeneration,and premature death (see Section II.3). Recently, it wasshown that the CNS accumulation of cholesterol in Npc1-deficient (Npc1−/−) mice can be partially reversed byLXR activation (Repa et al., 2007). Oral administrationof T0901317, a potent non-selective LXR agonist, inducedthe expression of APOE, ABCA1, and ABCG1 in the brain ofNpc1−/− mice, and significantly increased the exportation ofcholesterol, but not 24-(S)-hydroxycholesterol, towards thecirculation by undefined mechanisms (Repa et al., 2007).Importantly, although LXR activation was ineffective to

reduce hepatic cholesterol accumulation, it did extend theaverage lifespan of Npc1−/− mice significantly, suggestingthat LXR agonists could become a viable neuroprotectivealternative for NPC patients.

(d ) Relationship between CNS apolipoprotein E4, cholesteroltrafficking and Alzheimer’s disease

The pathogenesis of Alzheimer’s disease (AD) is char-acterized by the excessive production, deposition, andaggregation of extracellular amyloid (Aβ) fibrils, alongwith hyperphosphorylation and intracellular aggregation ofmicrotubule-associated protein tau in neurofibrillar tangles.It is now accepted that these abnormalities somehow leadto irreversible loss of synapses, neurodegeneration and cog-nitive deterioration characteristic of AD patients (Huang &Mucke, 2012).

Aβ is produced by proteolytic processing of the amyloidprecursor protein (APP), a single transmembrane polypeptidethat is cleaved by β− and γ−secretases (Zhang et al., 2012).Importantly, whereas just a few cases of AD are caused bygenetic defects of the APP processing machinery, the mostprevalent sporadic forms have a complex and multifactorialetiology.

Recent studies have suggested that neuron cholesterolcould be directly implicated in the pathogenesis and/orprogression of AD. It has been shown, for instance,that intramembrane γ−secretase-mediated proteolyticprocessing of APP preferentially occurs in cholesterol-richlipid rafts. Also, Aβ fibrillogenesis is significantly acceleratedby the enrichment of those membrane microdomainswith ganglioside GM1 (Yanagisawa & Matsuzaki, 2002;Vetrivel & Thinakaran, 2010; Di Paolo & Kim, 2011).Furthermore, the transmembrane domain of APP directlybinds cholesterol in a specific and dose-dependent manner(Barrett et al., 2012), suggesting that a pharmacologicalblockade of the APP-cholesterol interaction could preventits direction to lipid rafts and hinder its processing into Aβ.

So far, the only consistent risk factor for sporadic AD is theexpression of APOE ε4 isoform (APOE4) (Corder et al., 1993;Holtzman, Herz & Bu, 2012). However, given the multiplicityof functions of APOE and its receptors in the CNS, it hasbeen extremely difficult to pinpoint the exact mechanism(s)underlying this association (Hauser, Narayanaswami &Ryan, 2011; Schipper, 2011; Holtzman et al., 2012).

A number of potential mechanisms that could account forthe role of APOE4 in AD pathogenesis have been proposed.For example, it has been shown that all APOE variants canform protein-protein complexes with Aβ peptides during invitro fibrillogenesis assays, but APOE4 does so with the fastestkinetics, perhaps accelerating Aβ fibril assembly (Strittmatteret al., 1993; Sanan et al., 1994). This seems to be substantiatedby in vivo studies, in which apoE ablation (Bales et al., 1997) ora synthetic peptide that blocks apoE/Aβ binding (Sadowskiet al., 2006) significantly reduces the formation of amyloidplaques in the brain of transgenic mouse models of AD.

APOE also regulates soluble Aβ peptide clearance fromthe CNS through both proteolytic processing in microglia



(Jiang et al., 2008) and transcytotic traffic across the BBB(Deane et al., 2008). Both processes depend on the degree ofapoE lipidation and its specific binding capacity to membersof the LDLR family. In particular, apoE4/Aβ complexesbind to the LDL receptor-related protein 1 (LRP1) withmuch lower affinity than its apoE2 and apoE3 counterpartson the abluminal surface of mouse brain capillary cells.Since endocytosis through LRP1 is several times faster thanthrough other LDLR family members, apoE4 significantlydelays Aβ clearance from the CNS in vivo (Deane et al., 2008).

Collectively, these studies suggest that APOE4 maypromote the formation of extracellular amyloid fibrils inthe brain of carrier subjects by both binding to Aβ peptideand inhibiting its rapid export to the systemic circulation.

Importantly, APOE receptors are crucial mediatorsof cholesterol-independent signalling events that regulatemultiple cellular processes in the CNS, ranging from celldifferentiation and survival to synaptogenesis and synapticplasticity (for a recent review see Holtzman et al., 2012).Many of these signalling events can have a major impacton the neurodegeneration and cell loss characteristic ofAD patients. In fact, the Reelin/Disabled-1 pathwaythat works through APOE receptor 2 (APOER2) andthe very low-density lipoprotein receptor (VLDLR) hasbeen implicated in a phosphorylation cascade that inhibitsglycogen synthase kinase 3β (GSK3β), which, in its mostactive form, can mediate tau hyperphosphorylation, theother major hallmark of AD (Beffert et al., 2002).

A more recent hypothesis posits that APOE4 might alsobe deleterious for the structural integrity of the BBB, makingit more permeable to neurotoxic and pro-inflammatorycomponents of the plasma. In support of this idea, arecent study showed that APOE-deficient (Apoe−/−) micewith transgenic expression of human APOE4 show increasedBBB permeability relative to Apoe−/− mice expressing humanAPOE2 or APOE3, and that this correlates with acceleratedneurodegeneration (Bell et al., 2012). Again, it was suggestedthat, relative to APOE2 and APOE3, APOE4 binds poorlyto LRP1 on the cell surface of pericytes. These cellsare associated to the capillary endothelium and seal theBBB through close intercellular interactions. By unknownsignalling mechanisms, the lack of APOE-mediated LRP1activation triggers local inflammatory events that concludein the matrix metalloproteinase nine-dependent degradationof the pericytes’ tight-junctions, causing increased BBBpermeability (Bell et al., 2012).

Complementing these observations, a recent in vitro studyfound that excessive neuronal uptake of APOB-associatedlipoprotein cholesterol results in endolysosomal disruptionand increased Aβ production and aggregation (Hui, Chen& Geiger, 2012). Therefore, it is possible that a leakyBBB may allow APOB-lipoprotein access to the CNS andfavour amyloidogenic neuronal cell death by endolysosomal-dependent mechanisms. Notably, dyslipidemic states areassociated with increased BBB permeability in AD patients(Bowman, Kaye & Quinn, 2012), suggesting that thesephenomena could be relevant in humans.

Taken together, these findings suggest that, along withinterfering with the physical interaction between APOE andAβ, ameliorating brain local inflammation and loweringplasma LDL-cholesterol levels might be beneficial to delaythe onset and progression of AD in genetically susceptibleindividuals.

(3) Cholesterol in embryonic and fetal development

Several human malformation syndromes as well as animalmodels of pharmacological or genetic blockade of themevalonate pathway show that cholesterol is absolutelyrequired for normal prenatal development (Porter, 2003).These syndromes most likely result from structural, endocrineand/or signalling abnormalities derived from cholesteroldeficiency (Willnow, Hammes & Eaton, 2007; Steinhauer& Treisman, 2009; Santander et al., 2012). However, itis plausible that reduction and/or accumulation of othermevalonate pathway metabolites also play a role.

Because of their high proliferation and membraneformation rates, embryonic and fetal cells have elevatedcholesterol requirements. Interestingly, these cells do notsuppress cholesterol synthesis in response to rises in ER sterolconcentration (Schmid & Woollett, 2003; Yao et al., 2007),possibly as an adaptation to secure sufficient cholesterolsupply. Mechanistically, this lack of feedback regulationseems to result from SREBP constitutive processing andactivation, as a consequence of higher SCAP/INSIG ratiosin the ER membrane (Yao et al., 2007).

The most prevalent genetic disease involving reducedembryonic cholesterol levels is the Smith-Lemli-Opitz (SLO)syndrome, which is caused by a defective conversion of7-dehydrocholesterol (7-DHC) into cholesterol. This syn-drome is characterized by facial dysmorphia, micro-cephaly, syndactyly and variable degrees of mental retar-dation. Importantly, both mice lacking 3β-hydroxysteroid�

7-reductase (Dhcr7−/− mice) and rat embryos treated with theDHCR7 inhibitor AY9944, recapitulate human SLO (Roux& Aubry, 1966), establishing a causal link with cholesterolrestriction and/or 7-DHC accumulation.

In Drosophila, a genus that is incapable of cholesterolsynthesis (see Section II.1), the activities of HMG-CoAreductase, geranylgeranyl pyrophosphate synthase andgeranylgeranyl transferase are required for normal heartdevelopment (Yi et al., 2006). This suggests that metabolitesof the mevalonate pathway other than cholesterol are neededduring development. In support of this hypothesis, it has beenshown that statins induce an abnormal cardiac phenotypein flies, possibly due to the mislocalization of unprenylatedG proteins and a disruption of normal morphogenic cellsignalling (Yi et al., 2006). Remarkably, heart malformationshave also been reported after gestational exposure to statinsin humans (Edison & Muenke, 2005), suggesting that anormal metabolic flux through the mevalonate pathway isalso necessary for human embryonic development.

Nonetheless, cellular cholesterol build-up seems to be asembryotoxic as its deficiency. In fact, Insig1/Insig2 double-knockout mice, which have elevated SREBP levels and



therefore increased mevalonate pathway activity, developpalate or facial clefting because of defective midline fusion(Engelking et al., 2006). In these animals, statin administrationto pregnant females largely prevents the malformationphenotype, suggesting that an overflow of sterols or othermetabolites derived from the mevalonate pathway somehowpreclude the normal fusion of midline embryonic structures(Engelking et al., 2006).

(4) Origins of embryonic/fetal cholesterol

Experimental evidence indicates that cholesterol is activelytransferred from mother to embryo/fetus during pregnancy.First, fetuses with defective endogenous cholesterol synthesisstill have considerable amounts of this sterol in their tissues(Linck et al., 2000; Tint et al., 2006). Second, labeled LDLand/or HDL cholesteryl esters injected into the maternalcirculation are quickly found in fetal blood and tissues(McConihay et al., 2000; Yoshida & Wada, 2005; Burke et al.,2009). Finally, maternal lipoprotein metabolic abnormalitiesnegatively impact embryonic development. Indeed, lowmaternal plasma cholesterol concentration correlates withlow mass at birth and microcephaly in humans (Edisonet al., 2007), whereas hypercholesterolemia (beyond itsphysiological gestational increase) associates with fatty streaksin the fetal aorta (Palinski et al., 2007). Moreover, offspringof female mice lacking the endocytic HDL receptor megalin(Willnow et al., 1996) or apolipoprotein B, a key mediator ofLDL-LDLR interaction (Farese et al., 1995) develop cephalicand CNS abnormalities (Willnow et al., 2007).

From an anatomical point of view, maternal cholesterolmust cross two different cell layers before reaching thefetal circulation: the visceral endoderm in the yolk sacor trophoblast in the placenta, and fetal endothelial cells(Rossant & Cross, 2001). Although the precise molecularmechanisms underlying this phenomenon remain unknown,both trophoblast and placental endothelial cells expresslipoprotein receptors and sterol transporters that likelymediate cholesterol trafficking to the fetus in vivo (Stefuljet al., 2009; Woollett, 2011).

Since cholesterol is critical for normal embryonicdevelopment, improving mother-to-fetus cholesterol transfercould be envisioned as a promising in utero therapy forconditions in which embryonic cholesterol synthesis isrestricted, such as SLO syndrome. Indeed, pregnant micetreated with an LXR agonist showed increased placentalABCA1 expression with a concomitant acceleration ofcholesterol transport to the fetus. The same treatmentsignificantly increased tissue cholesterol content in murinefetuses that lack cholesterol synthesis (Lindegaard et al., 2008).

(5) Cholesterol cytotoxicity: implications foratherosclerosis

Atherosclerotic disease is the main cause of cardiovasculardeath worldwide and, although it is a complex process thatinvolves multifarious environmental and genetic factors, thedeposition of lipoprotein-derived cholesterol in the arterial

wall is the priming condition necessary for the progressionof more advanced lesions. Ironically, Anitschkow recognizedthe role of cholesterol in atherogenesis in 1913; unfortunatelyhis observations were dismissed by contemporary scientificauthorities, substantially delaying progress in the field bymany decades (Steinberg, 2004).

After the initial cholesterol accumulation in the arterialwall, local inflammation and endothelial dysfunction set off avicious circle leading to additional cholesterol deposition and,in some cases, atherosclerotic plaque rupture and thrombosis(Ross, 1999; Lusis, 2000). Importantly, acute ischemicatherothrombotic events invariably occur in cholesterol-richlesions; however, the mechanisms precipitating these plaquecomplications and its links to cholesterol remain unknown.Cell death of cholesterol-laden macrophages, mainly byapoptosis, has been proposed as a major triggering factor(Kellner-Weibel et al., 1998; Li et al., 2004; Tabas, 2004).Direct toxic effects of unesterified cholesterol on cellularmembranes or activation of death-promoting signallingmolecules, or a combination of both, are likely involvedin precipitating these complications. Consistently, theinhibition of acyl-coA:cholesterol acyltransferase (ACAT),an intracellular enzyme involved in cholesterol esterification,leads to macrophage cell death (Tardif et al., 2004; Su et al.,2005; Nissen et al., 2006), suggesting that free cholesterol isdirectly toxic for lesional macrophages. Conversely, NPC1mutant mice, which have defective cholesterol traffickingfrom lysosomes to the ER and accumulate unesterifiedcholesterol in endolysosomal compartments (see SectionII.3), are protected from cholesterol-induced macrophageapoptosis and atherosclerosis (Feng et al., 2003b), possiblybecause cholesterol in lysosomes is inaccessible to themachinery implicated in cell death.

Recently, activation of ER stress compensatory pathways,known as the unfolded protein response (UPR), has also beenimplicated in atherosclerosis (Feng et al., 2003a). In particular,high levels of cholesterol, oxysterols, and saturated fatty acidsdirectly activate the UPR in plaque macrophages (Tabas,2010), promoting its progression towards more advancedlesions. Chronic UPR activation, therefore, represents analternative cholesterol pathogenic pathway that opens newtranslational research possibilities.

IV. IS CHOLESTEROL ESSENTIAL FOR LIFE?

Challenging the conventional wisdom in the field, questionsabout the essential role of cholesterol for animal life wereraised after the generation of so-called ‘cholesterol-free’mice. Desmosterol reductase deficient mice (Dhcr24−/−) wereengineered to model the rare human disease desmosterolosis,in which cells cannot catalyze the final step of cholesterolbiosynthesis (Fig. 2) (Wechsler et al., 2003). Surprisingly, andin sharp contrast to human patients, these mice survived andreached adulthood in spite of virtually undetectable levelsof cholesterol in the blood, liver and central nervous system(Waterham et al., 2001; Andersson, Kratz & Kelley, 2002).



Why is cholesterol essential for some species (humans) but,at least apparently, not for others (mice)? The answer maylie in interspecies differences in the maternal availability ofcholesterol during embryonic development and/or in theability of desmosterol to fulfill the roles of cholesterol againstsuboptimal cholesterol concentrations. Indeed, althoughdesmosterol can replace cholesterol in membranes with nonegative biophysical effects (Huster et al., 2005), can regulatethe SREBP pathway, and does support cell proliferation inin vitro systems (Rodriguez-Acebes et al., 2009), it is unableto functionally replace cholesterol in whole human subjects,as shown by the lethality of desmosterolosis. However, bothmale and female Dhcr24−/− mice are infertile, suggestingthat, although cholesterol may not be essential for a normallifespan in rodents, it appears to be an absolute necessity forreproductive health across species.

For humans, it is in clinics where the question aboutcholesterol essentiality has more pressing relevance. Indeed,since all major therapeutic strategies to reduce cardiovascularrisk are centered on reducing LDL cholesterol levels, it isimportant to know the extent to which plasma cholesterol canbe safely reduced. Indeed, the American Heart Association(AHA)/American College of Cardiology Foundation (ACCF)guidelines currently recommend that in very high-riskcardiovascular patients, e.g. those that have previouslysuffered a myocardial infarction, LDL cholesterol should belowered to less than 70 mg/dl (Smith et al., 2011), implyingthe use of potent statins or mixed therapies to antagonize themevalonate pathway.

The answer to how low cholesterol can be before havingadverse clinical effects remains uncertain. In fact, apparentlycontradictory evidence complicates the matter even further.On the one hand, patients with nonsense mutations ofproprotein convertase subtilisin/kexin 9 (PCSK9), a proteaseinvolved in LDLR degradation (Horton, Cohen & Hobbs,2009), have a 28% reduction in mean LDL cholesterol(∼100 mg/dl versus ∼140 mg/dl) and an 88% reduction intheir cardiovascular risk relative to non-carrier individuals(Cohen et al., 2006). More importantly, in this cohort even themore intensely hypocholesterolemic-PCSK9 mutant carriers(with LDL cholesterol as low as ∼40 mg/dl) remainedfree of any health complications. In good agreement withthis patient study, undergoing clinical trials of statinscombined with PCSK9 blocking antibodies are reportingLDL-cholesterol reductions of up to 70%, or ∼40 mg/dl(McKenney et al., 2012; Stein et al., 2012). The safety ofsuch markedly reduced plasma cholesterol concentrationis supported by the fact that mean LDL cholesterol levelsmeasured in the cord blood of 2937 healthy newborns was∼50 mg/dl (Mishkel, 1974), suggesting that LDL cholesterolcould be sufficiently reduced as a cardiovascular therapeuticgoal with no adverse effects on general health.

On the other hand, previous experiences of potentsuppression of cholesterol synthesis with triparanol, aDCHR24 inhibitor drug (Fig. 2) raised serious concernsabout the safety of massive cholesterol reduction in humans.This compound drastically reduced plasma cholesterol in

human patients and was extensively used in the 1960s to treathypercholesterolemia (Steinberg, Avigan & Feigelson, 1961).Unfortunately, triparanol was also associated with severe sideeffects such as cataracts, intestinal lesions and teratogenesis(Modell, 1967), leading to the complete prohibition of thisdrug for clinical purposes in humans. It is noteworthy thatthe toxic side effects of triparanol can be alternativelyexplained by the excessive build-up of sterol precursors,including desmosterol, rather than by severe cholesteroldeficiency. Thus, triparanol’s hypocholesterolemic action,and possibly its toxicity, is not mechanistically equivalent tothat dependent on the PCSK9 pathway.

V. FUTURE DIRECTIONS

The long history of cholesterol is full of fascinating scientificchallenges and remarkable accomplishments. Starting withthe elucidation of the complex cholesterol structure and itsbiosynthetic pathway, followed by the study of its regulationin cells and whole organisms, cholesterol research has ledto some of the most significant biomedical advances of thelast century, providing effective therapeutic applications (e.g.statin development), and sparing possibly millions of livesworldwide. However, as summarized here, this knowledgeis far from complete. As our techniques for studying lipidmetabolism keep improving and our ability to inspect morevariables simultaneously across cell types, tissues, organs,and whole organisms increases, it is conceivable that newmolecular players and interactions will enter the cholesterolscene. Further research will then be needed to continueto deliver basic findings with high-stakes translationalimplications for human health. It is our hope that the materialpresented here will inspire scientists to explore cholesterolbiology in their favourite fields of interest.

VI. CONCLUSIONS

(1) Cholesterol has been historically linked to diversepathologies; however, this sterol is an essential componentof all metazoan cell membranes and is the precursor ofmany other biologically active compounds. Indeed, inborndefects that determine either excessive or reduced cholesterollevels in tissues are associated with embryonic lethalityand malformation syndromes, indicating that this sterol isessential for normal human embryogenesis.

(2) Given its insolubility in water (Fig. 1), cholesterol is onlyfound in lipid structures such as cell membranes, cytosoliclipid droplets and plasma lipoproteins. Inside these lattercomplexes, cholesterol mainly exists as cholesteryl esters. Inmembranes, cholesterol increases their stiffness along withtheir water and ion impermeability. Additionally, cholesterolis critical for lipid raft formation as well as transmembraneprotein conformation and function.



(3) Cholesterol can be synthesized by almost every animalcell and is the end product of the mevalonate pathway(Fig. 2). Other intermediates of this pathway are importantregulators of various biological processes, some of them onlyrecently uncovered, for example the role of isoprenoids onsynaptic plasticity and memory function.

(4) Whereas most prokaryotes do not synthesize oracquire cholesterol from the environment, some relevantintracellular pathogens do require sterols for their growthand tissue persistence. This knowledge may be useful for thedevelopment of new antibiotic drugs.

(5) Cellular cholesterol can also be obtained fromextracellular sources. In vertebrates, lipoprotein cholesterolis taken up by either endocytic or non-endocytic mechanismsvia cell-surface receptor-mediated mechanisms. The LDLRpathway was the first receptor-mediated endocytic system tobe described.

(6) Cells can efflux cholesterol towards extracellularacceptors. This is accomplished by active mechanisms thatinvolve members of the ABC transporter family, such asABCA1, ABCG1 and ABCG5/G8 (Fig. 3). Mutations inthese transporters can cause severe metabolic disorders suchas Tangier’s disease and sitosterolemia.

(7) The molecular machinery that regulates cellularcholesterol levels resides in the membrane of the endoplasmicreticulum (ER). Small variations in ER cholesterol contenttrigger the activation or inactivation of the SREBP system,which controls the transcription of LDLR and mevalonatepathway enzymes (Fig. 4).

(8) Because animal cells cannot break the cholesterolmolecule down to its original acetyl-CoA building blocks, thislipid is not an energy source and it can only be transformedinto other sterols/steroids and bile acids for ‘catabolism’.

(9) Whole-body cholesterol homeostasis is regulated atthe transcriptional level by members of the nuclearreceptor superfamily, namely, LXR and FXR. LXR isactivated by oxysterols and promotes the net movementof cholesterol from peripheral tissues to the liver where itis converted into bile acids for biliary secretion. FXR isactivated by bile acids and coordinates mechanisms thatprevent excessive bile acid accumulation in hepatocytes.Both LXR and FXR are also implicated in fatty acidand triglyceride metabolism, insulin action, and glucoseregulation. Thus, these nuclear receptors are promisingpharmacological targets for prevalent metabolic disorderssuch as atherosclerosis, gallstone disease and diabetesmellitus.

(10) The metabolism of cholesterol in the CNS isindependently regulated from the rest of the body. Thenormal blood–brain barrier is impermeable to plasmalipoproteins and there is no cholesterol exchange betweenthe CNS and the systemic circulation. Cholesterol has to beenzymatically converted to 24(S)-hydroxycholesterol in orderto be exported out of the CNS. Defects in this pathway areassociated with cognitive impairment in mice; its relevancein humans remains unknown.

VII. ACKNOWLEDGEMENTS

The authors thank Ashlee Stiles and Susan Smalley for theirkind help in editing the text and their valuable scientificsuggestions. V.A.C. is funded by Fondecyt # 11100168and Conicyt (Programa de Insercion en la Academia)# 79100018. D.B. is funded by Fondecyt # 11090064and Conicyt (Programa de Insercion en la Academia) #79090028. F.N. is funded by Fondecyt # 1100020. A.R. isfunded by Fondecyt # 1110712.

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(Received 22 June 2012; revised 22 January 2013; accepted 25 January 2013 )


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