The impairment of cholesterol metabolism in Huntington disease.

BBAMCB-57747; No. of pages: 11; 4C: Biochimica et Biophysica Acta xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Biochimica et Biophysica Acta journal homepage: www.elsevier.com/locate/bbalip

Review

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The impairment of cholesterol metabolism in Huntington disease☆

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Valerio Leoni a,b,⁎, Claudio Caccia b a

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Article history: Received 21 October 2014 Received in revised form 19 December 2014 Accepted 21 December 2014 Available online xxxx

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Keywords: Neurodegeneration Biomarker Mass spectrometry Lipid Metabolomics

Laboratory of Clinical Chemistry, Ospedale Causa Pia Luvini, Cittiglio, AO Ospedale di Circolo e Fondazione Macchi, Varese, Italy Laboratory of Clinical Pathology and Medical Genetics, Foundation IRCCS Institute of Neurology Carlo Besta, Milano, Italy

a b s t r a c t

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Huntington disease (HD), an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG trinucleotide repeat in the Huntingtin (HTT) gene, is characterized by extensive neurodegeneration of striatum and cortex and severe diffuse atrophy at MRI. The expression of genes involved in the cholesterol biosynthetic pathway and the amount of cholesterol, lanosterol, lathosterol and 24S-hydroxycholesterol were reduced in murine models of HD. In case of HDpatients, the decrease of plasma 24OHC follows disease progression proportionally to motor and neuropsychiatric dysfunction and MRI brain atrophy, together with lanosterol and lathosterol (markers of cholesterol synthesis), and 27-hydroxycholesterol. A significant reduction of total plasma cholesterol was observed only in advanced stages. It is likely that mutant HTT decreases the maturation of SREBP and the up-regulation LXR and LXR-targeted genes (SREBP, ABCG1 and ABCG4, HMGCoA reductase, ApoE) resulting into a lower synthesis and transport of cholesterol from astrocytes to neurons via ApoE. In primary oligodendrocytes, mutant HTT inhibited the regulatory effect of PGC1α on cholesterol metabolism and on the expression of MBP. HTT seems to play a regulatory role in lipid metabolism. The impairment of the cholesterol metabolism was found to be proportional to the CAG repeat length and to the load of mutant HTT. A dysregulation on PGC1α and mitochondria dysfunction may be involved in an overall reduction of acetyl-CoA and ATP synthesis, contributing to the cerebral and whole body cholesterol impairment. This article is part of a Special Issue entitled Brain Lipids © 2015 Published by Elsevier B.V.

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Abbreviations: 24OHC, 24S-Hydroxycholesterol; 27OHC, 27-Hydroxycholesterol; ABC, ATP-binding cassette transporter; ACAT, Acyl-Coa:cholesterol acyltransferase; AD, Alzheimer disease; ApoE, Apolipoprotein E; BACHD, Bacterial artificial chromosome HD; BDNF, Brain-derived neurotrophic factor; Cav1, Caveolin-1; CNS, Central nervous system; CSF, Cerebrospinal fluid; CYP7A1, Cholesterol 7α-hydroxylase; CYP27A1, Sterol 27-hydroxylase; CYP46A1, Cholesterol 24-hydroxylase; CYP51, Lanosterol 14-alpha demetylase; DHCR24, 24-Dehydrocholesterol reductase; ER, Endoplasmic reticulum; HD, Huntington disease; HDL, High density lipoproteins; HMGCoA, 3αHydroxy-3-methylglutarylcoenzyme A; HMGCoAR, HMGCoA reductase; HMGCoAS, HMGCoA synthetase; HTT, Huntingtin; Insig, Insulin induced gene; LDL, Low density lipoproteins; LDL-R, LDL-receptor; LRP, LDL-related protein; LXR, Liver X receptor; MBP, Myelin basic protein; MM, Mitochonrial membrane; MRI, Magnetic resonance imaging; MS, Multiple sclerosis; NPC, Niemann–Pick type C; NS, Neural stem; PGC1α, Peroxisome proliferator-activated receptor-gamma co-activator 1 alpha; PLP, Proteolipid protein; PPARγ, Peroxisome proliferator-activated recepetor gamma; SCAP, SREBP cleavage-activating protein; ST, Striatal; TCA, Tricarboxylic acid; SRE, Sterol responsive element; SREBP, Sterol responsive element binding protein; VLDL, Very low density lipoprotein; YAC, Yeast artificial chromosome ☆ This article is part of a Special Issue entitled Brain Lipids ⁎ Corresponding author at: Laboratory of Clinical Chemistry, Ospedale Causa Pia Luvini, AO Ospedale di Circolo e Fondazione Macchi, via Luvini 21033 Cittiglio (VA). Tel.: +39 0332 607285, +39 333 3818519 (mobile). E-mail addresses: [email protected], [email protected] (V. Leoni).

18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

1. Cholesterol

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Cholesterol is a structural element of mammal cellular membrane and regulates the fluidity of lipid bilayers. It is the precursor of bile acids, steroid hormones and oxysterols. The cellular needs are covered by de novo synthesis or by the uptake from circulating lipoproteins. All the cells are able to synthesize, release and take up cholesterol to maintain their cholesterol homeostasis: some produce an excess of cholesterol to provide other cells, some others need exogenous cholesterol because of limited synthetic capacity. In humans, under normal conditions, about the 60% of the body’s cholesterol is synthesized (about 700 mg/day) and the remaining is provided by the diet. Cholesterol, together with the other lipids, is absorbed by small intestine, loaded on chylomicrons and delivered to the liver. The exogenous cell supply is covered by very low density lipoprotein (VLDL)–low density lipoprotein (LDL) cycle. Since an excess of free cholesterol is toxic to the cells, a number of strategies have been evolved either to export it (via lipoproteins), to store it in an esterified form or release it after oxidation into oxysterols. A major fraction of the exceeding is exported by the reverse cholesterol transport mechanism involving the high density lipoprotein (HDL) and the ATP-binding cassette (ABC)-transporter family.

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http://dx.doi.org/10.1016/j.bbalip.2014.12.018 1388-1981/© 2015 Published by Elsevier B.V.

Please cite this article as: V. Leoni, C. Caccia, The impairment of cholesterol metabolism in Huntington disease, Biochim. Biophys. Acta (2015), http://dx.doi.org/10.1016/j.bbalip.2014.12.018

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by activation of more than 30 genes involved in the synthesis and uptake of cholesterol, fatty acids, triglycerides and phospholipids as well as NADPH [5]. SREBPs are expressed as inactive 120 kDa precursors (pSREBPs) integral to the ER membrane. When intracellular cholesterol levels are low, pSREBPs are translocated from the ER to the Golgi by an escort protein, SREBP cleavage-activating protein (SCAP), where they are cleaved into a 67 kDa active transcription factors, not membrane bound. These shorter mature SREBPs (mSREBPs) enter the nucleus and modulate transcription of genes containing a SRE in the promoter region. When intracellular cholesterol levels are in excess, SCAP, which has a cholesterol-sensing domain, binds insulin induced gene (Insig) and the Insig-SCAP-pSREBP is retained in the ER reducing cholesterol synthesis [6,7]. SREBPs exist in three isoforms: SREBP-1A activates cholesterol, fatty acid and triglycerides synthesis, SREBP-1C enhances fatty acid synthesis and SREBP-2 is primarily involved in cholesterol synthesis [6]. About 1 g of cholesterol is eliminated from the body every day. Approximately half of this is excreted into the feces after conversion into bile acids; the remainder is excreted as non-metabolized cholesterol

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Cholesterol is synthesized from acetyl-CoA that is converted into 3hydroxy-3-methylglutaril-CoA (HMGCoA) through two condensation steps (Figs. 1 and 2). Microsomal HMGCoA reductase (HMGCoAR) catalyzes the reduction of HMGCoA into mevanolate in endoplasmic reticulum (ER). Mevalonate is phosphorilated into isopentyl-pyrophosphate and other isoprenoids and then by condensation of six units is formed squalene which is cyclized into the parental steroid, lanosterol. Two alternative ways proceed with the cholesterol synthesis: the Block pathway (desmosterol as the main intermediate) and the Kandutsch– Russell pathway (lathosterol and 7-dehydrocholesterol, the two main intermediates). The quantification of the cholesterol precursors lanosterol, lathosterol and desmosterol is considered as surrogate marker for tissue or whole body cholesterol synthesis [1–3]. The HMGCoAR reaction is recognized as the rate limiting step of cholesterol synthesis [4]. Cholesterol and oxysterols are directly involved in a negative feedback mechanism of the enzyme regulation both at the protein and the transcriptional level. Oxysterols modulate lipid synthesis by acting on sterol responsive element (SRE) binding proteins (SREBPs). These transcription factors regulate lipid homeostasis in vertebrate cells

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V. Leoni, C. Caccia / Biochimica et Biophysica Acta xxx (2015) xxx–xxx

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Fig. 1. Simplified diagram of cholesterol metabolism in the cells. The formation of acetyl-CoA is the first step of cholesterol and fatty acids synthesis. The acetyl-CoA enters in cytosol in form of citrate by the tricaboxylate transport system. ATP-citrate-lyase (ACL) converts citrate into acetyl-CoA and oxaloacetate in an ATP-driven reaction. HMGCoAS catalyzes the condensation of 3 acetyl-CoA into HMGCoA. The rate limiting step occurs at the HMGCoAR followed by mevalonate formation. Phosphorylation is required to solubilize the isoprenoid intermediates in the pathway (the PP abbreviation stands for pyrophosphate). Intermediates in the pathway are used for the synthesis of prenylated proteins, dolichol, coenzyme Q and the side chain of Heme A. Pyrophosphated isoprenoids are condensed and cyclized by squalene synthetase (SQS) then the first sterol, lanosterol is formed. Two alternative pathways (Block and Kandush– Russel) lead to cholesterol formation. Liver CYP7A1 converts cholesterol into 7 α-hydroxycholesterol (7αOHC), the main precursor of the neutral bile acid pathway. Cholesterol 27hydroxylase (CYP27A1), expressed in different cell types, converts cholesterol into 27-hydroxycholesterol (27OHC), precursor of the acidic bile acid pathway. Neuronal specific cholesterol 24-hydroxylase (CYP46A1) is responsible for 24S-hydroxycholesterol (24OHC) formation.


81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99


3

astrocyte

neuron

oligodendrocytes

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HMGCoAR

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MPB

blood

100

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Fig. 2. A model for cholesterol metabolism in Huntington disease. In the mature brain, neurons down regulate their cholesterol (chol) synthesis and relay on the astrocytes delivery of cholesterol via ApoE lipoproteins. The expression of hydroxy-methyl-glutaryl-Coenzyme A reductase (HMGCoAR) is inhibited by cholesterol and oxysterols with a feedback regulation via the sterol-responsive element binding protein (SREBP) that binds to the sterol-responsive element (SRE-1) in the HMGCoAR gene. In astrocytes, cholesterol is loaded on ApoE by the ATP binding cassette (ABC) transporter A1 (ABCA1). The apoE-cholesterol complex is internalized via low-density lipoprotein receptors (LDLR). Neurons convert the excess of cholesterol into 24S-hydroxycholesterol (24OHC) via the cholesterol 24-hydroxylase (CYP46). 24OHC and other oxysterols are important ligands of the liver X-activated receptor (LXR), which translocates to the nucleus (as circle in the figure) and induces expression of both the APOE and the ABCA1 genes in astrocytes. Cholesterol and 24OHC are excreted from neurons via ABCG1/G4 to ApoE particles to the interstitial fluid and to cerebrospinal fluid. 24OHC through the blood–brain barrier can be delivered into plasma where it is esterified in lipoproteins (LDL and HDL) for further elimination by liver. In the oligodendrocytes, the cholesterol synthesis and myelin basic protein (MBP) are under control of peroxisome-proliferator-activated receptor gamma co-activator 1 alpha (PGC1α).

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2. Brain cholesterol metabolism

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About the 25% of the total body cholesterol is located in the brain (which represents just the 2% of the total body weight) [11], mainly in an unesterified form (N99.5%) [12] and distributed between myelin (about the 70%), glial cells (20%) and neurons (10%), as structural element of the cell membrane [13]. Cholesterol is organized in microdomains called lipid rafts which are involved in the maintenance of the properties of membrane proteins such as receptors and ion channels [14]. Brain cholesterol turnover is much slower than in periphery,

106 107 108 109 110 111 112 113 114 115

119 120 121 122 123 124 125

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104 105

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or the bacterial metabolite coprostanol. The bile acid synthesis begins with a cholesterol oxidation into oxysterols. There are two pathway described in humans (and rodents). The neutral pathway is initiated by the rate-limiting enzyme cholesterol 7α-hydroxylase (CYP7A1), which is mainly expressed in hepatocytes; under normal conditions the neutral pathway is prevalent in healthy adult humans and is under strict metabolic control [8]. In other cells and tissues cholesterol is eliminated by side chain oxidation as an alternative to the classical HDL-mediated reversed cholesterol transport. Almost all cells in the body contain the enzyme sterol 27-hydroxylase (CYP27A1) located in the inner membrane of the mitochondria and highly expressed by macrophages. At high levels of CYP27A1, 27OHC can be further oxidized by CYP27A1 into 3β-hydroxy-5-cholestenoic acid, which is converted into 7α-hydroxy-3-oxo-cholesten-4-cholestenoic acid to proceeds in the acidic pathway for bile acid synthesis, in the liver. This pathway is responsible for about 10% of the daily production of bile acids in humans [9,10].

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estimated to be about 4–6 months in rodents and more than 5 years in humans, confirming the importance of the structural role of this molecule [11,15,16]. The blood–brain barrier formed by the tight junction attachments between adjacent capillary endothelial cells prevents the diffusion of large molecules from circulation into the inner brain space and there is virtually no passage of lipoprotein-bound cholesterol from the circulation into the brain [11,15,16]. There is also no transvesicular movement of solution across the capillaries. It is likely that one or more members of the ABC-transporter family may be involved in the exclusion of circulating cholesterol from the brain. Therefore, more than 95% of cholesterol present in brain and in peripheral nervous system is produced by de novo synthesis. Cholesterol is involved in synaptogenesis, turnover, maintenance, stabilization and restore of synapses [17] and is a limiting factor for outgrowth of neuritis being involved in vesicle transport and exocytosis at synaptic levels [18–20]. Since cholesterol appears as a major regulator of neuronal activity, imbalances of its metabolism may potentially have severe consequences on brain function. Converging evidence in cellular and animal models and observations support the importance of disturbance of cholesterol metabolism from the pathogenesis of neurodegenerative diseases (Table 1). Cholesterol homeostasis is maintained by the dynamic equilibrium of de novo synthesis, transport, storage and removal. In the brain, cholesterol synthesis via the 7-dehydrodesmosterol pathway seems to be preferred over the 7-dehydrocholesterol pathway [21]. In neurons the main sterols found belong to the so-called Kandutsch–Russel pathway, whereas in astrocytes to the Bloch pathway. The accumulation of


126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153

4 t1:1 t1:2


Table 1 Cholesterol metabolism abnormalities in neurodegenerative diseases.

t1:3

Disorder

Pathogenesis

Cholesterol abnormalities

t1:4

Shmit-Lemli-Opitz- syndrome

t1:5 t1:6

Niemann–Pick type C disease Cerebrotendinous xantomatose

accumulation of 7-dehydrocholesterol: developmental deformities, incomplete myelination, and mental retardation endosome and lysosome cholesterol ester accumulation brain and tendon accumulation of cholesterol, high plasma cholestanol, very low 27OHC

t1:7

Spastic paraplegia SPG5

loss of function of 7-dehydrocholesterol reductase loss of function of NPC1 loss of function of sterol 27-hydroxylase CYP27A1 loss of function of sterol 7-hydroxylase (CYP7B1)

t1:8

Parkinson disease

degeneration of dopamine neurons in substantia nigra

t1:9

Alzheimer disease

abnormal cleavage of APP and Aβ accumulation (amyloid plaques). hyperphosphorylation of tau protein

t1:10

Multiple sclerosis

auto-immune demyelination by T helper cells against myelin antigens

t1:11

Huntington disease

toxic gain of function and loss of function by muted Huntingtin transcriptional dysregulation (LXR and PGC1α) reduced activation of SREBP mitochondria abnormalities

162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183

E

R

R

160 161

O

158 159

C

157

desmosterol in cultured astrocytes suggests that the desmosterol quantification allows to trace the sterol synthesis in this glial cell type. Since very low levels of lanosterol-converting enzymes, 24dehydrocholesterol reductase (seladin-1, DHCR24) and lanosterol 14-alpha demethylase (cytochrome P450, family 51, CYP51) were detected, the cholesterol synthesis is likely to end at lanosterol step in neurons [20]. The oligodendrocytes differentiate post-natally. The process of myelination in rodents and in humans occurs during the initial weeks (or months) with a coordinated accumulation of cholesterol and myelin basic protein (MBP) [11]. The rate of cholesterol synthesis is very high during brain maturation and gradually decreases over the time. It is also higher in regions with a higher fraction of myelin and white matter over gray matter, like mid brain and spinal cord compared to the cortex. The progressive accumulation of cholesterol ends in adulthood when the myelin formation is completed. The myelin sheath in the central nervous system (CNS) is formed by sections of oligodendrocyte plasma membrane repeatedly wrapped around an axon, with the extrusion of virtually all of the cytoplasm. One cell can wrap several axons. Myelin can thus be regarded as a discontinuous insulation that enables the saltatory conduction of the action potential. In addition to a large lipid component, myelin also contains many specific proteins such as the proteo-lipid protein (PLP) (about the 50% of the total myelin proteins) and the MBP (about the 30%) and they are essential to regulate the proper assembly of myelin [11,15]. Under in vitro conditions, the oligodendrocytes have the highest capacity to synthesize cholesterol, followed by the astrocytes which synthesize about five times more cholesterol than neurons [16,22]. The neurons appear to produce sufficient amounts of cholesterol to

N

155 156

U

154

C

T

E

D

P

R O

O

F

5 up to 10 fold increase of plasma and CSF 25OHC and 27OHC upper motor neuron degenerative diseases characterized by selective axonal loss in the corticospinal tracts and dorsal columns unknown mechanism for idiopatic PD. Neuronal loss and Lewy bodies in remaining neurons cholesterol and membrane lipids are involved in polymerization of Alpha-synuclein epidemiologic evidence relating high total cholesterol as risk factor for PD, high HDL-cholesterol is associated to longer disease duration, evidence for low LDL-cholesterol in PD reduced 24OHC and precursor sterols in patients ApoE4 risk factor related to a less efficient cholesterol transport between age membrane cholesterol accumulation increase amyloid generation and tau hyperphosphorylation high midlife cholesterol as a risk factor for late onset AD oxysterols 24OHC and 27OHC reduce Aβ generation favored by cholesterol accumulation cholesterol accumulation in plaques increased 24OHC in an early stage of disease (MCI) and reduction with disease progression proportionally to MRI brain atrophy inconsistent findings about beneficial effect of statins as therapy oligodendrocytes cholesterol metabolism is under LXR control and is increased in presence of LXR agonist plasma 24OHC reduced in MS patients proportionally to brain atrophy; in RR patients plasma 24OHC correlated with the space and time disease burden (T2 volumes at MRI) reduced precursor sterols in RR and PP patients reduced expression of biosynthesis genes (HMGCoAR, HMGCoAS, CYP51), cholesterol transporter genes (ABC-A1, -G1, -G4, APOE) and MBP reduced cholesterol synthesis in HD primary neurons, oligodendrocytes and astrocytes reduced total cholesterol, lanosterol, lathosterol and 24OHC in cortex, striatum and whole brain from HD rodent models accumulation of cholesterol in lipid droplets, caveolae and lipid rafts in neuronal HD cells reduced lipidation of ApoE reduced production of 24OHC reduced of plasma levels of lathosterol, lanosterol, 27OHC and 24OHC (proportionally to disease progression)

survive, to differentiate axons and dendrites and to form a few and inefficient synapses. The massive formation of synapses requires additional cholesterol delivered by astrocytes via apolipoprotein E (ApoE)containing lipoproteins [18–20]. Also the oligodendrocytes play an important role in cholesterol synthesis within the CNS and they seem to be the main cholesterol synthesizing cells able to supply cholesterol to neurons and to astrocytes by ApoE [23].

184

3. Brain cholesterol transport

191

When the process of brain maturation and myelin formation is concluded in the adulthood, the neurons down-regulate their cholesterol synthesis and rely on delivery of cholesterol from astrocytes which differentiate post-natally and release cholesterol rich lipoproteins (the “outsourcing” hypothesis of brain cholesterol metabolism) [20]. With this strategy the neurons are allowed to focus on generation of electrical activity rather than dispense energy on costly cholesterol synthesis: more than 100 mol of ATP are required for the synthesis of one mole of cholesterol [24]. This is of particular importance in presynaptic terminals and dendritic spines, which are distant from the soma [11,18–20]. ApoE is the main lipid carrier protein in CNS and is released by astrocytes in order to supply neurons and synaptogenesis with lipids and cholesterol [25–27]. The cholesterol delivered could be originated by de novo synthesis as well as by recycle of cholesterol released from degenerating axons [28]. Astrocytes release cholesterol on HDL-like particles with ApoE as apolipoprotein via ABC-A1 and ABC-G1 transporters [29,30]. The ApoE lipoproteins are internalized into neurons by the LDL-receptor (LDL-R) and the LDL-related protein (LRP), but also to some extent via VLDL receptor, ApoE receptor 2, and megalin [31–33]. The receptor–ApoE–

192


185 186 187 188 189 190

193 194 195 196 197 198 199 200 201 202 203 204 205 206 207 208 209 210 211


212 213

5

276 277

24OHC acts as an endogenous ligand of the nuclear receptor liver X receptor (LXR) and regulates the cholesterol and fatty acid synthesis pathways. In the adult brain, almost all the neuronal requirements of cholesterol are supplied by cholesterol transported on ApoE containing lipoproteins released from astrocytes [19]. Via LXR, 24OHC up-regulates the expression, synthesis and secretion of ApoE [56] and the expression of the sterol transporters ABC-A1, -G1 and -G4 on the astrocyte membranes, involved in the transport of cholesterol from glia to ApoE particles [57]. Thus, the turnover product of neuronal cholesterol seems to play a role as feedback regulator of astrocyte cholesterol metabolism [18,56,58]. Recently, the importance of this mechanism in vivo was challenged by results obtained in a study on mice overexpressing CYP46A1. Despite increased levels of 24OHC in the brain and in the circulation there was little or no increase in the expression of the different LXR target genes [52].

227 228

4. Turnover of brain cholesterol and conversion into 24S-hydroxycholesterol

5. Brain cholesterol in Huntington disease

291

The synthesis of cholesterol is balanced by the elimination via a hydroxylation in position 24 catalyzed by cholesterol 24-hydroxylase (CYP46A1) to form 24S-hydroxycholesterol (24OHC), in which the rate of translocation across the blood–brain barrier is thousand times higher than cholesterol and enters the circulation [14,38,39]. Under normal conditions, CYP46A1 is expressed only in neuronal cells, localized at the level of the neuronal cell body and dendrites, in cerebral cortex, hippocampus, dentate gyrus, amygdala, putamen and thalamus, i.e., associated with gray matter [40,41]. About 6–8 mg/24 h of cholesterol are released as 24OHC by the brain into the circulation [39]. In addition, there is a small efflux of cholesterol from the brain in the form of ApoE containing lipoproteins via the cerebrospinal fluid (CSF) [42]. It was hypothesized that the substrate availability is an important regulatory factor for the enzyme activity in vivo: the amount of free cholesterol in neurons should be balanced by the conversion into 24OHC which can also regulate the cholesterol synthesis and the secretion of ApoE from astrocytes [43]. Although CYP46A1 is primarily expressed by neuronal cells in the normal brain, also non-neuronal expression of CYP46A1 was found in the astrocytes of Alzheimer disease (AD) brains [44,45], after kainate injury [46] and after brain trauma [47], in the macrophage, in a model of multiple sclerosis (MS) [48] and in the microglial cells, in a rat model of trauma brain injury [49]. A modest reduction of HMGCoAR activity and cholesterol synthesis rate (as confirmed by observed reduced levels of brain lathosterol) was observed in CYP46A1(−/−) mice, while the total brain cholesterol levels were unaffected [42]. These mice presented severe deficiencies in spatial, associative and motor learning associated with a delay of long lasting potential and alterations in synaptic maturation [50]. When hippocampal slices collected from the wild-type animals were treated with an inhibitor of cholesterol synthesis, the effects observed in the CYP46A1(−/−) mice were essentially recapitulated [51]. On the contrary, when the CYP46A1 was overexpressed in a transgenic mouse model, cerebral and plasmatic 24OHC, cerebral lathosterol and lanosterol were significantly increased but total cholesterol was not. The brain cholesterol synthesis and elimination were closely related as demonstrated by a significant correlation between lathosterol and 24OHC [52]. When ACAT was ablated, a 13% reduction of the total brain cholesterol and a 32% increase of 24OHC were observed in a mouse model, while lanosterol and desmosterol were unchanged. Since less cholesterol may be esterified, more cholesterol was oxidized into 24OHC and removed from neurons [53]. In absence of an efficient cholesterol export from neurons (via ApoE or oxidation in position 24), cholesterol synthesis was reduced, while in absence of a cholesterol transporter involved in ApoE lipidation it was increased with a higher cholesterol uptake through blood–brain barrier. Thus, it is likely that a modification of cholesterol transport has a direct effect on its homeostasis [54,55].

Huntington disease (HD) (MIM #143100) is an autosomal dominant neurodegenerative disorder characterized by motor impairment, cognitive decline, psychiatric manifestation and progression to death 15–20 years from the time of symptomatic onset. HD is caused by an abnormal expansion of CAG trinucleotide repeat in the 5′ end of the IT15 gene (chromosome 4p16.3). Mutations code for expanded glutamine in huntingtin (HTT) protein [59]. The gradual atrophy of the striatum (caudate nucleus and putamen) together with astrogliosis [60] are neuropathological characteristics of the disease. According to MRI investigations there is also a severe cortical atrophy combined with striatal degeneration [61]. HTT is widely expressed in all tissues, especially in brain and testis; within the cell, it associates with various organelles and structures and has been ascribed in numerous roles in intracellular functions including protein trafficking, vesicle transport, endocytosis, postsynaptic signaling, transcriptional regulation and an anti-apoptotic function [62].

292

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5.1. Genomic and metabolomic impairment of cholesterol metabolism

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The expression of genes involved in the cholesterol biosynthesis, like HMGCoAR, CYP51, 7-dehydrocholesterol 7-reductase and DHCR24 were reduced in inducible mutant HTT cell lines, in striatum and cortex of transgenic R6/2 HTT-fragment mice and in post mortem cortical tissue collected from HD patients (for a brief description of HD animal models, see Table 2) [63–65]. In human derived fibroblasts and inducible mutant HTT cells exposed to delipidated medium, the rate of cholesterol synthesis was reduced according to the production of labelled cholesterol. The cellular survival of rat primary striatal neurons transfected with cDNA plasmids encoding the 480N-terminal amino acids of human HTT containing 68 CAGs was promoted in a dose dependent manner when supplied wit cholesterol [63]. The amount of total cholesterol was significantly reduced in primary neurons from Hdh (Q140/140) mice compared to wild-type mice Hdh(Q7/7). At an early differentiation stage, Hdh(Q140/140) neuronal stem (NS) cells had reduced cholesterol compared to Hdh(Q7/7); no reduction in cholesterol was found in NS cells from heterozygous knock-in HD mice Hdh(Q140/7), while Hdh(−/−) NS cells presented elevated cholesterol compared to Hdh(Q7/7) [66]. Reduced cholesterol synthesis was observed in immortalized knock-in cells derived from the embryonic striatum of mice carrying 109Q inserted in the mouse HTT gene (ST Kin(Q109/109)) [67], in astrocytes from R6/2 and YAC 128 mice [68] and in primary mouse oligodendrocytes with lenti-mutant HTT exon 1 (Q72) [69]. The cortical, striatal or whole brain amounts of lanosterol and lathosterol were reduced in several mouse models of HD. In case of yeast artificial chromosome (YAC) transgenic mice, the reduction of the precursor sterols was directly proportional to the length of CAG expansion (YAC46 N YAC72 N YAC128): longer was the CAG repeat, lower were the precursor sterols. A similar trend was also observed

309 310

236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275

O

R O

P

D

234 235

E

232 233

T

230 231

C

223 224

E

221 222

R

219 220

R

218

N C O

216 217

U

214 215

F

225 226

cholesterol complexes are delivered to the late endosomes/lysosomes where acid lipase hydrolyzes the cholesterol esters within the lipoprotein complexes, with the intracellular release of free cholesterol. This unesterified cholesterol exits the late endosomes/lysosomes via a Niemann–Pick type C (NPC) 1 and NPC 2 protein-dependent mechanism and is distributed to the plasma membrane as well as to the ER, which acts as a negative feedback sensor for the cholesterol homeostasis genes such as HMGCoAR and LDL-R. The cholesterol in excess, on the other hand, is esterified in the ER by acyl-Coa:cholesterol acyltransferase (ACAT) and stored in cytoplasmic lipid droplets as a reserve pool [17,34,35]. This intracellular pool of cholesterol is involved in the synaptic and dendritic formation and in the membrane remodeling [36]. There is also an exchange of cholesterol from the mature ApoE particles and the oligodendrocytes but this contribution to the general homeostasis of brain cholesterol is unclear [37].


278 279 280 281 282 283 284 285 286 287 288 289 290

293 294 295 296 297 298 299 300 301 302 303 304 305 306 307

311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329 330 331 332 333 334 335 336 337

6 t2:1 t2:2


Table 2 Rodent models of Huntington disease.

t2:3

Animal model

Model type

Transgene construct

Promoter

CAG repeats

Reference

t2:4 t2:5

Hdh (Q7/7) mouse R6/2 mouse

Wild-type Truncated N-terminal fragment

Mouse HTT Human HTT

CAG 7 CAG 144 (unstable)

[66,96] [63–65,68,69]

t2:6 t2:7 t2:8 t2:9 t2:10 t2:11

Hdh (Q140/140) mouse Hdh (Q109/109) mouse Hdh (Q72/72) mouse Hdh (Q111/111) mouse Hdh (Q150/150) mouse YAC 18-128 mouse

Full-length knock-in Full-length knock-in Full-length knock-in Full-length knock-in Full-length knock-in Full-length transgenic model

Endogenous HTT mouse gene 1,9 kb fragment from the 5' of human HTT Human HTT exon1 Human HTT exon1 Human HTT exon1 Human HTT exon1 Human HTT exon1 Full-length human HTT

BACHD mouse and BACHD rat

Full-length transgenic model

Full-length human HTT

CAG 140 CAG 109 CAG 72 CAG 111 CAG 150 CAG variable from 18 to 128 CAG 97

[66] [67] [69] [70,96] [70,72] [68,97]

t2:12 t2:13

Mouse HTT Mouse HTT Mouse HTT Mouse HTT Mouse HTT Human HTT and regulatory elements Human HTT and regulatory elements

366 367 368 369 370 371 372 373 374 375 376 377 378 379 380 381 382 383 384 385 386

C

E

360 361

R

358 359

R

356 357

O

354 355

C

352 353

N

350 351

U

348 349

F

O

In mHTT-expressing neurons isolated from the brain and grown in culture, an accumulation of free cholesterol in the plasma membrane and of esterified cholesterol in endosomes was observed. These processes also contribute to excitotoxicity [70–72]. In cultured Hdh(Q150/150) striatal neurons, the inhibition of intracellular trafficking resulted into a lysosomal accumulation of esterified cholesterol [72]. A concomitant higher amount of the ganglioside GM1 was observed, suggesting that membrane cholesterol accumulation is associated with an increased prevalence of lipid rafts. Caveolin 1 is a protein found in caveolae that binds cholesterol. Mutant huntingtin impairs the trafficking of post Golgi caveolin-1, leading to accumulation of cholesterol in membranes and lysosomes. The loss or reduction of caveolin-1 expression in Hdh(Q7/150) and in Hdh(Q150/150) mice models, suppresses these phenotypes with a reduction of cholesterol accumulation in striatal neurons [72]. At the moment, a line of evidences speaks in favor of a general inhibition to the cholesterol metabolism (i.e.: reduced gene expression, reduced cholesterol amount, reduced cholesterol synthesis detected with radionuclides and reduced amount of lathosterol and lanosterol as estimation of synthesis rate) and another in favor of an accumulation of cholesterol esters in mt-Htt expressing neuronal cells studied by a filipin or a colorimetric staining. The filipin staining, however, is increased both in case of cholesterol esters and phospholipids accumulation [73,74].

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In sub-confluent cultures with delipidated medium was observed a significant reduction in cholesterol synthesis and total cholesterol by gas chromatography–mass spectrometry (GC-MS), while filipin staining did not reveal any difference. No variation was detected in overconfluent cultures. No accumulation of lipid droplets was observed with Nile-red staining. A combination of genomic (real time-PCR) and metabolomic (isotope dilution mass spectrometry for sterols and oxysterols) methods resulted to be more accurate, reproducible and less prone to interferences in the study of cholesterol metabolism in HD [67]. It is likely that the procedure of sample preparation, the degree of confluence and clone properties, the use of lipidated versus delipidated serum, the methodology adopted to study cholesterol metabolism have a strong influence on the results and, in particular, on the level of total or intracellular cholesterol detected. 5.3. Molecular mechanism of cholesterol dysfunction

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for cholesterol but it was significantly reduced only in YAC72 and YAC128 mice. The rate of cholesterol synthesis decreases with brain maturation [11]: in the R6/2 mice, the observed reduction at any time point was larger compared to the wild-type mice at the same age [64]. Precursor sterols (and cholesterol) were also reduced in knock-in mice carrying the CAG expansion within the mouse HTT gene (Hdh(Q111/111)), proportionally to the load of mutant HTT (wt N Hdh(Q7/111) N Hdh(Q111/111)) [68]. Similarly, a decrease of cholesterol, zymosterol and lathosterol was observed in the Hdh(Q150/150) murine model [70]. In all the animal models reported above a reduction of 24OHC in whole brain, striatum and cortex was found, probably because an impairment of synthesis corresponded a parallel impairment of cholesterol elimination by metabolically active neurons [68]. The metabolomic markers of cholesterol synthesis, accumulation and turnover and the expression of genes involved in cholesterol synthesis, were reduced proportionally to the length of CAG repeats and to the load of mutant HTT (heterozygous N homozygous). In the wild type mice, the amount of cholesterol increases with maturation reaching a plateau in the adult animals. Conversely, the amount of precursor sterols decreases with maturation reaching a stable low level in the mature animals. In HD mice, the amount of brain cholesterol and precursors was lower compared to age matched littermates. The size of such differences was increased with the aging process [68]. These data suggest that HTT may play a specific role in cholesterol homeostasis and mutant HTT impairs brain cholesterol synthesis and turnover.

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To explain the impairment of cholesterol metabolism at a molecular level, a mutant HTT-dependent decrease in the amount of active SREBP was hypothesized. In both cellular models of HD and in samples of striatum collected from R6/2 mice, a reduced SREBP translocation was found. Such reduced entry of SREBP into the nucleus would result into decreased cholesterol synthesis [63]. A proper cholesterol supply is critical for the neurite outgrowth, the synapses and dendrites formation and for the axonal guidance [20,26, 75,76]: neurite loss is an early finding in various neurodegenerative disorders, including HD, in which, together to morphological abnormalities of the brain, defects in synaptic activity have been observed [77–79]. A cholesterol depletion leads to a synaptic and to a dendritic spine degeneration, to a failed neurotransmission and a decreased synaptic plasticity [80]. The fluidity of cell membranes and the distribution of microdomains such as lipid rafts affect the transfer of cholesterol between cells and cellular membrane structure and function. The spectroscopic Raman analysis of cellular membranes isolated from peripheral cells of HD patients showed an alteration of membrane properties and fluidity related to differences in cholesterol and phospholipid content [81]. Wild-type HTT is able to bind to some nuclear receptors involved in lipid metabolism: liver-X-receptor (LXR), peroxisome-proliferatoractivated receptor gamma (PPARγ) and vitamin D receptor [82]. Overexpression of HTT was shown to activate LXR while a lack of HTT led to an inhibition of LXR-mediated transcription. The possibility that mutant HTT is less able to up-regulate LXR and LXR-targeted genes, including SREBP, must be considered. Such a mechanism could be another possible link between the HTT-mutation and the disturbances in the cholesterol metabolism. As the reduced synthesis of cholesterol may contribute to the pathogenesis of HD, the dysregulation of cholesterol synthesis might be directly involved in the neurodegenerative process. In neuronal cells expressing mutant HTT, the observed reduction of cholesterol synthesis could be emended with cholesterol supply in the medium (up to 15 μM): cholesterol supply resulted in a progressive


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from a knock-in HD mouse model and in post-mortem human striatum [89]. These data confirm the importance of the PGC1α transcription disturbance in the HD pathogenesis. Together with an impairments of the mitochondrial biogenesis, alterations of the mitochondrial morphology, dynamics and functions were reported [90,91]. Particularly, defects in the respiratory chain (complexes I, II and IV) [92] and in the tricarboxylic acid (TCA) cycle [93] were found. The reduced mitochondrial energy supply to the metabolic pathways, the dysfunction of respiratory chain and oxidative phosphorylation (lower ATP production), the inefficient energy production and the reduced intermediate biosynthesis described in HD could be in part related to the PGC1α dysregulation [87,90,94,95]. Cholesterol synthesis begins with the free acetyl-CoA in cytosol released by the citrate lyase reaction which furnishes also the required ATP and NADPHH for lipid synthesis. In presence of a high energetic charge the TCA cycle is inhibited and the citrate in excess escapes the mitochondria for biosynthetic reaction. We could speculate that to a partial transcriptional silencing of the cholesterol genes may be also associated an inefficient production of metabolic intermediates for cholesterol synthesis via a reduced amount of Acetyl-CoA available in cytosol due to the mitochondrial dysfunction. Cholesterol is a major regulator of the membrane fluidity. Its amount in mitochondrial membranes (MMs) is very low compared with plasma membrane. Membrane fluidity was found to be significantly increased in MMs isolated from HD knock-in mice Hdh(Q111/111) compared to wild-type Hdh(Q7/7), in both adult and aged animals. Similar findings were obtained in striatal (ST) Hdh(Q111/111) cells and in BACHD rats. However, the changes in cholesterol content did not reflect the alterations in MM fluidity: a significant decrease of MM cholesterol was found only in BACHD rats, while cholesterol levels were unchanged in ST Hdh and in HD knock-in mice. These findings suggest that the reduced cholesterol synthesis might affect the structure of MM and thus the function of the proteins located on the MMs such as some of the TCA enzymes and some of the respiratory chain complexes (complexes I, II and IV). Olesoxime, a cholesterol oxime, is able to enter the cell and concentrate in the mitochondria showing a therapeutic efficacy for the treatment of mitochondrial dysfunction in cell and animal models of amyotrophic lateral sclerosis, MS and peripheral neuropathy. The treatment of HD cells with olesoxime led to a disease-unspecific and dose dependent decrease in MM fluidity. Moreover, an enhancement of cholesterol content in MMs isolated from HD and wild-type cells was observed. The effect of olesoxime in vivo was tested in treating BACHD rats for 12 months: MM fluidity and cholesterol content were found restored at wild-type levels [96].

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6. Study of cholesterol metabolism in HD patients

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A pathological alteration of the white matter may represent an early event in HD pathogenesis: MRI evidences of abnormalities in oligodendrocytes and white matter have been observed in pre-manifesting individuals [85,86]. In primary oligodendrocytes, mutant HTT inhibited the regulatory effect of peroxisome-proliferator-activated receptor gamma co-activator 1 alpha (PGC1α) on HMGCoA synthetase (HMGCoAS) and HMGCoAR, the expression of MBP and the cholesterol metabolism [69]. Brain samples from R6/2 and bacterial artificial chromosome HD (BACHD) mice had abnormal myelination and reduced expression of PGC1α and MBP. Similar findings were found in a PGC1α knock-out mouse model [69]. PGC1α is involved in mitochondria biogenesis, fatty acid oxidation and oxidative metabolism and regulates the expression of cytochrome C, component of respiratory chain complexes I–V [87]. Microarray studies found that the vast majority of PGC1α target genes were downregulated in striatal RNAs from asymptomatic and presymptomatic HD patients [88] and PGC-1α expression was reduced in medium spiny neurons from HD patients. PGC1α transcriptional activity was markedly decreased in HD striatal-like cells, in medium spiny neurons

About 50% of reduction of the cholesterol synthesis (as incorporation of 13C acetate into cholesterol) was observed in fibroblasts derived from HD patients compared to ones from healthy controls when grown in a lipid-free medium and much less (about 10%) in presence of medium with a source of cholesterol [63]. Plasma lanosterol, lathosterol and desmosterol (considered as markers of the whole body cholesterol synthesis) were reduced by ~25% in samples collected from 10-month-old YAC128 and increased by ~25% in YAC18 mice compared to wild-type mice [97]. The 24OHC was reduced by ~ 15% in YAC128 and increased by ~12% in YAC18 compared to wild-type animals [97]. In humans, a significant reduction of the total plasma cholesterol [98,99] together with a reduction of the HDL and the LDL cholesterol [99] were reported in premanifesting subjects and in HD patients compared to controls. In other studies, cholesterol was reduced proportionally to the disease stage but significantly only in advanced stages [100]. In a further study, lanosterol and lathosterol, together with 27OHC and 24OHC, were lower in plasma from HD patients [101]. These findings suggest that not only the cerebral synthesis but also the whole body cholesterol homeostasis is disturbed in HD patients.

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accumulation of total and esterified cholesterol in striatal cells [63], while a higher concentration of cholesterol in the medium (over 25 μM) had a cytotoxic effect with massive apoptosis [67]. An accumulation of cholesterol into caveolae and lipid rafts was also supposed, as previously discussed [72,83,84]: this could occur at an early stage of disease and contribute to an early neuronal loss being somehow masked by the general impairment of the brain cholesterol synthesis. By far, more than 90% of brain cholesterol is not located in neurons where the synthesis is also almost absent. According with this observation, significant reductions of cholesterol, desmosterol and lathosterol were observed in whole brain samples collected from 80 week old Hdh(Q150/150) mice compared to wt despite of cholesterol accumulation observed in cultured striatal cells from the same model [72]. The brain-derived neurotrophic factor (BDNF) is released by cortical neurons projecting into striatum and is involved in synaptic plasticity and neuronal survival. BDNF induces cholesterol synthesis in postsynaptic neurons. Mutant HTT affects the BDNF transport and release, presumably resulting into an inhibition of neuronal cholesterol synthesis further enhanced by an inefficient cholesterol removal due to lower expression of ApoE by astrocytes. In this scenario, the inefficient removal of cholesterol from neurons should lead to its accumulation in membranes and lysosomes involving caveoline-1. According to this hypothesis, the mRNA levels of cholesterol efflux genes (Abca1, Abcg4 and ApoE) involved were reduced (together with the biosynthesis genes) in primary astrocytes from both R6/2 and YAC 128 mice compared to the wild-type or YAC18. The astrocytes with mutant HTT synthesize and secrete less ApoE than wild-type cells and the ApoE-lipoproteins present in CSF collected from YAC128 mice were smaller and less lipidated than those collected from wild-type [68]. Taken together, these observations suggest that in HD there is a reduced ApoE mediated cholesterol transport and supply from astrocytes to neurons and an inefficient removal of cholesterol from neurons to astrocytes via ApoE. In theory, the impairment of the astrocyte cholesterol metabolism might be due to a combination of reduced activity of LXRs and a reduced SREBP activation [68]. It seems likely that there are other yet uncovered HTT-sensitive mechanisms affecting synthesis, transport and delivery of cholesterol from astrocytes to neurons. Finally, it may be possible that at an early stage of the disease there would be a neuronal accumulation of cholesterol in esterified form due to reduced CYP46A1 oxidation or, more likely, to a reduced removal via ApoE contributing to an alteration of the membrane structures and cellular death. Such neuronal process could not affect the whole brain cholesterol metabolism and the total amount of cholesterol and cholesterol synthesis since less than 10% of the total is located there.


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Huntingtin may play a regulatory role in the normal cerebral and extracerebral biosynthesis of cholesterol [84] which is modified in the presence of mutant HTT. Consistent findings about an impairment of synthesis, accumulation and turnover were reported in animal models of disease together with a reduction of the expression of key regulatory genes. Cellular experiments evidenced altered cholesterol homeostasis in neurons, astrocytes and oligodendrocytes. These dysfunctions all together are likely to explain the quantitatively significant reductions observed in striatum, cortex and whole brain samples from HD rodent models. The transcriptional dysregulation reduces the translocation of SREBP, the activation of LXR and LXR-dependent genes and it impairs PGC1α function. The dysfunction of the cholesterol metabolism is not restricted just to the CNS. Reduced cholesterol synthesis and turnover were also described in other neurodegenerative diseases [103]. The reduction of 24OHC depends on the decrease of the number of metabolically active neurons following the neurodegenerative process and it is related to brain atrophy. The role as biomarkers of 24OHC, is limited to give information about the brain cholesterol turnover. Cerebral and extracerebral factors should be carefully considered in the individual recruitment for cross sectional or longitudinal studies.

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The authors wish to gratefully acknowledge the collaboration along the years of Dr. C. Mariotti, Dr. L. Nanetti and Dr. S. Di Donato, Foundation IRCCS Institute of Neurology Carlo Besta, Milano, Italy; Dr. A. Nauti and P. Borroni, Ospedale di Circolo and Fondazione Macchi, University of Insubria, Varese; Dr. M. Valenza and prof. E. Cattaneo at University of Milano, Italy. A special mention is given to the scientific support and encouragement of Prof. I. Björkhem, Karolinska Insitutet, Stockholm, Sweden. Financial support: Italian Mininster of Health, Fondi per giovani Ricercatori 2008 (GR-2008-1145270), to V. Leoni.

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References

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hypercholesterolemia), metabolic syndrome and diabetes are associated with increased LDL and decreased HDL cholesterol, increased triglycerides, increased sterol precursors, decreased plantosterols and significant changes in plasma oxysterol concentrations [29,32,33,101,103, 106–108]. The treatment with inhibitors of cholesterol synthesis (i.e. statins), as the pravastatin at low dosage (40 mg/day), reduced cholesterol by ~20%, lathosterol ~20% and 24OHC ~14% but 24OHC/cholesterol ratio was increased (+15%) in healthy volunteers [123]. Even the collection time (after overnight fasting vs post-prandial blood collection) significantly affect the levels of oxysterols (as 24OHC) and sterols [32,33,39,100]. Finally, the selection criteria of the reference population seems to be very important in biomarker studies: we previously discussed that total, HDL and LDL cholesterol are reduced in HD subjects as well as in familial control when compared to healthy volunteers sampled from the general population [99]: these findings suggest that familial controls are not the more appropriate population for metabolomic or metabolic studies in HD. Despite the promising results about the 24OHC reduction in plasma, it is likely that the quantification of 24OHC as surrogate biomarker in the study of HD has a limited power due to the effect of extracerebral factors able to significantly affect its plasma levels which depend on the equilibrium between brain cholesterol turnover and excretion, plasma lipoproteins transport and turnover, liver clearance and metabolism.

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In a recent investigation [99], the plasma cholesterol levels were significantly reduced in both pre-manifest and manifest HD subjects when compared with a non-familial-aged matched control group representative of the general adult population. This significance was also seen in the manifest HD patients when compared with the familial controls. With respect to the HDL-cholesterol, the levels among the HD and familial control groups were comparable. The LDL-cholesterol was reduced in both pre-manifest and manifest HD subjects when compared to the general population but not to the familial group. No differences in triacylglycerols or free fatty acids levels were observed. These results support the hypothesis of an impairment of the whole body cholesterol metabolism in HD subjects and also suggest that a careful selection of the control population in metabolomic studies is required. Almost all the 24OHC in human plasma has cerebral origin. Its concentration depends on the cholesterol turnover in the brain neuronal cells, the functionality of blood–brain barrier, the metabolism of plasma lipoproteins and the liver clearance [102,103]: thus, 24OHC was proposed as a surrogate marker for the number of metabolically active neurons located in the gray matter of the brain [43,104–106]. According to this hypothesis, plasmatic 24OHC was decreased in AD, vascular dementia, Parkinson disease, MS and HD proportionally to the disease burden, the loss of metabolically active neuronal cells measured as MRI atrophy of gray structures, the functional impairment or the disease severity scores [36,100,101,106–114]. It was supposed that at an early stage of disease, a transitory higher membrane turnover may result into a larger amount of 24OHC secreted in plasma [115–118] and that in a more advanced stage, the plasma 24OHC will be reduced due to a progressive decrease of cholesterol turnover secondary to the neuronal loss. These two phases may be overlapped, resulting in an unclear profile [103,119]. The decrease of plasma 24OHC in the HD patients was proportional to the degree of caudate atrophy (measured as reduction of caudate volume at MRI) and to the motor impairment [100]. A progressive reduction of plasma 24OHC related to the HD progression was found in three progression groups (ranked as low, medium, high, calculated as CAP score = (Age at PREDICT-HD entry) × (CAG − 33.66) [120]) when compared to gene-negative control group. The patients in the low group had a mean plasma concentration of 24OHC higher than controls, medium and high groups. The differences for 24OHC concentration between groups showed a decreasing gradient, from the low group through the high group. The highest progression group had the most substantial (and significant) difference relative to the controls and to the lowest progression group. This evidence of a progression gradient is consistent with the pattern of findings in other studies adopting several psychological, neurological and MRI measurements of the structural atrophy to which 24OHC was significantly correlated [85,121,122]. The reduction of 24OHC observed does qualitatively mirror the progression of striatum atrophy and positively correlates with striatal volumes [118]: at an early stage the cyto-architectural and the synaptic rearrangements in the HD brain may result into a higher cholesterol turnover. These rearrangements include the prodromal participants for whom the morphology is altered compared with the control participants possibly because of pre-existing developmental abnormalities [86]. The different processes involved in pathogenesis of HD (toxic gain of function and loss of function of Huntingtin, mitochondria dysfunction, bioenergetic generation, transcriptional dysregulation) result into neuronal cell death, functional impairment and MRI brain atrophy [85]. Together with the activation of glia and extraneuronal expression of CYP46A1, the inflammatory response and the blood–brain barrier dysfunction may play a metabolic effect on brain cholesterol homeostasis [103]. The plasma 24OHC is very high in children, decreases in teenagers and becomes rather constant between the third to the sixth decade of life tending to increase constantly in more advanced age [106]. The concentration of 24OHC was reverse correlated to the body surface area and the liver dimension [11,106]. Diet, common forms of dyslipidemia (like familial combined hypercholesterolemia or polygenic

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Triheptanoin improves brain energy metabolism in patients with Huntington disease.

Study of cholesterol metabolism in Huntington's disease.

Role of cholesterol metabolism in the pathogenesis of Alzheimer's disease.

The role of cholesterol metabolism in Alzheimer's disease.

Huntington Disease in Asia.

Abnormalities in the tricarboxylic Acid cycle in Huntington disease and in a Huntington disease mouse model.

Aberrant palmitoylation in Huntington disease.

Clinical neurogenetics: huntington disease.

The end in sight for Huntington disease?

Early Detection of Huntington Disease.

Feasibility of Huntington disease trials in the disease prodrome.

Impairment of drug metabolism in polycystic non-parasitic liver disease.

Huntington disease and the abuse of genetics.

Genetic analysis of Huntington disease in Italy.

Regional impairment of cerebral oxidative metabolism in Parkinson's disease.

Letter: Schilder's disease: cholesterol metabolism in cultured fibroblasts.

Ubiquinone, dolichol, and cholesterol metabolism in aging and Alzheimer's disease.

The role of peroxisomes in cholesterol metabolism.

Cholesterol metabolism in man.

Complex patterns of linkage disequilibrium in the Huntington disease region.

Rectifying rectifier channels in Huntington disease.

Huntington disease in black African populations.

Predictive testing for Huntington disease.

Cloning of the Huntington disease region in yeast artificial chromosomes.