Cholesterol Metabolism in Huntington Disease

NATURE REVIEWS | NEUROLOGY VOLUME 7 | OCTOBER 2011 | 561

Centre for Molecular Medicine and Therapeutics, University of British Columbia, 950 West 28th Avenue, Vancouver, BC V5Z 4H4, Canada (J. M. Karasinska, M. R. Hayden).

Correspondence to: M. R. Hayden [email protected]

Cholesterol metabolism in Huntington diseaseJoanna M. Karasinska and Michael R. Hayden

Abstract | The CNS is rich in cholesterol, which is essential for neuronal development and survival, synapse maturation, and optimal synaptic activity. Alterations in brain cholesterol homeostasis are linked to neurodegeneration. Studies have demonstrated that Huntington disease (HD), a progressive and fatal neurodegenerative disorder resulting from polyglutamine expansion in the huntingtin protein, is associated with changes in cellular cholesterol metabolism. Emerging evidence from human and animal studies indicates that attenuated brain sterol synthesis and accumulation of cholesterol in neuronal membranes represent two distinct mechanisms occurring in the presence of mutant huntingtin that influence neuronal survival. Increased knowledge of how changes in intraneuronal cholesterol metabolism influence the pathogenesis of HD will provide insights into the potential application of brain cholesterol regulation as a therapeutic strategy for this devastating disease.

Karasinska, J. M. & Hayden, M. R. Nat. Rev. Neurol. 7, 561–572 (2011); published online 6 September 2011; doi:10.1038/nrneurol.2011.132

IntroductionHuntington disease (HD) is an autosomal dominant disease that leads to progressive neurodegeneration. The molecular cause of HD is the expansion of a CAG trinucleo tide repeat in the gene encoding huntingtin (HTT), resulting in a polyglutamine stretch in the Nterminus of the protein. The neuropathology of HD is charac terized by the selective loss of medium spiny neurons from the striatum, leading to neuropsychiatric changes and movement disorder, including chorea.1 HD pathogenesis has been under intense investigation since the discovery of HTT mutations around 20 years ago. Various cellular mechanisms underlying neuronal death have been identified, including Nmethyldaspartate receptor (NMDAR)mediated excitotoxicity,2,3 impaired axonal transport,4,5 and caspase activation,6 and these pathways are the focus of research into potential therapies. Recently, disturbances in cholesterol homeostasis have been described in patients with HD and animal models of the disease, raising the question of how changes in cholesterol metabolism might influence the development of HD.

Cholesterol is critical to brain development, myelination, and neuronal signaling and survival, and the maintenance of balanced cholesterol homeostasis is an important aspect of CNS function.7,8 The significance of brain cholesterol is emphasized by the detrimental impact of genetic defects affecting cholesterol synthesis or cellular transport on brain development and neuro degeneration, as manifest in Smith–Lemli–Opitz Syndrome (SLOS) and Niemann–Pick type C (NPC) disease. In SLOS, loss of enzymatic activity of 7 dehydrocholesterol (7DHC) reductase leads to failure of cholesterol synthesis, resulting in brain malformations and impaired cognitive function,

among other symptoms.9 NPC disease is characterized by lossoffunction mutations in the gene encoding NPC1, a protein involved in lysosomal cholesterol transport. This defect leads to progressive neurodegeneration.10 Disturbances in cellular cholesterol metabolism, plasma membrane cholesterol composition and intercellular transport have also been linked to the etiology of Alzheimer disease (AD; Table 1).11,12

Cellular cholesterol is derived from de novo syn thesis and the diet. Organ and blood cholesterol levels are maintained by a tightly regulated balance of syn thesis, catabolism, excretion and uptake.13 However, tissue and—in particular—plasma cholesterol levels can be dramatically disturbed by mutations in genes encoding proteins involved in one of the cholesterol metabolic pathways, or by high dietary cholesterol intake. For example, lossoffunction mutations in the gene encoding ATPbinding cassette transporter A1 (ABCA1), which facilitates cellular cholesterol efflux to apolipoprotein acceptors, reduce plasma HDL levels by up to 95%.14 Lossoffunction mutations in the PCSK9 gene, which encodes proprotein convertase subtilisin/kexin type 9 serine protease, lead to a decrease of up to 40% in plasma levels of LDL.15 A Westerntype diet high in fat raises plasma LDL. Fluctuations in blood and tissue cholesterol levels are evident in various human diseases, including atherosclerosis and diabetes.16,17

By understanding how changes in cellular cholesterol homeostasis affect CNS diseases, including HD, we will substantially enhance our knowledge of the role of cholesterol in influencing the delicate balance between life and death in neuronal cells, in addition to identifying potential new avenues for treatment. This Review will summarize the roles of cholesterol in the CNS and describe the current state of knowledge of cholesterol dysregulation in HD.

Competing interestsThe authors declare no competing interests.

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Brain cholesterol homeostasisUnesterified cholesterol accounts for most of the brain’s sterol content, twothirds of which is present in myelin and the rest of which resides in cellular plasma membranes.7,8 The brain has higher levels of cholesterol (15–20 mg/g) than any other organ, and in humans contains onequarter of the total body cholesterol, despite accounting for less than 5% of the total body weight.8

The protective blood–brain barrier (BBB) consists of tight junctions between the endothelial cells of brain capillaries that severely restrict intercellular and transcellular transport. The BBB separates the CNS from the circulation and is impermeable to plasma components including cholesterol (Figure 1),8,18 thereby preventing fluctuations in brain cholesterol in response to changing levels in the circulation. No significant cholesterol and lipoprotein transport occurs between the plasma and the brain under normal physiological conditions.8,19 How ever, the brain capillary endothelial cells express lipid transport proteins, including the cholesterol efflux transporters ABCA1 and ABCG1, the HDL receptor scavenger receptor class B member 1, and members of the LDL receptor (LDLR) family (Figure 1),20–23 and evidence is emerging from genetically manipulated mice that under some conditions cholesterol and lipoprotein

Key points

■ The brain is a cholesterol-rich organ, and cholesterol has a critical role in brain development, synaptogenesis, and neuronal activity and survival

■ Disturbances in brain cholesterol homeostasis are implicated in neurodegeneration

■ Attenuated plasma cholesterol levels, reduced brain cholesterol synthesis and cholesterol accumulation in neuronal plasma membranes are observed in patients with Huntington disease (HD) and in animal models of the disease

■ Multiple mechanisms are proposed to underlie cholesterol dysregulation in HD, including impaired activation of sterol regulatory element-binding protein, which regulates cholesterogenic gene expression, and reduced brain-derived neurotrophic factor signaling

■ Disturbances in cholesterol homeostasis may influence neuronal survival and susceptibility to excitotoxicity

■ Targeting of cholesterol metabolism represents a potential avenue for treatment to alleviate some neuronal impairments in HD

can be transported by the BBB.24–26 Although such transport may not markedly affect wholebrain cholesterol content, when one considers the extensive nature of the human brain vasculature (encompassing a 20 m2 surface area)18 the possibility arises that even small localized changes in brain cholesterol and lipoprotein uptake could have serious implications for neuronal health in affected brain areas.

Once the BBB is formed during embryonic development, the brain sterol pool becomes separated from the periphery, and brain cholesterol is synthesized in situ.7,8 All types of brain cells including neurons and glia (astrocytes, microglia and oligodendrocytes) synthesize cholesterol during development. It has been proposed, however, that cholesterol synthesis is attenuated in adult neurons, which then rely mostly on astrocytederived cholesterol.7,27 If cholesterol synthesis is ablated in adulthood, neurons can still survive, presumably by importing cholesterol from neighbouring glia.28 By supplying cholesterol, glial cells make a vital contribution to neuronal growth and activity. Astrocytederived cholesterol is required for optimal synaptogenesis and synaptic activity.29,30 Astrocytes synthesize and secrete cholesterol and apolipoprotein E (ApoE) to form discoidal lipoprotein particles in a process regulated by the activity of ABC transporters (Figure 1). These ApoEcontaining particles are lipidated and become similar in size and density to peripheral HDL,31 and can be taken up by neurons via members of the LDLR family, which recognize ApoE as a ligand.32 LDLRmediated uptake of ApoEcontaining particles facilitates axonal and dendritic growth33,34 and is implicated in the recovery from peripheral nerve injury, during which excess cholesterol from membrane debris is cleared or redistributed to regenerating nerves.35,36 In animal models of brain injury, ApoE and ABCA1 expression37,38 is increased, presumably to facilitate this transport.

Cholesterol synthesis slows down significantly in the adult brain, and cholesterol turnover in the human brain is estimated at approximately 5 years, compared with a few months in the rodent brain.7,8 CNS cholesterol levels are strictly maintained by de novo synthesis and excretion. Excess brain cholesterol is metabolized

Table 1 | Cholesterol abnormalities in neurodegeneration

Disorder Cholesterol abnormalities Mechanisms

Smith–Lemli–Opitz syndrome

Cholesterol synthesis failure, accumulation of 7-dehydrocholesterol9

Loss-of-function mutations in 7-dehydrocholesterol reductase, an enzyme that converts 7-dehydrocholesterol to cholesterol9

Niemann–Pick type C disease

Endosomal and lysosomal cholesterol accumulation10

Loss-of-function mutations in NPC1, a protein required for transport of cholesterol from endosomes and lysosomes10

Alzheimer disease

Risk associated with plasma cholesterol profile; cholesterol accumulation in plaques; increased production of 24-hydroxycholesterol11,48

APOE ε4 allele risk factor; increased amyloid generation due to membrane cholesterol accumulation; reduced amyloid degradation due to decreased Apo-E lipidation11,12,42,43

Huntington disease

Attenuated brain sterol biosynthesis; reduced plasma cholesterol; reduced production of 24-hydroxycholesterol;51–53,59 cholesterol accumulation54

Reduced activation of SREBP leading to reduced HMG-CoAR transcription; reduced BDNF signaling;5,53 caveolin sequestration at plasma membrane; increased lipid rafts; neuronal cholesterol accumulation54,66,67

Abbreviations: Apo-E, apolipoprotein E; BDNF, brain-derived neurotrophic factor, HMG-CoAR, 3-hydroxy-3-methylglutaryl-CoA reductase, NPC1, Niemann–Pick C1 protein; SREBP, sterol regulatory element-binding protein.

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by the brainspecific enzyme cholesterol 24hydroxylase (CYP46, encoded by the CYP46A1 gene) to 24hydroxycholesterol (24OHC),39 which crosses the BBB.40 CYP46 is highly expressed in neurons, suggesting that 24OHC production is the main pathway for cholesterol excretion by neurons.7,8,39 The precise control of brain cholesterol content is evident in mice in which Cyp46a1 is disrupted: although CYP46deficient mice are unable to convert cholesterol to 24OHC, their brain cholesterol levels remain constant owing to compensatory suppression of synthesis.41 The rigorous control of brain cholesterol levels demonstrates the need to maintain optimal cholesterol content and indicates that any imbalance of cholesterol homeostasis in the HDaffected brain could influence neuronal function and survival.

Cholesterol and neurodegenerationAbundant evidence links changes in cholesterol metabolism to neuropathological features of AD.11,12 The APOE ε4 allele is a strong risk factor for lateonset AD,42 indicat ing that pathways regulating brain lipoprotein and cholesterol transport influence the pathogenesis of AD. Degradation of amyloidβ (Aβ) peptide, the main constituent of plaques found in the brains of indivi duals with AD, is facilitated by lipidated ApoE,43 suggesting that efficient transport of cholesterol to ApoE helps

prevent plaque deposition. Cholesterol also influences the processing of amyloid precursor protein (APP). Low plasma membrane cholesterol is associated with reduced Aβ generation through inhibition of the activity of βsecretase, the enzyme responsible for the generation of Aβ from APP.12 ABCA1 polymorphisms are associated with an altered risk of AD.44,45 Recently, genomewide association studies reported that variants at ABCA7, which encodes another ABC transporter expressed in the brain and involved in lipid efflux, are associated with AD,46,47 providing further evidence for lipid abnormalities in patients with AD. Interestingly, higher plasma HDL levels are associated with a reduced risk of developing this disease.48 The mechanisms underlying the hypothetical link between altered plasma lipid profiles and neurodegeneration are unclear, but dyslipidemia may coexist with other risk factors for dementia.

The evidence that cholesterol abnormalities markedly influence AD underscores the importance of investigating cholesterol metabolism in other neurodegenerative diseases, including HD.

Cholesterol metabolism in HDEvidence from human studiesInterest in the role of cholesterol homeostasis in HD is a relatively recent phenomenon. Early investigations in

Figure 1 | Cholesterol metabolism and transport in the CNS. Cellular cholesterol is synthesized from acetyl-CoA in a multistep pathway, with HMG-CoAR representing the rate-limiting step. In the adult brain, cholesterol synthesis is attenuated in neurons that rely on astrocyte-derived cholesterol. Cholesterol and Apo-E synthesized in astrocytes are secreted in an ABCA1-dependent process, forming discoidal lipoprotein particles, which can be further lipidated. Apo-E is a ligand for LDLR family members, which mediate neuronal lipoprotein uptake, thereby providing a supply of cholesterol to neurons. Excess cholesterol is metabolized to 24-OHC, which passes into the circulation. 24-OHC is a ligand for LXRs, which activate Apo-E and ABCA1 expression. No appreciable transfer of cholesterol from plasma HDL or LDL occurs under normal conditions, but the BCECs express cholesterol uptake proteins and transporters, including ABCA1, SRB1 and LDLR. mHtt inhibits HMGCoAR expression and activity, resulting in reduced cholesterol synthesis. Consequently, 24-OHC production might be reduced and expression of Apo-E and ABCA1 might be attenuated in an attempt to maintain cellular cholesterol levels. Abbreviations: 24-OHC, 24-hydroxycholesterol; ABCA1, ATP-binding cassette transporter A1; Apo-E, apolipoprotein E; BCEC, brain capillary endothelial cells; CYP46, cholesterol 24-hydroxylase; HMG-CoAR, 3-hydroxy-3-methylglutaryl-CoA reductase; LDLR, LDL receptor; mHtt, mutant huntingtin; LXR, liver X receptor; SRB1, scavenger receptor class B member 1.

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small groups of patients with HD did not indicate any lipid abnormalities. Specifically, no significant changes were observed in plasma concentrations of lipids, including cholesterol.49 In addition, fibroblasts from patients with HD showed no evidence of alterations in cholesterol content and activity of 3hydroxy3methylglutarylCoA reductase (HMGCoAR), the ratelimiting step enzyme in the sterol synthesis pathway (Figure 2).50

By contrast, morerecent studies, which demonstrated altered cellular cholesterol synthesis and metabolism in HD patients and cell and animal models of HD, suggest that cholesterol homeostasis is impaired in HD. Blood

cholesterol levels are reduced in patients with latestage HD,51 although whether this a direct effect of the HTT mutation or a result of latestage metabolic changes remains to be established. Since direct measurements of brain cholesterol content in living individuals are not feasible, data are mostly generated from cell lines and postmortem tissue. Expression of mRNA for cholesterol biosynthetic genes, including those encoding HMGCoAR and 7DHC reductase, was reported to be reduced in a mutanthuntingtinexpressing cell line.52 A subsequent study reported reduced mRNA levels of HMGCoAR, lanosterol 14 αdemethylase (CYP51) and 7DHC reductase in fibroblasts and postmortem striatal and cortical tissue from patients with HD, and found that HD fibroblasts have a reduced ability to stimulate de novo cholesterol synthesis (Figure 2).53 Although sterol biosynthesis is reduced in the brains of patients with HD, one group reported elevated cholesterol content in the caudate in this disorder.54

Two mechanisms could account for increased cholesterol content in the presence of reduced synthesis: first, a reduction in cholesterol conversion to 24OHC, and second, an increase in brain cholesterol uptake from plasma to compensate for decreased cholesterol availability from de novo synthesis. Whether sterol uptake from plasma is altered in patients with HD is not known. Brain cholesterol turnover to 24OHC is estimated by measuring plasma 24OHC levels, in view of the brainspecific distribution of CYP46, the enzyme responsible for 24OHC generation.39,41 Plasma and cerebrospinal fluid (CSF) 24OHC levels are reported to be altered in patients with AD or multiple sclerosis, raising the possibility that circulating 24OHC levels may act as a biomarker for disease severity and progression in these conditions.55–58 CYP46 is expressed predominantly in neurons, and increased plasma 24OHC in AD and multiple sclerosis signifies induction of neuronal cholesterol catabolism, possibly owing to increased cholesterol excretion by degenerating neurons. By contrast, plasma 24OHC levels are reduced in patients with HD who exhibit motor symptoms and normal in premanifest patients.51,59 One explanation for reduced 24OHC in HD is a decrease in total neuronal cholesterol turnover as a result of reduced neuronal volume. However, the reduction in 24OHC does not change across different HD stages, despite a progressive decrease in caudate volume.59 Hence, the more likely explanation is that attenuated production of 24OHC in HDaffected brains is a result of inhibition of cholesterol excretion to compensate for reduced synthesis, in order to maintain normal brain cholesterol homeostasis.

Overall, human patient and tissue data demonstrate changes in body cholesterol metabolism and attenuation of brain cholesterol biosynthesis in HD, as well as a reduction in cholesterol excretion in the late stages of the disease.

Evidence from HD modelsUndoubtedly, the largest volume of data supporting a role for altered cholesterol metabolism in HD has been generated using cellular and animal models. However,

Acetyl-CoA+Acetoacetyl-CoA

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24-Hydroxycholesterol

Cholesterol 24-hydroxylase

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Desmosterol

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Figure 2 | Influence of mutant huntingtin on the cholesterol biosynthesis pathway. Endogenous cholesterol is synthesized from acetyl-CoA. The rate-limiting step involves the synthesis of mevalonate by HMG-CoA reductase. After the synthesis of the first sterol precursor lanosterol, the pathway diverges and cholesterol can be synthesized from the precursors desmosterol or lathosterol. In the brain, cholesterol is mainly metabolized by cholesterol 24-hydroxylase to 24-hydroxycholesterol. Mutant huntingtin leads to a reduction in the expression of some enzymes and in the levels of some sterols, all of which are indicated in blue. By contrast, both a reduction and an accumulation in cholesterol have been reported in cellular and mouse models of Huntington disease. Abbreviations: HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; PP, pyrophosphate.

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the interpretations offered by numerous studies create a complicated picture, with different groups reporting seemingly conflicting results.

One line of evidence implicates reduced sterol biosynthesis leading to reduced brain cholesterol content in HD animal models. The R6/2 transgenic mouse, which expresses exon 1 of the human HD gene including the polyglutaminecontaining domain, and the yeast artificial chromosome 128 (YAC128) transgenic mouse, which expresses the full mutant Htt gene with 128 CAG repeats, both exhibit decreased brain cholesterol synthesis.53,60–62 These two models differ substantially in disease onset and lifespan, with R6/2 mice developing progressive motor deficits from about 9 weeks of age and dying before 16 weeks of age,63 and YAC128 mice showing motor abnormalities starting at 2 months, neuro pathology at 9 months and, in male mice, a reduced life span of 12 months.64,65 Hence, decreased cholesterol syn thesis can be attributed to the presence of mutant huntingtin rather than a secondary effect of longterm neuronal or metabolic changes.

HMGCoAR, 7DHC reductase and CYP51 expression levels, and HMGCoAR activity were found to be diminished in the brains of symptomatic R6/2 mice, leading to reduced levels of cholesterol precursors including lanosterol and lathosterol (Table 2).53,61 However, striatal cholesterol content analysis yielded inconsistent results, with no change in levels until the late symptomatic stage in this mouse strain.53,61 The turnover of total cholesterol content in the brain takes several months in the mouse,7,8 and the absence of robust cholesterol changes in R6/2 mice is probably explained by their short lifespan. In the longerliving YAC128 mice, brain HMGCoAR activity, cholesterol precursors, cholesterol and 24OHC levels are reduced during the symptomatic stage,60,62 indicating that cholesterol synthesis and turnover are attenuated at a time when striatal atrophy is observed. Lathosterol, choles terol and 24OHC levels are also decreased in symptomatic HD knockin mice carrying the CAG

expansion within the endogenous Htt gene and a rat HD model carrying a truncated Htt fragment with 51 CAG repeats.62 However, cholesterol accumulation has been reported in YAC72 mice, which express the full mutant Htt gene with 72 CAG repeats and have milder motor behavioral and neuropathological abnormalities than YAC128 mice,65 and in HD knockin mice.54,66

The detailed analysis of several animal models mirrors data from human samples demonstrating reduced sterol biosynthesis in the presence of mutant huntingtin. However, another line of evidence implies a functional role for neuronal cholesterol accumulation in HD. When mutanthuntingtinexpressing neurons are isolated from the brain and grown in culture, they accumulate free cholesterol at the plasma membrane, and this process has been shown to contribute to excitotoxicity.54,66,67 Although the idea that reduced cholesterol synthesis and increased cellular cholesterol levels occur concomitantly and contribute to HD pathogenesis might seem counterintuitive, several mechanisms could explain these observations. As most cholesterol synthesis in the adult brain is thought to occur in glia, reduced sterol synthesis in HD brains may reflect cholesterol status in this particular cell type. Indeed, levels of HMGCoAR and 7DHC reductase are attenuated in HD astrocytes.62 In addition, reduced brain sterol synthesis does not preclude accumulation of cholesterol in single cells, especially if cellular membrane properties are altered (as discussed in the next section), or if cholesterol trafficking is impaired. For example, ABCA1 and ApoE, which are critical in cellular cholesterol efflux in the brain, are reduced in mutanthuntingtinexpressing astrocytes.62 Failure to efficiently remove cholesterol from the plasma membrane owing to reduced efflux could lead to neuronal cholesterol accumulation.

Several techniques can be used to assess sterol content in tissues and cells. Differences in methodologies affect the sensitivity of the measurements, and could underlie some of the variations in cholesterol levels reported by

Table 2 | Changes in plasma and brain cholesterol metabolism in HD

Study population or model Plasma Brain

Patients with HD Reduced levels of cholesterol, precursors lanosterol and lathosterol, and cholesterol metabolite 24-OHC51,59

Postmortem brain tissue:Reduced expression of enzymes involved in cholesterol synthesis, including HMG-CoAR, CYP51 and 7-DHC reductase53

Increased caudate cholesterol content54

R6/2 mouse model NA Reduced expression of HMG-CoAR, CYP51 and 7-DHC reductase53

Reduced HMG-CoAR activity61

Reduced levels of lanosterol and lathosterol61

Reduced cholesterol in late neuropathological stages53

YAC128 mouse model Reduced 24-OHC, reduced total cholesterol and precursors60

Reduced HMG-CoAR activity60

Reduced levels of lanosterol, lathosterol, cholesterol and 24-OHC60,62

YAC72 mouse model NA Cholesterol accumulation in neuronsIncreased striatal cholesterol content66

Hdh knock-in mouse model NA Reduced lathosterol, cholesterol and 24-OHC62

Increased cholesterol54

Rat model NA Reduced lathosterol, cholesterol and 24-OHC62

Abbreviations: 7-DHC, 7-dehydrocholesterol; 24-OHC, 24-hydroxycholesterol; CYP51, lanosterol 14-α demethylase; HD, Huntington disease; Hdh, murine HD homolog; HMG-CoAR, 3-hydroxy-3-methylglutaryl-CoA reductase; NA, not available.

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separate groups. Striatal cholesterol was decreased in YAC72 mice as analyzed by gas chromatography–mass spectrometry,62 but increased cholesterol levels in the striatum were reported in YAC72 mice when measured by filipin staining and thinlayer chromatography (TLC),66 as well as in HD knockin mice when measured by an enzymatic assay.54 In general, filipin staining is difficult to quantify and, owing to rapid photobleaching, is the least reliable method to measure changes in cholesterol content.68 Gas chromatography, TLC and enzymatic assays can also differ in their measurements of free and esterified cholesterol and cholesterol oxidation products.69–71 To gain a better understanding of specific changes in cholesterol metabolism in HD, therefore, it is important not only to assess brain and neuronal cholesterol content in HD models, but also to study other markers of cholesterol turnover such as sterol pre cursors and 24OHC, as well as intracellular cholesterol distribution.

Taken together, human and animal data demonstrate reduced cholesterol biosynthesis and turnover in HDaffected brains, and cholesterol accumulation in mutanthuntingtinexpressing neurons. Most of the changes in cholesterol synthesis are described in symptomatic animals, suggesting that they occur as a result of mutanthuntingtininduced cellular damage rather than acting as a contributing factor to neuronal death. However, support for involvement of huntingtin in choles terol synthesis is provided by the finding that in contrast to YAC128 mice, YAC18 mice, which over express wildtype human HTT with 18 CAG repeats, show increased HMGCoAR expression and activity.60,62 These observations suggest that the cholesterol synthesis pathway is facilitated by wildtype huntingtin but impaired by mutant huntingtin.

Mechanisms of cholesterol change in HDHuntingtin is a ubiquitously expressed, 348 kDa intracellular protein, the highest levels of which are detected in neurons and the testes. Huntingtin is found in several intracellular compartments, including the nucleus, endoplasmic reticulum, Golgi complex and mitochondria, and it also associates with clathrincoated vesicles, caveolae and microtubules.72,73 This extensive intra cellular distribution suggests involvement in many cellular processes. Huntingtin also has many interacting partners,73 which further broadens its potential influence on cell function. Huntingtin has been implicated in embryonic development, neurogenesis and neuronal survival, and at the molecular level it influences gene transcription, neuronal vesicle transport and endocytosis, and post synaptic activity.72,74

Huntingtin may influence the expression of cholestero genic genes through its interaction with specific transcription factors. For example, huntingtin interacts with Sp1,74 a transcription factor also known to co operate with sterol regulatory elementbinding protein (SREBP),75 which activates gene transcription when cellul ar ch olesterol is depleted.

Reduced cholesterol synthesisLow levels of cellular cholesterol lead to induction of sterol synthesis by activating SREBPs, which control the express ion of genes encoding cholesterogenic enzymes, includ ing HMGCoAR.76 The SREBP family has three mem bers, SREBP1a, SREBP1c and SREBP2, which form a complex with the SREBP cleavageactivating protein (SCAP) in the endoplasmic reticulum mem brane (Figure 3). Under lowcholesterol conditions, the SREBP–SCAP complex translocates to the Golgi apparatus

SREBPSCAP

Cytoplasm

Golgi

High cholesterol

ER lumen

SRE

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SREBP

SCAP

Cytoplasm

Golgi

Low cholesterol

mHtt

INSIG

ER lumen

SRE

NucleusSREBPactive

INSIG

Increasedcholesterolsynthesis

Figure 3 | Influence of mHtt on SREBP-dependent cellular cholesterol synthesis. When cellular cholesterol is high, the SREBP–SCAP complex is retained in the ER membrane by the INSIG proteins, leading to inhibition of sterol synthesis. Under low-cholesterol conditions, INSIG dissociates from the SREBP–SCAP complex, which is then transported to the Golgi membrane. The active form of SREBP is cleaved and translocates into the nucleus, where it activates the expression of genes containing the SRE in their promoter region, including genes involved in cholesterol synthesis. In the presence of mHtt, inhibition of SREBP activation is observed when cellular cholesterol levels are low, resulting in reduced cholesterol synthesis. The details of the mechanism underlying reduced SREBP activation are unknown. Abbreviations: ER, endoplasmic reticulum; INSIG, insulin-induced gene; mHtt, mutant huntingtin; SCAP, SREBP cleavage-activating protein; SRE, sterol regulatory element; SREBP, sterol regulatory element-binding protein.

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membrane, where SREBP is activated by proteo lysis, releasing the active Nterminal domain into the cytosol.76,77 This protein fragment enters the nucleus and induces transcription of genes containing the sterol regulatory element (SRE) in their promoter region. Levels of the active SREBP fragment are reduced in the nucleus and SRE induction is impaired in mutanthuntingtin expressing cells cultured in delipidated serum, suggesting that mutant huntingtin interferes with SREBP activation.53 Reduced SREBP activation leads to decreased expression of HMGCoAR and, consequently, attenuated cholesterol synthesis, as observed in HD. INSIG1 and INSIG2 are SCAPinteracting proteins that retain the SREBP–SCAP complex in the endoplasmic reticulum in the presence of high cellular sterol concentrations. Excess cellular cholesterol inhibits HMGCoAR activity in two ways: by promoting the interaction of INSIG1 and INSIG2 with SCAP, thereby retaining the SREBP–SCAP complex in the endoplasmic reticulum and inhibiting HMGCoAR transcription, and through INSIG1mediated degradation of HMGCoAR protein.77,78 Hence, cholesterol accumulation in mutanthuntingtinexpressing cells may lead to reduced sterol synthesis through both impairment of SREBP activation and INSIGmediated HMGCoAR inhibition (Figure 4).

Cholesterol synthesis is also influenced by brainderived neurotrophic factor (BDNF), which is secreted by cortical neurons projecting to the striatum and has an important role in neuronal survival (Figure 5). BDNF

stimulates neuronal de novo cholesterol synthesis.79 Huntingtin facilitates vesicular transport of BDNF along microtubules,5 and BDNF levels are reduced in postmortem striatal tissue from patients with HD and brains of HD mice.80 Hence, attenuated BDNF levels may contribute to decreased striatal cholesterol synthesis—a model that is consistent with the preferential reduction in sterol levels in the striatum of HD mice.60,62

Cellular cholesterol accumulationNeurons expressing mutant huntingtin accumulate choles terol despite downregulation of cholesterol synthesis, indicating that mechanisms other than increased choles terol production are in operation. As already discussed, reduced efflux and decreased conversion to 24OHC may partially contribute to cellular cholesterol accumulation. Data from in vitro experiments demonstrate that specific changes at the plasma membrane also lead to elevated cholesterol content (Figure 4). Lipid rafts are cholesterolrich membrane domains that cluster the distribution of proteins, including neuro transmitter receptors, thereby forming signaling platforms that are essential for intracellular signal transduction.81 Caveolae, which are also rich in cholesterol, are membrane invaginations involved in cell signal ing and endocytosis.82 Mutanthuntingtinexpressing neurons show enhanced levels of the lipid raft marker ganglioside GM1, suggesting that membrane cholesterol accumulation is associated with an increased prevalence of lipid rafts.54 Caveolin1


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Figure 4 | Neuronal cholesterol accumulation in the presence of mHtt. Caveolin-1 is expressed in caveolae, cholesterol-rich plasma membrane invaginations involved in endocytosis. Caveolin-1 binds cholesterol, and endocytosis of caveolin-1 is inhibited by mHtt, possibly leading to membrane cholesterol accumulation. In addition, mHtt increases the presence of lipid rafts—membrane signaling platforms enriched in cholesterol. Membrane cholesterol accumulation in mHtt-expressing neurons may lead to increased cholesterol in the endoplasmic reticulum, resulting in retention of the SREBP–SCAP complex and inhibition of cholesterol synthesis. The details of the mechanism, however, remain to be established. Abbreviations: HMG-CoAR, 3-hydroxy-3-methylglutaryl-CoA reductase; INSIG, insulin-induced gene; mHtt, mutant huntingtin; SCAP, SREBP cleavage-activating protein; SREBP, sterol regulatory element-binding protein.

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is a protein found in caveolae that binds cholesterol.83,84 Mutant huntingtin impairs the internalization of caveolin1, increasing its distribution at the plasma membrane and thereby leading to accumulation of membrane cholesterol.66 However, whether concentration of caveolin1

results in increased membrane cholesterol in vivo is questionable, as increased cholesterol distribution to membrane compart ments in response to elevated membrane caveolin1 should stimulate cholesterol efflux in the presence of extra cellular acceptors.85 Efflux may relieve membrane choles terol excess, as indicated by reduced sterol content in synaptosomes—plasma membrane fragments containing presynaptic and postsynaptic components —isolated from symptomatic R6/2 mouse brains.62 The presence of smaller, lesslipidated lipoprotein particles in the CSF of HD mice,62 however, suggests that cho lesterol efflux to ApoE is reduced in HD brains, which may contribute to membrane cholesterol accumulation.

The evidence to date demonstrates that reduced choles terol synthesis and cellular cholesterol accumulation are both occurring in HD, and are connected through pathways that link cellular cholesterol levels to the regulation of its synthesis, catabolism and transport. One of the key challenges is to determine which aspect of cho les terol dysregulation predominantly influences neuronal function in HD.

Cholesterol and neuronal function in HDAn important question is whether changes in cho lesterol metabolism contribute to the pathogenesis in HD by influencing neuronal survival or neuroinflamma tion, or whether they exacerbate the consequences of known pathological mechanisms, including glutamate receptormediated excitotoxicity and disruption of axonal transport (Figure 6).

Neuronal function and survivalChanges in cellular and plasma membrane cholesterol content can have a marked impact on neuronal activity and survival under basal and excitotoxic conditions. Reduced cholesterol synthesis in HD could impair synapse maturation, neurotransmitter vesicle generation and synaptic activity, all of which strongly depend on the presence of cholesterol.27,30 ABCA1 and ApoE expression is decreased in YAC128 astrocytes, possibly to preserve intracellular cholesterol stores.62 However, the decrease in expression of these two proteins, which are critical for cholesterol transport, may compromise the astrocytetoneuron cholesterol shuttle, thereby limiting the amount of cholesterol delivered to neurons. ApoEcontaining lipoprotein particles are abnormally small in size in the CSF of YAC128 mice, indicating reduced cholesterol content.62 In addition to influencing basal neuronal activity, a decrease in ApoE and ABCA1 levels could impair cholesterol redistribution to injured neurons, which may render these cells more vulnerable to death.

The cholesterol content of synaptic vesicles is higher than that of other intracellular organelles.27 A reduction in cholesterol availability for optimal synaptogenesis could result in decreases in synaptic vesicle numbers and synaptic activity. Presynaptic activity, as measured by pairedpulse facilitation, is reduced in HD mice, indicating reduced neurotransmitter release.86,87 Dopa mine release is attenuated in brain slices from sympto matic R6/2 mice owing to a depletion of reserve

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Motorcomplex

BDNF vesicle

P


Cholesterol synthesis

Increased HMG-CoARexpression

Neuronal survival

Synaptic plasticity

Figure 5 | Huntingtin, BDNF trafficking and neuronal cholesterol synthesis. BDNF is released by cortical neurons projecting into the striatum. In addition to its recognized role in neuronal survival and synaptic plasticity, BDNF stimulates neuronal cholesterol synthesis via activation of Trk-B. Huntingtin promotes vesicular transport of BDNF vesicles along microtubules. This process is inhibited by mHtt, resulting in decreased BDNF levels in the striatum, which may be one of the pathways that contributes to reduced neuronal cholesterol synthesis in Huntington disease. Abbreviations: BDNF, brain-derived neurotrophic factor; HMG-CoAR, 3-hydroxy-3-methylglutaryl-CoA reductase; mHtt, mutant huntingtin; P, phosphate; Trk-B, BDNF/NT-3 growth factors receptor.

mHtt-inducedcellularcholesterolmetabolismchanges

In�uence oncellularfunction

In�uence onneuropathology

Glia

■ Reduced cholesterol synthesis■ Reduced cholesterol ef�ux

Neurons

■ Reduced cholesterol synthesis■ Reduced cholesterol ef�ux■ Reduced BDNF release■ Membrane cholesterol accumulation

■ Impaired cholesterol supply to neurons■ Impaired cholesterol- dependent response during neuronal injury■ Altered in�ammatory response

Neuroin�ammation

ExcitotoxicityReduced neuronalsurvival

■ Impaired generation of synaptic vesicles■ Increased NMDA receptors in lipid rafts■ Impaired caveolin-dependent endocytosis■ Impaired BDNF-dependent cholesterol synthesis

Figure 6 | Potential cholesterol-dependent pathways influencing neuropathology in Huntington disease. Several functions in glia and neurons are known to be dependent on cholesterol homeostasis. Changes in cellular cholesterol in mHtt-expressing glia and neurons can lead to discrete impairments in cell-type-specific functions (yellow-shaded boxes). Consequently, these impairments may influence the neuropathological features of Huntington disease, including neuronal survival, susceptibility to excitotoxicity, and neuroinflammation. Abbreviations: BDNF, brain-derived neurotrophic factor; mHtt, mutant huntingtin; NMDA, N-methyl-d-aspartate.

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vesicles available for mobilization.88,89 One should note that although vesicle generation and fusion depend on choles terol,27,84,90 vesicle content and release is also likely to be affected by the direct role of huntingtin in vesicular trafficking. Huntingtin interacts with proteins involved in endocytosis and vesicle trafficking, including HIP1, HAP1, kinesin heavy chain, and the p150Glued subunit of dynactin,5 and mutant huntingtin results in defective axonal vesicle transport.4

NeuroinflammationCellular cholesterol metabolism is controlled by liver X receptors (LXRs)—ligandactivated transcription factors that increase the expression of sterol transport genes in response to cellular cholesterol accumulation.91 LXR activation represses inflammation via inhibition of the nuclear factorκB pathway.92,93 LXR agonists inhibit microglial activation, reduce neuro inflammation and improve outcomes in mouse models of AD, NPC disease, experimental autoimmune encephalo myelitis, and focal cerebral ischemia.94–99 APOE and ABCA1 are LXR target genes, and reduced APOE and ABCA1 mRNA in astrocytes expressing mutant huntingtin, as well as reduced brain production of 24OHC, an endogenous LXR agonist, suggest that LXR activation is attenuated in HD. The presence of mutant huntingtin has also been associated with reduced LXR activation.100 Reduced LXR signaling leads to increased susceptibility to neuro inflammation and exacerbates neuropathology in AD mice,99 and may also play a part in neuronal death in HD, particularly since neuroinflammation is present in HD brains.101,102

ExcitotoxicityPlasma membrane cholesterol accumulation in mutanthuntingtinexpressing neurons is likely to disrupt signaling of neurotransmitter receptors distributed in lipid rafts. One proposed mechanism underlying neuronal death in HD is excitotoxicity mediated by NMDARs —ionotropic glutamate receptors that associate with lipid rafts.103 Increased distribution of NMDAR subunits to lipid rafts is observed in HD neurons,54 and post synaptic NMDAR activity is augmented in various HD mouse models.3,104–106 Mutant huntingtin also accumulates in lipid rafts with glycogen synthase kinase3β (GSK3β),107 which has been linked to tau phosphorylation, one of the neuropathological hallmarks of AD.108 GSK3β activity promotes neuronal death, which can be attenuated by membrane cholesterol depletion.109–111 GSK3β inhibitors reduce death of mutanthuntingtinexpressing neurons, suggesting that increased GSK3β activity contributes to neuronal demise in HD.107 Taken together, these findings indicate that increased presence of membrane cho lesterol and lipid rafts may directly contribute to glutamate receptormediated excitotoxicity as well as GSK3β activation in HD neurons.

Cholesterol as a therapeutic targetAnticonvulsants, neuroleptics and antidepressants are used to alleviate motor and behavioral symptoms in patients with HD,112 but no effective treatment that delays

onset or progression of the disease is currently available. After two decades of intense research into endogenous huntingtin function and mechanisms underlying the pathology of HD, however, specific targets for potential therapy are being identified. These targets include inhibition of NMDAR and caspase6 activity, as well as regulation of vesicular transport.6,112 NMDAR antagonism improves outcome in HD mice,113 and caspase6 inhibitors for testing in mouse models are under development.

Animal studies using drugs aimed at altering brain cholesterol synthesis or reducing membrane cholesterol accumulation should establish whether targeting of choles terol metabolism represents a potential avenue for treating some of the neuronal impairments in HD. If the main culprit influencing the disease is reduced cholesterol synthesis, compounds that are capable of stimulating the sterol synthesis pathway or increasing uptake of cellular cholesterol may prove beneficial to neurons. However, given that increased membrane cholesterol is found in HD neurons, stimulation of synthesis or uptake of the sterol would potentially result in further membrane cholesterol accumulation.

The use of cholesterolreducing agents also has potential caveats. Statins, which inhibit the activity of HMGCoAR, are widely used in the treatment of hypercholesterolemia, and have shown some neuro protective effects in neuro degenerative conditions, including AD, although the evidence from prospective studies is inconsistent.12,114 Simvastatin protects neurons from NMDAinduced excitotoxicity115 and reduces NMDA excitotoxicity in mutanthuntingtinexpressing cells by decreasing the prevalence of lipid rafts.54 Although statins have beneficial effects in neuronal culture experiments, however, a further decrease in HMGCoAR activity resulting in attenuated sterol synthesis may exacerbate neuro pathology in HD. Another major caveat is that although drugs such as statins show effects in vitro, their direct effects on cholesterol synthesis in the CNS have not been proven. In fact, the neuroprotective function of statins is sometimes attributed to their pleiotropic effects other than inhibition of cholesterol synthesis.116

The effectiveness of any drug designed to target brain cholesterol will depend on its ability to cross the BBB and influence brain cholesterol metabolism. The different manifestations of cholesterol dysregulation in HD suggest that one approach to reestablishing brain cholesterol homeostasis is to restore efficient cholesterol transport out of cells as well as between cells in the CNS. Activation of LXRs, which include the LXRα and LXRβ isoforms, increases expression of genes encoding proteins involved in cholesterol transport, including ABCA1, ABCG1 and ApoE.91 In cells that express mutant huntingtin, LXR activation should stimulate cholesterol efflux thereby normalizing membrane cholesterol content, facilitate cholesterol transport on ApoE between astrocytes and neurons, and stimulate cholesterol synthesis if low sterol levels are reached. These potential effects of LXR activation, as well as its recognized antiinflammatory function in neuro degenerative disease, indicate that the use of selective LXR agonists may be of interest in HD therapy.

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However, the benefits of longterm LXR activation with currently available agents are hindered by their induction of liver steatosis—a serious adverse effect—owing to increased lipogenesis via SREBP1c.117 One way to overcome this problem is to develop agonists that selectively target the LXRβ isoform, which is widely expressed in the brain. Mice lacking LXRα, which is expressed predominantly in the liver, do not exhibit LXRagonistinduced hepatic steatosis.118 The therapeutic potential of selective LXRβ activation is recognized in the treatment of athero sclerosis as well as neurodegeneration, but LXRβ agonists are not yet available for testing.

Recently, a novel approach to modification of cholesterol metabolism has been reported. MicroRNAs (miRNAs) are endogenous, small, noncoding RNA molecules that bind to target mRNAs, leading to translational repression, and are abundantly expressed in the brain.119 miR33 has recently been described as a regulator of cellular cholesterol metabolism because its activity reduces levels of the transporters ABCA1 and ABCG1.120,121 miR33 inhibitors increase HDL formation—a finding that has raised substantial interest in miR33 as a potential target in the treatment of atherosclerosis. Although most interest so far has been in the action of miR33 in the liver, miR33 is also highly expressed in the brain,120 suggesting that it could regulate cholesterol metabolism in the CNS. The widespread role of miRNAs in physiological processes makes them promising targets for drug develop ment, but whether miRNA targeting in the brain represents a valid therapeutic approach in neuro degeneration remains to be established.

ConclusionsData obtained over the past few years from humans and animal models indicate that cholesterol metabolism is altered in HD. Emerging evidence suggests that cholesterol changes in the presence of mutant huntingtin influence intercellular cholesterol transport, neuronal excitotoxicity and apoptosis. Changes in brain cholesterol homeostasis in HD underscore the importance of cholesterol in neuronal function and survival, and provide another example of the involvement of cholesterol dysregulation in neurodegeneration. As the detailed role of cholesterol metabolism in HD emerges, it becomes clear that future studies should aim to resolve the question of whether reduced sterol biosynthesis or neuronal cholesterol accumulation is the predominant mechanism contributing to HD pathogenesis in vivo. Studies in whole animals, investigating the effects of cholesterol regulation on neuronal survival, are needed to provide support for research into the potential benefits of cholesterol targeting therapy in HD.

Review criteria

PubMed and Google Scholar databases were searched using the terms “Huntington disease cholesterol”, “huntingtin cholesterol”, “brain cholesterol” and “cholesterol neurodegeneration”. Publications from all years were searched and were limited to full-text papers written in English. Additionally, the bibliographies of selected papers were searched for further references on specific subjects for background information where necessary.

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AcknowledgmentsThis study was supported by grants from the Canadian Institutes of Health Research (CIHR MOP-106684 and MOP-84438).

Author contributionsJ. M. Karasinska researched data for the article and wrote the text. J. M. Karasinska and M. R. Hayden contributed equally to discussions of content, and review and editing of the manuscript before submission.

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Cholesterol Metabolism in Huntington Disease