The Role of Brain Cholesterol and its Oxidized ...

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The Role of Brain Cholesterol and its Oxidized Products in Alzheimer's Disease Anna Maria Giudettia, Adele Romanob, Angelo Michele Lavecchiab and Silvana Gaetanib,* a

Department of Biological and Environmental Sciences and Technologies, University of Salento, Lecce, Italy. bDepartment of Physiology and Pharmacology “Vittorio Erspamer,” Sapienza University of Rome, Rome, Italy Abstract: The human brain is the most cholesterol-rich organ harboring 25% of the total cholesterol pool of the whole body. Cholesterol present in the central nervous system (CNS) comes, almost entirely, from the endogenous synthesis, being circulating cholesterol unable to cross the blood-brain barrier (BBB). Astrocytes seem to be more active than neurons in this process. Neurons mostly depend on cholesterol delivery from nearby cells for axonal regeneration, neurite extension and synaptogenesis. Within the brain, cholesterol is transported by HDL-like lipoproteins associated to apoE which represents the main apolipoprotein in the CNS.

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Although CNS cholesterol content is largely independent of dietary intake or hepatic synthesis, a relationship between plasma cholesterol level and neurodegenerative disorders, such as Alzheimer’s disease (AD), has often been reported. To this regard, alterations of cholesterol metabolism were suggested to be implicated in the etiology of AD and amyloid production in the brain. Therefore a special attention was dedicated to the study of the main factors controlling cholesterol metabolism in the brain. Brain cholesterol levels are tightly controlled: its excessive amount can be reduced through the conversion into the oxidized form of 24-S-hydroxycholesetrol (24-OH-C), which can reach the blood stream. In fact, the BBB is permeable to 24-OH-C as well as to 27-OH-C, another oxidized form of cholesterol mainly synthesized by non- neural cells. In this review, we summarize the main mechanisms regulating cholesterol homeostasis and review the recent advances on the role played by cholesterol and cholesterol oxidized products in AD. Moreover, we delineate possible pharmacological strategies to control AD progression by affecting cholesterol homeostasis.

Keywords: Alzheimer’s disease; brain, blood-brain barrier, cholesterol metabolism, oxysterols, statins, antioxidants. 1. INTRODUCTION Although blood components are maintained separated from the central nervous system (CNS) by the blood-brain barrier (BBB) there is evidence that plasma cholesterol may be involved directly or indirectly in the etiology of Alzheimer’s disease (AD) [1]. However, the relationship between cholesterol levels in plasma and the risk of developing AD is still a matter of discussion. In the present review, we summarize the main mechanisms regulating brain cholesterol homeostasis and the newly described relationship between cholesterol and its oxidized forms in the development of AD, emphasizing the possible effects of antioxidant and hypolipidemic drugs in this process. 1.1. Circulating Cholesterol and Lipoproteins Cholesterol is a lipid molecule that can be found in all animal tissues, especially in the brain, spinal cord and adi*Address correspondence to this author at the Department of Physiology and Pharmacology “V. Erspamer”, Sapienza University of Rome Address: Piazzale Aldo Moro, 5 – 00185 Rome, Italy; Tel.: +39 06 49912520; Fax: +39 06 49912480; E-mail: [email protected] 1567-2050/16 $58.00+.00

pose tissue and that plays numerous essential roles in the body [2]. Due to the lipophylic structure, cholesterol is transported in the blood mainly by lipoproteins, which are micellar aggregates formed by protein and lipid components [3]. The lipoproteins have a globular structure constituted by an outer sheet mainly made of phospholipids, cholesterol, and apoproteins and a hydrophobic lipid core containing neutral lipids such as esterified cholesterol and triglycerides [3]. These particles are stabilized by non-covalent bonds between the protein moiety and the lipids. The presence of non-covalent bonds allows the exchange of constituent lipids and proteins between the various types of lipoproteins and between the latter and cell membranes. Lipoproteins may differ in size and density depending on the quantitative protein/lipid ratio. An increased density corresponds to a decreased protein content and size. Based on increased their density plasma lipoproteins are grouped into four classes: a) Chylomicrons, originating from the intestine after a meal and containing high percentages of fats, with larger dimensions than the other classes;

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b) Very low density lipoproteins (VLDL) that have a high fat content (especially triglycerides) and a large size; c) Intermediate density lipoproteins (IDL) that derive from VLDL; d) Low density lipoproteins (LDL) which derive from IDL; e) High-density lipoprotein (HDL) with protein content higher than the other lipoproteins. The proteins associated to lipoprotein particles are known as apolipoproteins. There are 4 main types of apolipoproteins known as apolipoprotein (apo): A, B, C, and E; within each of these groups different subgroups have been described. Minor apolipoproteins include apo D, F, G, H, and J [3]. Apolipoproteins may function as cofactors (such as C-II for lipoprotein lipase - LPL and A-I for lecithin cholesterol acyltransferase - LCAT), as lipid transfer proteins, and ligands for lipoprotein receptors expressed by several tissues (such as apo B-100 for the LDL receptor, apo E for the remnant receptor, and apo A-I for the HDL receptor). Dietary lipids, in the form of chylomicrons, are transported to the liver where triglycerides, esterified cholesterol, and phospholipids are assembled with the apolipoprotein B100 (apoB100) and excreted in the plasma as VLDL. In the blood, lipoprotein lipase (LPL) transforms VLDL in LDL carrying about 70% of plasma cholesterol. The size of VLDL plays a role in the subsequent catabolic fate of VLDLs that can be transformed into LDL of heterogeneous composition and size. The cholesterol of LDL is both free and esterified. The latter one is more hydrophobic, and typically less toxic to the cell. The enzyme acyl-CoA cholesterol acyltransferase (ACAT) is responsible for the synthesis of cholesteryl esters [4]. “Nascent HDLs” are synthesized in the liver and intestine. In the circulation HDLs receive phospholipids and cholesterol interacting with ATP binding cassette transporter protein family member A1 (ABCA1) [5] present on the cell surface of hepatocytes and enterocytes. This process leads to the formation of "mature HDLs" rich in lipids. Unesterified cholesterol present on the surface of these lipoproteins is esterified by the enzyme LCAT [4] with formation of HDL with spherical shape. An aliquot of esterified cholesterol present on HDL is transferred to VLDL and LDL by that cholesteryl ester transfer protein (CETP) [6]. Liver is the main site of cholesterol metabolism; lipoproteins synthesized in the liver are mainly distributed to peripheral tissues and organs except for the brain. In fact, brain cholesterol is considered as a distinct pool from body cholesterol [7]. 1.2. Brain Cholesterol The importance of cholesterol in the brain comes from the consideration that although brain represents only the 2% of the entire body mass, it contains approximately 25% of the total amount of free cholesterol in the whole body pool [7]. Being cholesterol almost unable to cross the blood-brain barrier (BBB) the majority of brain cholesterol is exclusively derived from the de novo biosynthesis, rather than from plasma lipoproteins [8].

Although neurons can synthesize cholesterol, it has been suggested that neurons rely on delivery of cholesterol from nearby cells such as astrocyte, which are the main source of cholesterol in the central nervous system (CNS) [9]. Interactions between neurons and astrocytes in cholesterol homeostasis have been reported [10, 11] and are supported by the observation of a cholesterol shuttle from astrocytes to neurons [9]. Cholesterol delivered to neurons may support synaptogenesis and may be incorporated into synaptic vesicles [8] (Fig. 1). The synthesis of cholesterol in the brain starts from acetyl-CoA and involves the subsequent action of at least 20 enzymes [12]. The 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase; Hmgcr; EC 2.3.3.10), which converts 3-hydroxy-3-methylglutaryl-CoA in mevalonate, is the rate limiting enzyme in cholesterol biosynthesis and also the target of statin pharmacotherapy [13]. It has been demonstrated that similarly to other cells, HMGCoA reductase activity and sterol synthesis are inversely related to the levels of LDL cholesterol in the incubation medium [14, 15] of glial cell cultures. To maintain brain cholesterol steady-state, the excess of cholesterol in neurons can in part be stored within lipid droplets after esterification by ACAT1, or streamed from the brain [9] (Fig. 1). Although a little amount of cholesterol can be directly secreted into the cerebrospinal fluid (CSF) bound to apoE, a more quantitatively important mechanism of cholesterol efflux involves the conversion of cholesterol into the oxidized form of 24-S-hydroxycholesterol (24-OH-C) which can cross the BBB and reach the general circulation [16-19] (Fig. 1). 24-OH-C is synthesized by CYP46A1, an enzyme that, by adding a hydroxyl group to the 24 position, converts the hydrophobic molecule of cholesterol into a more lipophilic product that can cross the BBB by diffusion [9]. Brain is the major source of 24-OH-C, being CYP46A1 almost exclusively expressed by neuronal cells in the brain [20] and in the retina [21]. 24-OH-C is a potent activator of liver-X receptors (LXR), which play an important role in the regulation of cholesterol homeostasis [22, 23]. Moreover, these receptors have been shown to be also expressed in the brain [24] where they can stimulate the efflux of cholesterol from astrocytes by inducing the activation of ABCA1 [24] (Fig. 1). In addition, LXR in association with retinoid X receptors (RXR) and peroxisome proliferator-activated receptor gamma (PPAR-gamma) can transcriptionally regulate apoE [25] that, as above mentioned, represents the main apolipoprotein that conveys cholesterol in the CNS. Interestingly, the secretion of 24-OH-C seems to regulate the rate of new cholesterol synthesis in the brain, as suggested by the observation that in 24-hydroxylase knockout mice the synthesis of brain cholesterol is reduced by about 40% [26]. 1.3. Cholesterol Flux Among CNS Cells It has been reported that in the CNS the only lipoproteins present are HDL-like lipoproteins [9]. Moreover, unlike plasma HDLs that contain apoA-1 as major apolipoprotein, the predominant apolipoprotein of HDL in the CNS is apoE,

The Role of Brain Cholesterol and its Oxidized Products in AD

which mediates the primary mechanism of cholesterol transport and is involved in the receptor-mediated endocytosis of lipoprotein particles within the brain [27]. Furthermore, in the brain, besides apoE, HDL-like lipoproteins are also associated with, apoJ and apoA-I [27]. Brain and liver show the highest apoE expression. Within the brain the highest expression of apoE was observed in astrocytes and to some extent in microglia [27, 28]. Cholesterol synthesized by astrocytes is secreted in the form of HDL-like lipoproteins with apoE as the major apolipoprotein and the flux of cholesterol among CNS cells occurs via these lipoproteins. ABCA transporters, particularly ABCA1, are required for the shuttling of cholesterol from glial cells to neurons [9] (Fig. 1). Cholesterol uptake by neuronal cells is mediated by receptors of the LDL receptor family such as the LDL-receptor and LDL receptor-related protein (LRP) [9]. These receptors mediate the endocytosis of the apoEcontaining lipoprotein and the cholesterol released from these lipoproteins is used to support synaptogenesis, maintenance of synaptic connections and repairing damaged neuronal membranes [10] (Fig. 1).

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1.4. Brain Cholesterol in AD Increasing body of evidence suggests that alterations of cholesterol levels and cholesterol turnover in the brain might be involved in the etiopathology of neurodegenerative disorders, such as Alzheimer’s disease (AD) [8]. In particular, ApoE seems to represent the major link between alterations of cholesterol homeostasis in the brain and neuropathology development in AD [29]. One of the major neuropathological hallmarks of AD is the presence of neuritic plaques due to amyloid-beta peptide (Abeta) accumulation in parenchyma and cerebral blood vessels [30]. Abeta is a product of a sequential proteolytic cleavage of the amyloid precursor protein (APP) mediated by the beta-secretase 1, also known as beta-site APP cleaving enzyme (BACE-1) and a gamma-secretase [30]. A large body of evidence demonstrates that, besides controlling cholesterol homeostasis in the brain, apoE is able to regulate Abeta aggregation and clearance in the brain [29]. In humans, the polymorphic apoE gene is present in three different allelic isoforms (2, 3, and 4) [29]. Among these isoforms, the apoE ε4 allele leads to the production of an apoE isoform (apoE4) that has a low efficiency in binding and transporting Abeta [31-34]. At least 50 % of patients with AD possess at least one apoE ε4 allele [35, 29] and apoE4 has been shown to increase the risk of developing AD with each inherited copy [35, 36]. Studies conducted on murine models of AD have shown that ABCA1 plays an important role in the etiopathology of AD: in fact, the whole degradation pathway of the Abeta peptide slows down in the absence of ABCA1 [37]. Interestingly, the clearance rate of Abeta is strongly affected by the amount of lipid carried on apoE3. Thus, saturation of apoE3, by increased ABCA1 activity, could induce a more rapid degradation of Abeta peptides and less amyloid formation [38].

Fig. (1). Interactions between neurons and astrocytes in cholesterol homeostasis. Cholesterol synthesized in the astrocytes from acetyl-CoA throughout the HMG-CoA reductase pathway is transported to neurons. The mechanism of cholesterol delivery requires the intervention of ATP binding cassette transporter protein family member A1 (ABCA1) and the association of cholesterol to apoEcontaining HDL. The uptake of cholesterol by neurons is mediated by LDL-receptor (LDLR). Cholesterol in the neurons supports synaptogenesis maintains synaptic connections and can be used to repair damaged neuronal membranes. Moreover, neuron cholesterol can be in part accumulated in intracellular droplets or expelled from neurons essentially in the form of 24-S-hydroxycholesterol (24-OHC), which is synthesized by CYP46A. 24-OH-C is a potent activator of liver-X receptors (LXR), which in turn are able to stimulate the efflux of cholesterol from astrocytes by inducing the activation of ABCA1. 24-OH-C, 27-OH-C, whose synthesis takes place out of brain via CYP27A, and 7α-hydroxy-3-oxo-4-cholestenoic acid (7αOH), which is produced by neuron metabolism of 27-OH-C, can cross the brain blood barrier and be eliminated in the bile products.

In contrast to what happens in the whole body, about 99% of cholesterol in the CNS is present in an unesterified form [9]. A little amount of cholesterol in the CNS may be esterified by ACAT1 and, in the form of cholesteryl esters, can either accumulate in intracellular droplets or can efflux through the plasma membrane into the extracellular environment [39]. Interestingly, it has been demonstrated that the balance between free and cholesterol ester can control amyloidogenesis. To this respect increasing levels of cholesterylesters enhances Abeta release in cultured cells, while pharmacological inhibition or genetic ablation of ACAT1 leads to the reduction of Abeta [40-42]. A possible interpretation could be that an increased availability of free cholesterol, caused by ACAT1 inhibition, can produce more 24-OH-C, thus increasing cholesterol clearance from the CNS [43]. Most of the brain free unesterified cholesterol is localized in the specialized membrane of myelin sheaths of oligodendroglia, whereas the remaining it is found in the plasma membrane of neurons, astrocytes and extracellular lipoproteins [8]. The particular high level of cholesterol in myelin is consistent with the pivotal role that this membrane plays in the neuronal transmission, i.e. allowing a rapid saltatory conduction throughout the axon [9].

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Plasma membrane composition of neuronal cells is generally maintained broadly constant by homeostatic mechanisms that can be anyway altered by aging, environmental and genetic factors [44]. It has been demonstrated that cholesterol is asymmetrically distributed in plasma membranes with the exofacial leaflet containing substantially less cholesterol than the cytofacial and mostly condensed in lipid rafts [44, 45]. Studies suggest a critical and decisive role for lipid rafts in Abeta generation. APP associated with lipid rafts, mainly causes Abeta generation, whereas localization of APP in non-lipid raft compartments favors the activity of alphasecretase, an enzyme involved in the non-amyloidogenic process [46]. Moreover, the raft-derived Abeta has been demonstrated to promote fibrillogenesis of soluble Abeta and amyloid plaque formation, whereas cholesterol depletion reduces the seeding properties of Abeta [46]. Interestingly, it has been demonstrated that Abeta signaling, regulated by cholesterol level, can in turn control Abetainduced tau proteolysis by calpain, but also by caspases. Intraneuronal neurofibrillary tangles, formed from hyperphosphorylation of tau protein is, together with amyloid deposits, the second defining element of AD [43]. A pool of hyperphosphorylated tau is present in lipid rafts, along with APP metabolites, BACE1, the gamma-secretase complex and apoE [43]. 1.5. Relationship Between Dietary Cholesterol and AD The level of cholesterol in the brain is likely to be affected only by the de novo synthesis and recycle, rather than by dietary cholesterol intake [47]. However, the transfer of small amount of cholesterol, in the form of LDL cholesterol, from the periphery to the CNS has been demonstrated [48]. The transport seems to be mediated by receptors of luminal endothelial cells of BBB and seems not to be relevant for brain cholesterol steady-state [48]. Although brain cholesterol level does not respond to mechanisms controlling body cholesterol homeostasis [47] a number of epidemiological studies suggest an association between plasma total cholesterol and increased risk for AD [reviewed in 48]. Hypercholesterolemia was associated to a greater number of senile plaques in the brain of non-demented patients [49] and drug lowering serum cholesterol levels were observed to reduce AD risk both in animals [50, 51] and humans [52-55]. Similarly, in animals fed with a high cholesterol diet a greater number of senile plaques were measured in the brain [56]. In vivo studies have reported that BACE1 expression and/or activity could be also influenced by dietary cholesterol [57]. However, it seems likely that the association between plasma cholesterol level and AD is not as explicit as suggested by these observations: the age of the subjects might also be implicated [48]. In fact, hypercholesterolemia in midlife, during a presymptomatic stage, is associated with an increased risk of dementia, whereas in older adult blood cholesterol does not seem to correlate with this risk and statin treatment failed to show any benefit [48]. A possible explanation might be linked to a different age-dependent organiza-

tion of the raft areas, which in turn, can affect the activity of beta-secretase [48]. Generally, the age-associated decrease in membrane cholesterol content, causing the disorganization of rafts, may induce an amyloidogenic pathway of APP processing, increasing Abeta generation [48]. 1.6. Oxysterols and AD In the brain, although in a lower amount with respect to 24-OH-C, another oxysterol, the 27-OH-C, can be also found [58]. The synthesis of 27-OH-C is catalyzed by CYP27A1, an enzyme ubiquitously expressed within the human body, with macrophages showing a particularly high activity [59]. The pattern of distribution indicates that the influx from the periphery is more prominent than the in situ synthesis in the brain [17]. Thus, while 24-OH-C mainly comes from endogenous brain synthesis, 27-OH-C enters the brain after passing the BBB [58]. It has been demonstrated that CYP27A1 expression is decreased in neuron and astrocytes in AD brains [59]. Despite this decrease, higher levels of 27OH-C were observed in AD brain and in a mouse AD-model [60] suggesting that the influx of 27-OH-C to the brain may be the link between dietary cholesterol and the risk of developing AD. In the CNS 27-OH-C can be converted into the major steroidal acid 7α-hydroxy-3-oxo-4-cholestenoic acid by CYP7B1, which is able to cross the BBB [61]. Both 24-OHC and 27-OH-C, as well as the 7α-hydroxy-3-oxo-4cholestenoic acid, are eliminated as bile acids within the liver [9] (Fig. 1). Although plasma level of 24-OH-C reflects the balance between cerebral production and liver metabolism, this value might be interpreted as a marker for brain cholesterol homeostasis and risk of AD [62]. In support of this hypothesis, experimental evidence suggest that plasma 24-OH-C may be higher at the early stages of cognitive impairment and lower at more advanced stages of AD, when compared to cognitively normal control subjects [62]. Interestingly, the secretion of 24-OH-C seems to regulate brain rate of new cholesterol synthesis as demonstrated by experiments in 24-hydroxylase knockout mice, in which the synthesis of brain cholesterol was reduced by about 40% [26]. The pharmacological treatment of AD mice with drugs increasing mRNA expression of CYP46A, reduced Abetaproduction, neuritic plaque formation and behavioral deficits [63]. A possible explanation might be that an increased synthesis of 24-OH-C, by reducing the level of membrane cholesterol, can increase alpha-secretase activity, thus favouring the non amyloidogenic pathway of APP metabolism [63]. Conversely, it has been reported that higher levels of 27-OHC are associated to an increased Abeta production and BACE1 activity [64]. In accordance with this finding, postmortem analysis of brains collected from AD patients showed reduced levels of CYP7B1, the enzyme responsible for 27-OH-C metabolism [65]. On the basis of these considerations, the involvement of cholesterol metabolism in AD needs to be re-examined. It is likely that fluctuations in oxysterols, rather than cholesterol per se, may better correlate with AD. Hypercholesterolemia,

The Role of Brain Cholesterol and its Oxidized Products in AD

by increasing the turnover of cholesterol to 27-OH-C, may induce an increase of this metabolite in the circulation and potentially in the brain, thus suggesting a possible role of oxysterols in AD. 1.7. Pharmacological and Nutraceutical Intervention in AD Based on the possible link between cholesterol metabolism and AD it has been hypothesized that drugs affecting cholesterol homeostasis, from a quali-quantitative point of view, could provide therapeutic options for this pathology. By acting as competitive inhibitors of HMG-CoA reductase, statins are able to reduce cholesterol synthesis [66]. Moreover, they induce the over-expression of LDL receptors, decreasing the circulating level of LDL-cholesterol [67]. Experimental studies conducted to test the possible protective effects of statins in AD did not produce univocal positive results [43]. The elements that could be taken into account for the conflicting conclusions within the published statin studies are correlated both to the intrinsic properties of the statins used and to differences in the methodological design (dosage, duration of treatment etc.). Considering that statins can exert their hypolipidemic role in the brain after crossing the BBB, it follows that only lipophilic statins may be good candidates. Owing to the different structures, statins can have also different binding affinities for HMG-CoA reductase and therefore have different effects on AD. Moreover, the lack of LDL receptors in the CNS may warrant certain failure of statin response in CNS [9]. In addition, studies reporting some effects of statins in AD should consider the impact of these compounds on the BBB integrity and, therefore, a probable indirect effect on brain cholesterol metabolism should be also taken into account [9]. However, the mechanism of action by which statins mediate their potentially beneficial effects are still under debate. Probably these benefits might be not directly associated with the wellknown ability of statins to lower cholesterol but rather to their so called pleiotropic effects. Statins in fact, can modulate several cellular pathways, independent of their ability to inhibit HMG-CoA reductase. Indeed, pre-clinical studies in aged beagle with atorvastatin were encouraging [68-72]. Another pharmacological approach with promising results involved the use of inhibitors of ACAT1 [57] able to cross BBB. As reported previously, ACAT1 is an enzyme involved in the cholesteryl-ester synthesis and its activity has been correlated to the etiology of AD. However, also this approach needs further investigation to better understand the molecular mechanisms underlying the improvement of AD phenotypes by these compounds. A possible link between promising novel pharmacological interventions for AD and central cholesterol metabolism might exist for other emerging targets, such as drugs acting as activators of PPARs. These drugs have been classically used as anti-diabetic and anti-dyslipidemic agents, but an increasing body of evidence demonstrated that they show also anti-amyloidogenic, anti-inflammatory, insulinsensitizing, and cholesterol-lowering properties, suggesting that they could provide potential new therapies for AD [73]. In particular, it has been demonstrated, that through the activation of PPAR-alpha, palmitoylethanolamide (PEA) is able

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to exert a robust neuroprotective action toward the neurotoxic effects induced by Abeta [74-76]. The mechanisms of PEA’s action has been mostly linked to the ability of modulating neuroinflammatory processes triggered by Abeta accumulation, however we cannot exclude a possible involvement of cholesterol metabolism. This hypothesis arises from the observation of the effects produced by oleoylethanolamide (OEA), a monounsatured analogue of PEA that acts as an endogenous ligand of PPAR-alpha [77]. OEA was shown to decrease hypercholesterolemia in obese animals [77] and to exert anti-atherosclerosis functions by suppressing the synthesis of oxidized LDL in mouse macrophages and reversing the endothelial pathological effects induced by oxidized LDL observed in mice lacking apoE [78]. Several studies have demonstrated a link between high dietary cholesterol and/or fat and oxidative stress in the brain of mice and rats [79]. Given that oxidative stress is a principal cause of neurodegenerative disorders, in the last few years a considerable attention has been given to nutraceuticals with an antioxidant functions [80, 81]. A slower cognitive decline was observed in patients taking vitamin E supplement with respect to a control group who did not take the supplement [816]. Moreover, vitamin D has demonstrated neuroprotective properties in subjects with AD [82]. Amongst the most studied categories of dietary antioxidants, polyphenols such as quercetin, resveratrol, curcumin and other natural antioxidants have rapidly gained attention as viable candidates for clinical testing in neurodegeneration and acute neuronal injury such as stroke [83-86]. Resveratrol, a red wine extract, and quercetin, found mainly in green tea, are two natural polyphenols presenting antioxidant properties in a variety of cellular paradigms. In vitro studies have demonstrated that quercetin protects C6 glioma and PC12 cells from oxidative stress caused by different oxidants [87] whereas in vivo quercetin expressed neuroprotective effect improving cognitive impairments [87]. Similarly to quercetin, resveratrol and curcumin show a strong neuroprotective effect both in vitro and in vivo studies essentially decreasing the production of reactive oxygen species and pro-apoptotic signals [87]. Besides the most popular compounds a number of other nutraceutical belonging to the family of phenolic acids and diterpenes, have provided neuroprotective effects as molecules able to scavenging free radicals [88]. Furthermore, a beneficial effect in the stabilization of cognitive function in AD patients was also measured giving alpha lipoic acid which is considered as a potent antioxidant [89]. CONCLUSION Brain cholesterol plays a pivotal role both at cerebral and body level. Although the link between cholesterol and AD has still many unresolved aspects, studies have found strong evidences that cholesterol has a role in the pathogenesis of AD. Current data provide tantalizing indications that modulation of intracerebral cholesterol levels may be a possible strategy for protection against AD. However, it is not clear

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yet whether the use of statins can be a useful strategy for treating the onset of clinical dementia. Novel therapeutic strategies should be considered and deserve further investigation. Oxysterols, normal lipid components in the brain, can regulate Abeta. Contrary to cholesterol, these oxidized products can cross the BBB and then interact with the rest of the body. To this end, whole CNS cholesterol production can be very elegantly studied by analyzing the concentrations of 24OH-C, the exclusive metabolite of CNS metabolism. Moreover, a relationship between brain oxysterol level and AD has been demonstrated. Thus, oxysterols more than simple cholesterol can furnish important target for the regulation of brain cholesterol homeostasis. Keeping these concepts in mind, the involvement of cholesterol metabolism in AD needs to be re-examined. Thus, in addition to HMG-CoA reductase many other possible targets for the modulation of cholesterol homeostasis in the brain should be taken into account.

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CONFLICT OF INTEREST The author(s) confirm that this article content has no conflicts of interest.

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ACKNOWLEDGEMENTS All authors have contributed substantially to the preparation of the manuscript

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Received: March 27, 2015

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Revised: ????????????????

Accepted: ????????????????