Cellular Cholesterol Homeostasis in Alzheimer's ...

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including 24S-hydroxy, 25-hydroxy, and 27-hydroxy cholesterol (8). Oxysterols can be ... To cite a few examples: 25-hydroxycholesterol is produced mainly by.
Cellular Cholesterol Homeostasis and Alzheimer’s Disease By

Ta-Yuan Chang*1, Yoshio Yamauchi†, Mazahir T. Hasan§, and Catherine Chang*2



Nutri-Life Science Laboratory, Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan §

Laboratory of Memory Circuits, Achucarro Basque Center for Neuroscience, Bizkaia Science and Technology Park, Building 205, E-48170 Zamudio, Spain

To whom correspondence should be addressed: 1

Ta-Yuan Chang, Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, 7200 Vail

Bldg. Room 304, Hanover, NH 03755, USA, tel.: 603-650-1622; Fax: 603- 650-1128; E-mail: [email protected]. 2

Catherine C.Y. Chang, Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth,

7200 Vail Bldg. Room 304, Hanover, NH 03755, USA, tel.: 603-650-1709; Fax: 603- 650-1128; E-mail: [email protected].

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*Department of Biochemistry and Cell Biology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA

ABSTRACT Alzheimer’s disease [AD] is the most common form of dementia in older adults. Currently, there is no cure for AD. The hallmark of AD is the accumulation of extracellular amyloid plaques composed of amyloid beta peptides [Abeta; especially Abeta1-42], and neurofibrillary tangles, composed of hyper-phosphorylated tau, accompanied with chronic neuroinflammation. Abeta are derived from the amyloid precursor protein APP. The oligomeric form of Abeta is probably the most neurotoxic species; its accumulation eventually forms the insoluble and aggregated amyloid plaques. ApoE is the major apolipoprotein of the lipoprotein (s) present in the CNS. ApoE has three alleles, of which the Apoe4 allele constitutes the major risk factor for late onset AD [LOAD]. Here we describe the complex

parts: 1. Key elements involved in cellular cholesterol metabolism and regulation; II. Key elements involved in intracellular cholesterol trafficking; III. Links between ApoE4, Abeta, and disturbance of cholesterol homeostasis in the CNS; IV. Potential lipid based therapeutic targets to treat AD. At the end, we recommend several research topics that we believe would help in better understanding the connection between cholesterol and AD for further investigations.

Running Title: Cholesterol and AD Abbreviations: Abeta, amyloid peptides; AL, acid lipase; ABCA1, ATP-binding cassette transporter A1; ACAT1SOAT1, acyl-coenzyme A:cholesterol acyltransferase 1sterol O-acyltransferase 1; AD, Alzheimer’s disease; ApoE/PL/C, ApoE lipidated with phospholipids and cholesterol; APP, amyloid precursor protein; CD, cyclodextrin; CEH, cholesteryl ester hydrolase; CLU, clustrin; CNS, central nervous system; Cyp46A1, cholesterol 24S-hydroxylase; EE, early endosome; ER, endoplasmic reticulum; ERC, endocytic recycling compartment; GM1, monosialotetrahexosylganglioside; HMGR, HMG-CoA reductase, KI, knock-in; LE, late endosome; LDL, low-density lipoprotein; LOAD, late onset Alzheimer’s disease; MAM, mitochondrial associated membrane; NPC1, Niemann-Pick type C1, NPC2, Niemann-Pick type C2; PL, phospholipids; PM, plasma membranes; SCAP, SREBP cleavage activating protein, CE, cholesteryl esters; LCAT, lecithin: cholesterol acyltransferase; LBP, lipid binding protein; NRs, nuclear receptors; OSBP, oxysterol

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relationship between ApoE4, oligomeric Abeta peptides, and cholesterol homeostasis. The review consists of five

binding protein; ORPs, oxysterol binding protein related proteins, PL, phospholipids; 24S-OH, 24Shydroxycholesterol; Lxrs, liver X receptors; MCS, membrane contact sites; NFTs, neurofibrillary tangles; NMDAR, N-methyl-D-aspartate receptor; PS1, presenilin 1; Rxrs, retinoid X receptors; SCAP, SREBP cleavage activating protein; SREBP, sterol-regulatory element binding protein; StAR, the steroidogenic acute regulatory protein; STARDs, the steroidogenic acute regulatory protein (StAR)-related lipid transfer domain proteins.

Keywords: Alzheimer’s disease; aging; cholesterol metabolism; ApoE; lipid trafficking; ABCA1; Brain lipids; membrane contact sites; Acyl-CoA:cholesterol acyltransferase; lipid raft; amyloidopathy; taupathy. Downloaded from www.jlr.org at Dartmouth College, on May 3, 2017

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Part I. Cellular cholesterol metabolism. A. Key enzymes in the early stage of cholesterol biosynthesis. Essentially all mammalian cells, including cells in the CNS, possess the capacity to biosynthesize cholesterol de novo. Sterol biosynthesis begins by using acetyl CoA (1) present in the cytoplasm [instead of in the mitochondria] as the building block. The enzyme acetyl CoA acyltransferase 2 [abbreviated as ACAT2 footnote 1; also known as cytosolic acetoacetyl-CoA thiolase] utilizes acetyl CoA to produce acetoacetyl-CoA. Acetoacetyl-CoA is then converted by the cytosolic HMG-CoA synthase to produce HMG-CoA. The next step, the reduction of HMG CoA to mevalonic acid, is catalyzed by HMG CoA reductase, and is the rate-limiting step in sterol synthesis. Mevalonic acid contains 5 carbons, and is the obligatory precursor for producing many naturally

modifications on many macromolecules via enzymatic prenylation. Most of the mevalonic acid is converted through a series of biochemical reactions to lanosterol, which is the first sterol precursor with 30 carbons. Through several additional enzymatic reactions that occur mostly at the ER membrane, lanosterol is converted to cholesterol, a sterol with 27 carbons. Malfunctions in late stages of the sterol biosynthetic pathway can lead to serious human diseases, such as the malformation syndrome (2). A detailed review covering various aspects of sterol biosynthetic pathway is available (3). B. Modes of regulation of HMG-CoA reductase (HMGR). An important feedback control in cholesterol biosynthesis involves sterol and non-sterol metabolite [geranylgeraniol; derived from mevalonate] mediated rapid degradation of HMGR [reviewed in (4), (5)]. HMG-CoA reductase is a multi-span membrane protein at the ER. The degradation occurs when the sterols in the ER membranes accumulate, which triggers binding of reductase to a pair of ER membrane proteins called Insig-1 and Insig-2. Insig binding leads to the ubiquitination of HMGR, which subsequently undergoes proteasomemediated degradation in the cytosol. In addition to control at the posttranslational level, the gene that encodes HMGR, along with many other genes in sterol biosynthesis pathway, as well as the low-density lipoprotein receptor [LDLR; also see Part III section A on Lipoproteins], is transcriptionally activated by the sterol dependent transcription factor sterol regulatory element binding protein 2 [SREBP2] [reviewed in (6)]. The precursor SREBP2 is a membrane-bound protein located at the ER membrane, where it forms a complex with another membrane protein called SREBP cleavage activating protein [SCAP]. When the sterol content at the ER falls to a certain threshold, SCAP undergoes conformational changes to allow the precursor SREBP2/SCAP complex to exit the ER and move to the Golgi. At the Golgi, the precursor of SREBP2 undergoes sequential cleavages by two specific proteases designated as S1P and S2P. These actions liberate the

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occurring organic substances, including sterols and non-sterols. It is also a needed precursor to produce covalent

active, soluble form of SREBP2, which travels to the nucleus to increase expressions of many genes in sterol biosynthetic pathway, as well as the gene that encodes LDLR. Conversely, high levels of cellular cholesterol prevent activation of SREBP2, causing down regulation in sterol biosynthesis and in LDL uptake. HMGR activity is also controlled by cellular metabolic state in sterol-independent manner: the enzyme can be phosphorylated/dephosphorylated in a reversible manner through the actions of a protein kinase and a phosphoprotein phosphatase respectively, resulting in enzyme inactivation or activation, respectively (7). C. Metabolites of cholesterol. Cholesterol is the obligatory precursor for biosynthesis of various key oxysterols, including 24S-hydroxy, 25-hydroxy, and 27-hydroxy cholesterol (8). Oxysterols can be produced from various cell types. Multiple oxysterols can mediate down regulation of cholesterol biosynthesis (9). Multiple oxysterols can also serve as

oxysterols may act in tissue specific manner. To cite a few examples: 25-hydroxycholesterol is produced mainly by macrophages and play important roles in mediating innate and adaptive immunity (11). 27-hydroxycholesterol is the most abundant oxysterol in the plasma, and can be biosynthesized from cholesterol by the enzyme Cyp27A1 present in multiple systemic tissues. Recently, it was shown that the most aggressive form of human breast cancer expresses very high level of Cyp27A1; this and other evidence suggest that 27-hydroxycholesterol can contribute to tumor genesis (12). 24Shydroxycholesterol is the most abundant oxysterol in the brain, with the enzyme responsible for its biosynthesis CYP46A1 playing a key role in the degradation and excretion of cholesterol from the brain (13). Due to the tight junction that exists between the endothelial cells of the blood brain barrier [see (14) for a review], the efflux of macromolecules between the brain interior and the blood vessels within the brain is severely limited; a much higher concentration of 24Shydroxycholesterol is found in the brain than in the blood, while the opposite is true for 27-hydroxycholesterol. However, under condition when the BBB becomes leaky, more 24S-hydroxycholesterol may enter the blood more readily, while 27hydroxycholesterol may move into the brain interior more readily, as suggested by Bjorkhem and colleagues (15), (16). Under various oxidative conditions, cholesterol can be auto-oxidized and converted to 7-ketocholesterol. In steroidogenic tissues such as the adrenal glands, testes, ovaries (17), and the hippocampi of the brain (18), cholesterol is first converted to pregnenolone which can then serve as the precursor for the synthesis of many steroid hormones. In the animal models of various neurodegenerative diseases, neurosteroids (such as those produced in the brain hippocampus) are shown to exhibit neuroprotective activities (19). In the liver, cholesterol serves as the precursor for bile acid biosynthesis. Bile acids are the major catabolites of cholesterol, and play important roles in lipid digestion and absorption. Shown in Fig. 1 are

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activating ligands for the liver X receptors [LXRs], which play important roles in lipid metabolism (10). Individual

chemical structures of cholesterol, various oxysterols, pregnenolone, and cholesteryl ester [shown here as cholesterol oleate as an example]. D. Macromolecules involved in cellular uptake, storage and removal of cholesterol. In addition to synthesizing cholesterol de novo, essentially all mammalian cells can acquire cholesterol from exogenous sources. Cells in systemic tissues acquire their cholesterol mostly from low-density lipoprotein, the major cholesterol-carrier in the blood, via its internalization through the LDLR (20). Cells in the CNS on the other hand acquire their cholesterol mainly from ApoE lipidated with phospholipids and cholesterol [ApoE/PL/C), the major lipoprotein in the CNS (21), through its internalization via multiple ApoE receptors (Also see Part III, section A on Lipoproteins). To prevent the excessive accumulation of free (i.e., unesterified) cholesterol in cells, the excess cholesterol gets either esterified to form cholesteryl

coenzyme A:cholesterol acyltransferases, also called sterol O-acyltrsansferases [ACATSOAT]; these enzymes use various long chain fatty acyl-CoAs, and various sterols with 3-beta –OH, including cholesterol and various oxysterols (22), (23) as their substrates. There are two ACATSOAT genes (24), (25). The ACAT1SOAT1 gene is ubiquitously expressed in essentially all cells, including cells in systemic tissues and in the CNS; while the ACAT2SOAT2 gene is expressed in intestinal enterocytes and in hepatocytes; low levels of ACAT2SOAT2 are also detectable in various peripheral tissues examined. Both ACAT1SOAT1 and ACAT2SOAT2 are integral membrane proteins located at the ER region, and both are allosterically activated by their own substrate [cholesterol or oxysterols] (26, 27). Neither Acat1Soat1 nor Acat2Soat2 is controlled by the transcription factor SREBP2. Regarding the cellular sterol removal process: It involves the release of sterols and certain phospholipids at the cell surface. This process depends on the ATP cassette binding protein A1 [ABCA1], which is a multi-span membrane protein mainly enriched at the PM [reviewed in (28)]. The sterol efflux process also requires the presence of certain helical apolipoproteins located at the cell exterior, such as ApoA-I, the major apolipoprotein in high-density lipoprotein [HDL] in the blood, or ApoE, the major apolipoprotein in the CNS (Also see Part III, section A on Lipoproteins). ABCA1 and a related protein ABCG1, which is a separate member of the ABC transporter family (29, 30), work in concert to prevent the built-up of excess cholesterol (31, 32), (33). ABCA1 exhibits preference to use newly biosynthesized sterols, especially lanosterol, as its sterol substrate (34). The expression of ABCA1 gene is positively controlled by the liver X receptors (LXRs) and retinoid X receptors (RXRs), with oxysterols and retinoid as the main activating ligands; these ligands act in synergistic manner [For details, see review by Landreth

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esters for storage, or gets removed from the cell altogether. Cholesterol esterification is catalyzed by the enzymes acyl-

and colleague in this series]. In addition, the Abca1 gene is negatively regulated by several microRNAs including miR106b, miR-758, and miR-33, which are expressed abundantly in the brain [Reviewed in (35)].

Part II. Intracellular cholesterol trafficking A. Lipid raft domains. The lipid and protein compositions in various membrane organelles are highly heterogeneous; however, they all contain cholesterol as a ubiquitous lipid ingredient. The cholesterol that binds tightly with sphingolipids form a sterol-rich, sphingolipids-rich domain, referred to as the lipid raft domain. These domains are present in various cell membranes, and serve as platforms to host various membrane proteins involved in many cell-signaling processes (36). Lipid-based platforms that are independent of cholesterol are also present in cell membranes (37). Within a given

example, by using different approaches, different investigators reported that cholesterol is either enriched at the inner leaflet (38), (39), or enriched at the outer leaflet of the plasma membrane (40). B. The concept of “active cholesterol”. Within the membrane, cholesterol interacts with sphingolipids as well as phospholipids to form complexes. The cholesterol content in excess of those that form complexes with phospholipids/sphingolipids can be considered as “active cholesterol”; active cholesterol has higher tendency to move away from the membrane than the “bound” cholesterol (41). Cholesterol is practically insoluble in water; various transport mechanisms are needed in order to catalyze the movement and the recycling of cholesterol within the cell. C. Non-vesicular lipid transport. The transfer and/or exchange of cholesterol and other lipids between two adjacent subcellular membrane organelles are facilitated mostly via non-vesicular mechanisms. Unlike vesicular transport [that account for the vast majority of intracellular protein transport (42), (43)], the non-vesicular transport occurs without involving membrane fusion. This process is governed by various lipid-binding proteins (LBP) through membrane contact sites (MCS). For examples, ER membranes form MCS with the membranes of mitochondria, endosomes, the PM, and the Golgi (44). MCS also exist between other membrane pairs. Various tethering factors are involved in producing MCS (45), (46), (47). Two classes of LBPs, the steroidogenic acute regulatory protein (StAR)-related lipid transfer domain protein family [STARDs], and the oxysterol binding protein related protein family [ORPs], have been studies extensively. STARDs [which include 15 members in mammals] (48), (49), including StAR as its first member (50), are involved in transferring cholesterol and/or other lipids (51), (52), (53). ORPs, including its first member OSBP (54), have ten

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membrane, whether the distribution of cholesterol across the lipid bilayer is asymmetrical or not remains unclear. For

members in mammals (55). ORPs contain multiple domains that enable them to transfer different classes of lipids between two adjacent membranes in asymmetric manner (56) (57, 58), (59). D. The three cholesterol trafficking routes in a mammalian cell. The transport and movement of cholesterol through various subcellular compartments can occur by at least three distinct metabolic pathways, resulting in three major cholesterol pools not in rapid equilibration with another. This concept is outlined in Fig. 2, and is elaborated as follows. 1. Exogenous cholesterol is supplied to the recipient cells mainly by cholesterol-rich lipoproteins, which enter the cell by endocytosis; the best example being the clathrin mediated endocytosis of low-density lipoprotein [LDL] via the LDL receptor (20) footnote 2. After endocytosis, LDL first enters a distinct early endocytic compartment enriched in the acid lipase (AL) that catalyzes the hydrolysis of the cholesteryl esters (60). Cholesterol released from LDL-derived cholesteryl esters

soluble protein present in the luminal region of the endo/lysosomes (61), bind to cholesterol and transfers it to NPC1 (62), (63), a protein that contains multiple transmembrane domains, including the “sterol-sensing domain” (64). The sterolsensing domain is also present in HMGR, in SCAP, as well as in several other membrane proteins involved in sterol dependent cell signaling processes. NPC1 then exports cholesterol to the exterior of the late endo/lysosome, in manners that are not well understood at present. Cholesterol exiting from the late endosomes arrives at other membrane compartments, via various transport mechanisms. These recipient membrane compartments include the PM (65), (66), the ER (67), (68), (69), the TGN (70), (71, 72), the mitochondria [described in the next section], and the peroxisomes [described in the next section]. Mutations in NPC1 or in NPC2 can cause the Niemann-Pick type C [NPC] disease in humans and in certain other animal species. Children affected with NPC disease almost invariably die before reaching adulthood. 95% of NPC cases are caused by mutations in the Npc1 gene, with the rest caused by mutations of the Npc2 gene (5%). Currently, there is no cure for this disease. In NPC patients, unesterified cholesterol accumulate within the endo/lysosomes of various organs, including the liver, the spleen (73), and various regions of the brain (74-76); reviewed in (77). 2. In addition to exogenous uptake, cells also acquire cholesterol through endogenous biosynthesis de novo. The majority of sterols, including cholesterol, lanosterol, and multiple other precursor sterols newly synthesized at the ER quickly moves to the PM within a few minutes; this movement is not yet well understood at the molecular level, but is independent of NPC1 (78), (79). In yeast, several StAR-like proteins have been shown to participate in the sterol movement from the ER to the PM (49). Upon arriving at the PM, a significant amount of the newly synthesized sterols,

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then moves to the endosomes that contain a pair of cholesterol binding proteins designated as NPC1 and NPC2. NPC2, a

especially lanosterol, is released to the cell exterior by the previously described sterol efflux process that depends on ABCA1 and helical apolipoproteins (34). Cholesterol, lanosterol, and other precursor sterols remaining at the PM recycles between the PM and various internal compartments, including the endo/lysosomes (80) (81). Interestingly and unexpectedly, in the absence of sterol acceptors in the media, deficiency in ABCA1 delays sterol sensing and delays sterol esterification at the ER (82). This result implicates that the retrograde movement of cholesterol and other sterols from the PM to the ER partially depends on ABCA1. The molecular nature of the ABCA1 assisted retrograde sterol movement is not clear at present; but this process requires the intrinsic ATPase activity present in ABCA1 (82), and involves clathrinindependent endocytosis (82), (83). ABCA1 maybe located within the lipid raft domain as shown in Fig. 2; however, this assignment is only tentative (34). In addition to ABCA1, two soluble lipid binding proteins, oxysterol binding protein

ER (57). In addition, the StAR-like lipid transfer domain protein 4 (STARD4) has also been implicated in moving cholesterol from the PM to various destinations including the ER (52, 84). 3. Cholesterol that builds up at the ER gets esterified for storage by the enzyme acyl-CoA:cholesterol acyltransferase 1 [ACAT1SOAT1]. Both cholesterol derived from lipoprotein and cholesterol synthesized endogenously serve as the substrate for ACAT1SOAT1. Cholesterol delivery to ACAT1SOAT1 is partially dependent on NPC1/NPC2 (85, 86), and partially dependent on the ABCA1 (82). Cholesteryl esters sequester as cytoplasmic lipid droplets. These lipid droplets are subject to hydrolysis by enzymes that are collectively designated as cholesteryl ester hydrolases [CEHs]. The expressions of CEHs are tissue and cell-specific (87). Neutral cholesteryl esterase and hormone sensitive lipase play key roles in macrophages (88), while in rat hepatocytes, a member of the carboxylesterase family called ES-4 accounts for the majority of cholesteryl ester hydrolysis (89). Under cholesterol rich condition, a cholesterol-CE cycle occurs continuously (90); the majority of cholesterol in this cycle originates from sterols synthesized endogenously, rather than derived from lipoproteins (91). When ACAT1SOAT1 is inhibited, the cholesterol pool destined for storage rapidly reaches the PM to serve as a substrate for ABCA1 mediated lipid efflux (92). There are at least three different cholesterol pools at the PM (66); one of these pools is recently shown to be highly enriched at the microvilli region of the PM (93). The relationship between the PM cholesterol pools and the three cholesterol trafficking routes described here requires further investigation. 4. Cholesterol flux in the mitochondria, and in peroxisomes. Mitochondria produce ATP to meet the cellular demand for energy. In steroidogenic cells, cholesterol in the inner membranes of mitochondria serves as the precursor for biosynthesis of pregnenolone. In non-steroidogenic cells, cholesterol in mitochondria serves as the precursor for biosynthesis of 27-

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related protein 1 and protein 2 [ORP1 and ORP2], have been implicated in the cholesterol movement from the PM to the

hydroxycholesterol, which is the most abundant oxysterol in the blood. Cholesterol in mitochondria comes from at least three sources: 1. PM. The molecular nature of the PM-mitochondria cholesterol movement process is not clear at present, but this process does not require NPC2 (94). At mitochondria, the transfer of cholesterol from the outer membrane to the inner membrane is mainly mediated by the protein steroidogenic acute regulatory protein StAR (95). 2. In cells that express low level of StAR, a StAR- like protein MLN64 [also called STARD3] present in the late endosomes (96), (97) works along with the NPC2 protein to transport cholesterol from the late endosome/lysosome to the mitochondria (98), (99). This process explains the unusual finding that in mutant NPC1 cells, cholesterol overloading occurs in mitochondria (100), (101). 3. The third source of mitochondrial cholesterol comes from a specialized membrane region designated as the mitochondrial associated membranes [MAM], which are part of the ER membranes in close physical contact with the

and in the protein content of ACAT1SOAT1 (104). It is also rich in cholesterol and in the simple sphingolipid ceramide (105). An outer mitochondrial membrane adaptor protein HUMMR [standing for hypoxia-upregulated mitochondrial movement regulator] increases the ER-mitochondria membrane contact sites to facilitate cholesterol flux to mitochondria (106). Interestingly, studies in mouse model for AD showed that, in early stage of AD development, synaptic mitochondria exhibit significant functional deficits; the degree of deficits correlates positively with Abeta peptides accumulation (107). In the future, it would be interesting to test if mitochondrial cholesterol overload may execrate Abeta toxicity in synaptic mitochondria. Peroxisomes play important roles in the biosynthesis and catabolism of various lipids. Disrupting the contact sites between peroxisome and lysosome impairs the LDL-dependent decrease in SREBP processing and increase in cholesterol esterification; these findings implicate peroxisomal cholesterol transport as an important intermediate step in delivering LDL derived cholesterol from the late endo/lysosome to the ER (108).

Part III. Disturbances of CNS cholesterol homeostasis in AD A. Lipoproteins in the blood versus lipoproteins in the CNS. Lipoproteins transport lipids and fat-soluble vitamins to various cells for utilization and for storage. In the plasma, lipoproteins include chylomicrons, very-low-density lipoproteins (VLDL), low-density lipoproteins (LDL), and highdensity lipoproteins (HDL). These lipoproteins differ in apolipoprotein/lipid composition, in size and density, and in function. Chylomicrons deliver dietary triglycerides to body cells; they contain ApoB48 as the major apolipoprotein. VLDLs deliver triglycerides synthesized in the liver to body cells, and contain ApoB100 as the major apolipoprotein.

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mitochondrial membranes (102). These membranes are enriched in enzymes that biosynthesize phosphatidyl serine (103),

LDLs deliver cholesterol to body cells, and contain the ApoB100 as the only apolipoprotein. HDLs play important roles in cellular efflux of cholesterol and phospholipids; they contain apoA1 and ApoAII as the major apolipoproteins. Chylomicrons, VLDLs, and HDLs also contain ApoE and ApoCs as the minor apolipoproteins. To address a key function of ApoE in lipoprotein metabolism in systemic tissues: chylomicrons circulating in the blood are rapidly catabolized by lipoprotein lipase, and converted to remnant particles of much smaller sizes, designated as chylomicron remnants. ApoE present in chylomicron remnants serves as the key ligand, such that the remnant particles can be recognized by the receptor present at the cell surface, the chylomicron remnant receptor, also called low-density lipoprotein related protein 1 (LRP1). LRP1 is highly expressed in several tissues, including hepatocytes, macrophages, and adipose tissue, as reviewed in (109), and by Shinohara, Bu, and colleagues in this series. Through LRP1, chylomicron remnants undergo endocytosis

blood brain barrier, the vast majority of plasma lipoproteins cannot readily enter the brain interior. In the brain, ApoE is the major apolipoprotein and is produced within the brain. The ApoE is present as lipid-rich ApoE particles in the interstitial fluid and cerebral spinal fluid, and exhibit- density and size similar to those of plasma HDL. Similar to the ApoE present in chylomicron remnants, the lipid-rich ApoE also uses LRP1 as one of the main receptors for binding and endocytosis, and LRP1 is highly expressed in various brain cells within the CNS. Humans contain three slightly different alleles of the Apoe gene, e2, e3, and e4. The most common allele is e3, and is found in more than half of the population. Apo E2 binds poorly to chylomicron remnant receptor/LRP1. Humans homozygous for ApoE2 are deficient in the clearance of chylomicron remnants, and tend to have hypercholesterolemia and premature atherosclerosis. Instead, the ApoE4 isoform confers significantly increased susceptibility to late-onset Alzheimer disease (LOAD; see below). In addition to producing ApoE, brain cells produce a different apolipoprotein Apo J (also named as clustrin and abbreviated as CLU); polymorphism in the Apoj gene also confers a risk factor for LOAD [reviewed by Rebeck in this series]. Cells in the brain do not produce ApoB48, ApoB100, or ApoA1. However, the cerebral spinal fluid (that circulates between the subarachnoid space of the brain and the spinal cord) can acquire a significant amount of ApoA1 from the blood via unknown mechanism(s) [reviewed in (110)]. Under condition of certain chronic vascular diseases, such as hypertension, hypercholesterolemia, and diabetes, the BBB becomes partially leaky, and may allow plasma lipoproteins (as well as other macromolecular components present in the blood) to become more readily accessible to the brain interior [reviewed in (110)]. Recently, Zlokovic and colleagues showed that unlike normal mouse, the ApoE KO mouse exhibits a leaky BBB phenotype; expressing human ApoE2 or human ApoE3, but not human ApoE4, rescued the leaky BBB phenotype of the

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to deliver lipids for utilization and storage in various tissues. To address the function of ApoE in the CNS: Due to the

mouse. In addition, in a cultured BBB model, Michikawa and colleagues showed that ApoE plays an important role in controlling the integrity of tight junction in isoform-specific manner (111). Together, these results support the hypothesis that the inability of ApoE4 to maintain the integrity of BBB could be a causative factor that leads to various neurodegenerative diseases, including AD (112). B. The involvement of ApoE4. 1. The hallmark of AD consists of extracellular amyloid plaques, mainly composed of amyloid beta peptides (Abeta; especially Abeta1-42), neurofibrillary tangles, mainly composed of hyper-phosphorylated tau, accompanied with chronic neuroinflammation. The current review focuses only on the link between cholesterol and Abeta. Abeta are mainly produced in neurons, and are derived from the amyloid precursor protein APP by sequential proteolytic cleavages; with

also occur within the smooth muscle cell layer of the arterial blood vessels within the brain, producing a pathological condition designated as cerebral amyloid angiopathy [reviewed in (113), and by Holtzman and colleagues in this series]. Defects in the clearance of Abeta from the brain through cellular degradation mechanism(s), and/or by transport pathway(s) across the blood-brain barrier underlie many cases of late onset AD [LOAD] [reviewed in (114), (112)]. Tau is a soluble, microtubule binding protein produced abundantly in neurons. Various forms of tau [i.e., soluble, and/or misfolded, and/or mislocalized, and/or hyperphosphorylated forms] may eventually aggregate and form neurofibrillary tangles [reviewed in (115)]. Abeta peptides and tau can interact with each other synergistically to cause neuronal dysfunctions and trigger the disease [reviewed in (116)]. Dysfunctional neurons and misfolded proteins can cause activations in microglia and in astrocytes that lead to neuroinflammation [reviewed in (117), and by Landreth and colleagues in this series]. 2. The brain is an organ very rich in cholesterol; it contains 23% of the body’s total cholesterol, though it constitutes only 2.1% of total body weight (118). Due to the blood brain barrier, cholesterol within the brain does not readily equilibrate with cholesterol bound to lipoproteins in the blood. Thus essentially all the cholesterol in the brain is derived from biosynthesis within the brain. Both astrocytes and neurons have high demand for cholesterol, and both cell types can synthesize cholesterol, as discussed in detail by Pfrieger and colleague (119). Studies in mouse show that neurons need to synthesize their own sterols during prenatal stage (120), but may be able to acquire enough sterols from other sources during adult life (121). In the central nervous system [CNS], cholesterol transport between different cell types occur, and the lipid rich ApoE, i.e., ApoE lipidated with phospholipids and cholesterol [abbreviated as lipoprotein that plays the

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time, Abeta oligomerizes to form the insoluble and aggregated amyloid plaques in neurons. Deposition of Abeta peptides

major role in transporting cholesterol (21). ApoE is a 36,000-dalton protein present in the plasma and in the brain. As mentioned earlier (Part III, section A), in humans, ApoE is polymorphic, with three major alleles, ApoE2 [cys112, cys158], ApoE3 [cys112, arg158], and ApoE4 [arg112, arg158]. For LOAD, besides aging, the ApoE4 allele is the most important risk factor (122), (123). Conversely, the ApoE2 variant appears to have a protective role in LOAD. ApoE affects Abeta aggregation and clearance in the brain in isoform-specific manner (124), (125). ApoE in the CNS is mainly produced by astrocytes (126). Under brain injury condition, neurons also produce ApoE (127). Once synthesized, ApoE is secreted as a complex of ApoE protein rich in phospholipids and cholesterol (ApoE/PL/C). The formation of the ApoE/PL/C depends on ABCA1 (128), (129), (130). Once secreted, a portion of cholesterol in ApoE/PL/C is esterified by the enzyme lecithin: cholesterol acyltransferase [LCAT], in ABCA1 and ApoE dependent manner (131). ApoE/PL/C

ApoE receptors, including LRP1, LDLR, apolipoprotein E receptor 2 [ApoER2], and very low-density lipoprotein receptor [VLDLR]; etc. [Reviewed by Shinohara, Bu and colleagues, and by Herz and Colleague in this series]. ApoE consists of a receptor-binding region and a lipid-binding region; the different ApoE isoforms have different lipidation states; which influence their binding properties to the cognate receptors (132), (125). ApoE4 is less lipidated than ApoE2 or ApoE3, and undergoes more rapid degradation within the CNS (133). In neurons, once internalized, ApoE is thought to participate in regulation of lipid metabolism, including phospholipid metabolism (134), intracellular cholesterol transport and lipid efflux (135), as well as cholesterol esterification (136), in ApoE isoform specific manner. In neurons, cholesterol can be esterified by ACAT1SOAT1 (137); it can also be converted to the major oxysterol in the brain 24Shydroxycholesterol (24S-OH) (138). The lipidated ApoE also participates in the efflux of phospholipids and cholesterol in isoform-specific manner; this process also depends on ABCA1 (139), (140). A related protein ABCG1 works in concert with ABCA1 to control the effluxes of cholesterol and 24S-hydroxycholesterol (141), (142). At present, the mechanisms that govern the fate of ApoE derived cholesterol in neurons and in astrocytes are not well understood. To stimulate further investigation, here we provide a working model that depicts ApoE mediated cholesterol homeostasis in astrocytes and neurons (Fig. 3). This model is based largely on the results described in Refs. 130-140. In this model, we also hypothesize that 24S-OH, secreted by neurons in an ApoE and ABCA1 dependent manner (141), (142), may reach astrocytes, to down regulate sterol biosynthesis, and to activate gene expressions of ApoE, ABCA1, and other key proteins involved in lipid homeostasis in astrocytes. The validity of the model depicted in Fig. 3, especially at the in vivo level, requires further investigations. This topic is also reviewed in detail by Rebeck in the current series.

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delivers lipids from the astrocytes to the neurons and other CNS cells through internalization after binding to various

3. In addition to ABCA1 and ABCG1, a separate ABC family member called ABCA7, has been identified as one of the AD susceptibility genes (143). Deleting Abca7 increases Abeta accumulation in a mouse model for AD (144). Although the precise function of ABCA7 remains not well understood, it is probably involved in lipid transport. A role of ABCA7 related to Abeta biogenesis in the brain has been reported (145). 4. In addition to affecting cholesterol homeostasis in astrocytes and neurons, ApoE also affects the functions of various receptors present in the membranes of postsynaptic neurons, in an isoform specific manner. For an example, the Nmethyl-D-aspartate receptor (NMDAR) is a glutamate receptor and an ion channel protein. This receptor is important for controlling synaptic plasticity and memory formation [reviewed in (146)]. Studies in cell culture show that ApoE4 causes malfunction of the NMDAR mediated signaling in the hippocampus and in the cortex (147), (148) (149). [Reviewed by

is very sensitive to cholesterol and to other lipids (150). Many other membrane receptors, channels, and transporters are also significantly affected by cholesterol content in the neuronal membrane [reviewed in (41)]. In the future, it would be interesting to test whether ApoE affects the activities of membrane receptors/channels/transporters and different signaling pathways through its ability to control lipid homeostasis. 5. Studies in vivo have shown that human ApoE4 causes age-dependent learning and memory impairments in mouse without amyloidopathy (151), (152) [Reviewed by Rebeck in this series]. It also exacerbates neuroinflammation [Reviewed in (153); by Rebeck, as well as by Mary Jo Ladu and colleagues in this series]. C. The Abeta/lipid rafts connection In AD, oligomeric forms of Abeta peptides are probably the most toxic molecular species that cause synaptic loss (154) (155). The Abeta monomer/oligomer conversion is affected by ApoE in isoform specific manner; Abeta peptides can enter the cells via multiple mechanisms; some of them depend on the ApoE and the ApoE receptors, especially the LDLR and LRP1. The interactions between Abeta peptides and ApoE/PL/C are complex [reviewed in (125), and by Holtzman and colleagues in this series]. Inside the neurons and other cell types, the oligomeric Abeta are reported to cause numerous functional disturbances such as: alterations in mitochondrial morphology and oxidative stress (156),(157), (158); alterations in Golgi morphology, causing fragmentation and malfunctions (159), (160); alterations in mitochondria associated membranes [ER/mitochondria contact sites] (161), (162); alterations in cellular cholesterol metabolism (163); alterations in synaptic organelle transport, including the transport of recycling endosomes, mitochondria, etc. (164). Abetas are highly concentrated at the lipid raft region (165). Specifically, Abeta preferentially bind to the GM1 [a

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Herz and colleague in this series]. NMDAR is concentrated at the plasma membranes of postsynaptic neurons; its function

prototype of gangliosides] in membranes (166); the interaction between Abeta and GM1 is much enhanced when cholesterol is present (167, 168). In addition, Abeta may also bind to cholesterol directly (169). Thus, it has been proposed by several investigators that the toxicity of Abeta may be produced in part by disturbing the lipid raft domains present in various membrane organelles. While Abeta can affect the structure and function of raft domains, the converse is also true: the enzymes involved in the APP processing pathway [i.e., the alpha, beta, and gamma secretases (170-172),(173), as well as APP itself (174)], have all been reported to be affected by the composition of the lipid raft domains where they reside in. The fate of APP itself is a complicated matter: In addition to its cleavages by the secretases, a significant fractions of APP and its C-terminal fragments can also degraded by lysosomal hydrolases (175), (176). To summarize, the Abeta/lipid raft connections are complex and need further investigations.

Here we discuss targets and candidate drugs being tested at clinical stage first, and discuss those being considered at preclinical stage. A. Statins and HMG-CoA reductase. The statin drugs have been successful in treating patients with dyslipidemic cardiovascular diseases (177). They work by inhibiting the key enzyme in endogenous sterol biosynthesis HMGR (178), (179). In general, statin drugs inhibit HMGR with an inhibitor constant Ki at less than 1 nM (179). When used at superhigh concentrations (1000X higher than the Ki value), statins have been shown to exert off-target effects that are independent of HMGR inhibition; for example, at 2-10 micro M, statins can act as PPAR alpha ligands (180). Here we focus on the effects of statins on inhibiting HMGR. Studies in vitro and in animal models showed that statins strongly reduce the levels of Abeta peptides Aβ 42 and Aβ 40 (181), (182). Thus, several clinical trials have been conducted to see if the statin drugs may benefit AD patients. Unfortunately, the results have been inconclusive (183). To speculate the actions of statins on AD patients: HMGR produces mevalonate as its enzymatic reaction product. Mevalonate is an essential precursor for the biosyntheses of sterols as well as several non-sterol metabolites, including coenzyme Q, dolichol; etc. These metabolites are essential for cell growth and maintenance. Mevalonate is also needed for the enzymatic modification (by prenylation) of numerous proteins; these proteins depend on prenylation for their biological functions (184). Statins used at low dosage may reduce cholesterol content present in the lipid raft, thereby decreasing the interaction between lipid raft and Abeta, and diminishing the toxicity produced by Abeta; on the other hand, statins may affect various channels/receptors that depend on optimal membrane cholesterol content to maintain their functionalities,

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Part IV. Lipid based potential targets to treat AD

especially those channels/receptors involved in the learning and memory process. Statins used at high dose may significantly decrease the levels of mevalonate derived non-sterol metabolites, in addition to decreasing the cholesterol content present in the lipid raft; the long-term consequence of treating AD patients with high concentrations of statins cannot be predicted at present. B. Nuclear receptor agonists: The nuclear receptor agonists are promising agents to treat AD. These agonists activate several ligand dependent transcription factors, including LXRs, RXRs, and peroxisome proliferator-activated receptor (PPARs), to induce the gene expressions of ApoE, ABCA1, and ABCG1. This topic is reviewed in detail by Landreth and colleague in this series. C. Omega-3 fatty acids and other nutritional supplements. Omega-3 fatty acids are essential fatty acids, and include

plant oil. They are being tested as an AD prevention strategy in humans. This topic is reviewed in detail by Hartmann and colleagues in this series. D. Cyclodextrin (CD). CDs are water-soluble oligomers of glucose. They do not elicit immune responses and have low toxicities in animals and humans. CDs can form water-soluble inclusion complexes with hydrophobic small molecules, such as cholesterol and other lipid molecules (185). Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) is an FDA approved drug delivery vehicle for various pharmaceutical purposes. In NPC disease, treating animal models for NPC disease with HP-beta-CD overcome the cholesterol transport defect caused by mutations in NPC in various organs, and produced significant improvement in delaying the disease onset, in ameliorating the disease progression, and in prolonging the life span (186, 187), (188), (189); reviewed in (77). Currently, HP-beta-CD is under clinical trial to treat children affected with NPC disease (190), (77). CD enters the cell interior rapidly and acts by mobilizing cholesterol within the endo/lysosomal compartment, thus facilitating the cholesterol transfer from the endo/lysosomes to other cellular compartments, including ER and PM; etc. (191, 192). Based on the results of using CD to treat NPC disease, CD has been tested in a mouse model for AD, and the results were interesting: beginning at postnatal day 7, continuous intravenous injections of CD for 4 months led to the reduced Abeta accumulation, diminished tau immunoreactive dystrophic neuritis, and rescued cognitive deficits; HP-beta-CD may act by increasing the APP processing, and by increasing the gene expression of ABCA1 (193). One needs to be aware that when CD is used to treat animal model for NPC disease, the effect of CD is most effective when CD is administered at a very young age. In addition, treating CD at high concentration causes hearing loss in cats (194), and in mice (195).

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docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). They are richly present in deep- sea fish oil but absent in

E. MiR-33. MicroRNAs (miRNAs) are short noncoding regulatory RNAs. They specifically bind to various target mRNAs, repressing the expression of the corresponding target genes through translational repression, and/or through mRNA decay. In animal studies, a specific miRNA miR-33, present within intron 2 of the gene that encodes SREBP2, down regulates the gene expression of ABCA1, and causes decrease of the HDL level in vivo (196). Interestingly, in mouse brain, overexpression of miR-33 suppresses ABCA1 expression and causes impaired cellular lipid efflux, as well as increased extracellular Abeta accumulation; conversely, inhibition of miR-33 induces ABCA1 and causes increased lipidation of ApoE, and reduced Abeta levels (197). Furthermore, pharmacological inhibition of miR-33 by using an antisense oligonucleotide specifically in the brain markedly decreased Abeta levels in the cortex of a mouse model for AD (197). In the brain, ABCA1 is redundantly targeted by miR-106b, miR-758, and miR-33 (35). These results suggest that

F. ApoA1 mimetic peptides. In cell culture and in systemic tissues, the synthetic, ApoA1 mimetic peptides are known to mimic the effects of ApoA1 and ApoE in stimulating ABCA1-dependent cellular cholesterol efflux as reviewed in (198). Michaelson and colleagues (199) injected one of these mimetic peptides CS-6253 directly into the brains of young ApoE4 KI mice, and showed that this peptide increased the lipidation of the ApoE4 associated lipoproteins. CS-6253 also reversed many of the ApoE4 associated pathology, including Abeta accumulation and tau hyperphosphorylation in hippocampal neurons, as well as synaptic impairments and cognitive deficits. These results show that increasing the lipidation of ApoE4 associated lipoproteins is a promising strategy to combat AD. G. Cyp46a1 and Cyp46a1 activators. The enzyme Cyp46A1, also called 24S-hydroxylase (200), converts cholesterol to 24S-hydroxycholesterol, which is the most abundant oxysterol in the brain (201). Study in mouse showed that Cyp46a1 gene knock out reduced cholesterol excretion from the brain by more than 50%, reduced cholesterol biosynthesis rate in the brain by 40%, without altering the overall brain cholesterol content. These results show that Cyp46A1 is responsible for the turnover of at least 40% of brain cholesterol; in the absence of Cyp46a1, synthesis of cholesterol de novo is reduced in order to maintain cholesterol homeostasis (202). The cyp46a1 KO mouse was used to test the effect of reducing 24S-hydroxycholesterol in AD pathology. The result showed that Cyp46a1 gene knock did not affect the amount of the insoluble amyloid plaques (203). The AD mice employed in this study had a shorter life span (for unknown reason); Cyp46a1 gene knock prolonged the life span of these AD mice, but did not affect the life span of the non-AD mice (203). To test the effect of increasing 24S-hydroxycholesterol in the AD mouse brain, the Cyp46a1 overexpression experiments were carried out next. The results showed that in two different mouse models for AD, overexpressing Cyp46a1

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inhibition of these miRNAs may provide a novel therapeutic strategy to treat AD.

ameliorated amyloid pathology (204), or ameliorated taupathy (205). In addition, overexpressing Cyp46a1 in a mouse model for Huntington disease decreased neuronal atrophy and improved motor neuron deficits (206). These results showed that overexpressing Cyp46a1 in the brains with certain neurological diseases is neuro-protective, and suggest that specific Cyp46A1 activators may provide a novel therapeutic strategy to treat AD and other related neurodegenerative diseases. The mechanistic basis for the neuroprotective effect(s) of increasing 24S-hydroxycholesterol remains to be clarified. H. ACAT1SOAT1 inhibitors. In the disease atherosclerosis, cholesteryl esters produced by ACATSOAT accumulate in macrophages and smooth muscle cells, causing them to become foamy. For this and other reasons, ACAT SOAT inhibitors were produced for anti-atherosclerosis purpose. Several ACAT SOAT inhibitors including CI1011 [an inhibitor that inhibits

to stage 2 or stage 3 clinical trials. Due to lack of efficacy, none became a medicine. Regarding the CEs levels in mouse and human brains: in normal states, the values are very low; making up less than 1% of the free, unesterified cholesterol. However, in the vulnerable (entorhinal cortex) regions of brain samples from AD patients, CE levels increase by 1.8 fold (209). In the brains of 3 different AD mouse models (that express mutant human APP, or express mutant APP and mutant presenilin 1), the CE levels rose to values 3 to 11 fold higher than those in the control mice (209), (210). In addition, under high fat diet, the brain CE content in the ApoE4 knock-in (KI) mouse is significantly higher than that in the ApoE3 KI mouse (211). Together, these results suggest that increases in CE content correlate positively with AD development. In mouse models for AD, both the pharmacological approach (212), (213) and the molecular genetic approach (137), (214) showed that inhibiting ACAT1SOAT1 significantly reduced amyloid plague load and restored cognitive deficits. The mechanism(s) underlying the beneficial effects seen with blocking ACAT1SOAT1, as summarized in Fig. 4, include: In cell culture, blocking ACAT1SOAT1 increases autophagy mediated lysosomal biogenesis, and increases the capacity to degrade oligomeric Abeta in microglia (215), and increases the capacity to degrade the soluble form of mutant tau in neurons (216). In vivo, blocking ACAT1SOAT1 increases the content of the major oxysterol 24S-hydroxycholesterol and decreases the content of the full-length human APP in the brain of AD mice (137). In addition, when the isoform-non-specific ACATSOAT inhibitor CP-113,818 (212) or CI-1011 (217) was employed, the results showed that APP maturation was inhibited, resulting in reduced Abeta production. Together, these results suggest that the ACATSOAT inhibitors can provide benefits to AD through multiple mechanisms. A detailed review on this topic is available (218). It is known that similar to the brains of AD patients, increases in amyloid plaques have been observed in the aging normal brains, though the

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ACAT1SOAT1 and ACAT2SOAT2 at equal potency (207)], and K604 [an ACAT1SOAT1 specific inhibitor (208)], had advanced

accumulations are much less than those in AD patients (113). In addition, dysfunctions in microglia have been linked with AD (219) and with aging (220). It is thus tempting to speculate that inhibiting ACAT1 SOAT1 in myeloid cells may benefit aging, in addition to benefiting AD. Would inhibiting ACAT1 SOAT1 affect the progression of other human diseases? Recent results in mouse models show that ACAT1SOAT1 is also a potential target for treating various forms of cancer (221), (222), (223). In addition, in a mouse model for atherosclerosis, a recent study showed that, in contrast to the result of knocking out ACAT1SOAT1 in the whole body, knocking out ACAT1SOAT1 in the myeloid cell lineage (including monocytes/macrophages, neutrophils, and eosinophil) actually reduces atherosclerotic lesions (224). Thus, ACAT1SOAT1 may be a potential target to treat multiple diseases. Regarding the toxicity issue-Feeding certain very hydrophobic and highly potent ACATSOAT inhibitors, such as CP-113,818 or ATR101 (225) to guinea pigs or to dogs, caused ER-stress that

tissues examined. The ACATSOAT inhibitors that passed clinical phase 2, including CI 1011 and K604 described earlier, are less hydrophobic than ATR101; these inhibitors do not cause adrenal toxicity. Thus, the adrenal toxicity seems to be caused by using extremely hydrophobic ACATSOAT inhibitors at high doses. Part V-Future Perspectives LOAD is a disease with complex etiology. We consider AD as a special lipid disease. In this review, we chose topics that we are familiar with for in depth discussions. Regretfully, a number of important research topics were left with little or with no discussion. Below we recommend several research areas (A to I) that relate cholesterol metabolism with AD for further investigations. A. The roles of cholesterol and other lipids in affecting the integrity and function of the blood brain barrier. B. The in vivo significance of the ApoE mediated cholesterol homeostasis in the CNS, in cell-type specific manner (depicted in Fig. 3 as a working model). C. The specific functions of ApoJ, ABCA7, or ORPs, or STARDs in affecting cellular cholesterol homeostasis in the CNS, in cell-type specific manner. D. The in vivo significance of APP/Abeta mediated disturbance in cellular cholesterol homeostasis and in membrane biology. E. The possible link between cholesterol overload, lipid raft domain, and oligomeric Abeta accumulation at the synapse mitochondria. F. The roles of various oxysterols in controlling brain cholesterol metabolism in vivo. G. The roles of neurosteroids in affecting brain cholesterol metabolism, and in affecting cognition and behavior.

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led to cell apoptosis in adrenals; the toxicity was restricted to adrenals and did not occur in macrophages, or in other

H. The in vivo significance of the nonvesicular and vesicular cholesterol movements in affecting the lipid raft domains in various cells of the CNS. I. The possible link between tau and cellular cholesterol homeostasis in the CNS. AD is a disease that affects the CNS. To be effective as a primary therapy to treat AD, the candidate drug needs to enter the brain interior. Therefore, we also recommend the development of methods for facile CNS drug delivery as a top priority research endeavor.

Acknowledgment The research conducted in the Chang laboratory is supported by an NIH RO1 Grant AG 037609. Y.Y. is supported by

The authors declared no conflict of interests. We thank members of the Chang Lab for discussion, and thank Bryan Newmann and Rami Ballout for careful editing of this manuscript. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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an AMED-CREST program from Japan Agency for Medical Research and Development (AMED).

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Footnotes The acetyl CoA acetyltransferases are not to be confused with acyl-CoA: cholesterol acyltransfearases 1 and 2, which are abbreviated as ACAT1/2SOAT1/2 in this review.

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In the CNS, ApoE, instead of LDL, is the major cholesterol carrier in the interstitial fluid and cerebral spinal fluid.

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