Neurochemical Research, Vol. 26, Nos. 8/9, September 2001 (©2001), pp. 1045–1068

Intracellular Cholesterol and Phospholipid Trafficking: Comparable Mechanisms in Macrophages and Neuronal Cells* Gerd Schmitz1,2 and Evelyn Orsó1 (Accepted May 31, 2001)

During the past ten years considerable evidences have accumulated that in addition to monocytes/ macrophages, that are implicated in innate immunity and atherogenesis, neuronal cells also exhibit an extensive cellular metabolism. The present study focuses on the major protein players that establish cellular distribution of cholesterol and phospholipids. Evidences are provided that neuronal cells and monocytes/macrophages are equipped with comparable intracellular lipid trafficking mechanisms. Selected examples are presented that trafficking dysfunctions lead to disease development, such as Tangier disease and Niemann-Pick disease type C, or contribute to the pathogenesis of diseases such as Alzheimer disease and atherosclerosis.

KEY WORDS: Monocytes; macrophages; neuronal cells; membrane traffic; cholesterol; phospholipids.

INTRODUCTION

phospholipids are compartmentalized are still not precisely characterized.

Cholesterol and phospholipids are essential cellular constituents of mammalian tissue. They are intimately involved in a broad spectrum of distinct biological processes listed in Table I. Within each cell, complex regulatory and transport mechanisms are present that control levels and appropriate intracellular distribution of cholesterol and phospholipids (Fig. 1). Feedback of synthesis and uptake of the major lipid classes is achieved by transcriptional, translational and post-translational processes and these mechanisms have been extensively investigated. Despite of extensive research in this field however, the exact mechanisms how cholesterol and

Major Players in Membrane Lipid Translocation Many textbooks imply that cellular membranes represent a continuous system, in which considerable lateral movements for membrane soluble or anchored molecules occur. Since the lumenal cavities of membrane-bound subcellular compartments can communicate with each other and with the intracellular space via transport vesicles or tubular membrane connections, all lumenal faces of the membranes in this system can be regarded as topologically equivalent to each other (Fig. 1). The inner/cytoplasmic and outer/lumenal leaflets of membrane bilayers however, exhibit different lipid compositions and most membranes display some degree of lipid asymmetry (1). Furthermore, different subcellular organelles contain unique sets of lipid classes in their membranes and multiple molecular mechanisms maintain this specific lipid distribution (1). Endoplasmic Reticulum Membranes. Membrane(s) of the endoplasmic reticulum (ER) are known to exhibit

1

Institute for Clinical Chemistry and Laboratory Medicine University of Regensburg, Regensburg, Germany. 2 Address reprint request to: Gerd Schmitz, M.D., Ph.D., D.Sc., Institute for Clinical Chemistry and Laboratory Medicine, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, Germany. Tel: ⫹49 941 9446200; Fax: ⫹49 941 9446202; E-mail: [email protected] * Special issue dedicated to Prof. E. Sylvester Vizi.

1045 0364-3190/01/0900–1045$19.50/0 © 2001 Plenum Publishing Corporation

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Schmitz and Orsó Table I. Selected List of Distinct Biological Pathways in which Cholesterol and Phospholipids Are Essentially Involved. ( The References Indicate Some of the Most Updated Comprehensive Reviews and Not Primary Contributions) Function Membrane biogenesis Formation and maintenance of specific plasma membrane microdomains Cell growth and migration Membrane ruffling and formation of ‘podia’ Differentiation of the endoplasmic reticulum and Golgi complex Metabolism of lipoproteins Synthesis of (neuro)steroids Vesiculogenesis (budding and fission) and vesicular traffic Cellular signaling

Reference(s) 1–3 4 –6 7 8,9 10–12 13,14 15 16–18 19,20

Fig. 1. Cellular lipid trafficking pathways and associated transport complexes. The defective routes in two disorders of intracellular cholesterol movement, namely Niemann-Pick type C disease (→ NPC1 pathway) and Tangier disease (→ ABC1 pathway), and their putative compensatory pathways to and from the medial-Golgi complex are indicated (thick arrows). Modified from ref. 21. Abbreviations: ABCAJ: ATP-binding cassette transporter 1, ACAT: acylCoA:cholesterol acyltransferase, ACBP: acylCoA-binding protein (identical with DBI), ALDP: adrenoleukodystrophy protein, ALDR: adrenoleukodystrophy-related protein, BDZ: peripheral type benzodiazepine receptor, CE: cholesteryl ester, Cer: ceramide, CURL: compartment of uncoupling receptors and ligands, DBI: diazepam binding inhibitor, 1,2-DAG: 1,2-diacylglycerol, E: endosome, eE: early endosome, FA: fatty acid, FA-CoA: fatty acyl coenzyme A, HMG-CoA: 3-hydroxy-3-methylglutaryl coenzyme A. HSL: hormone sensitive lipase, L: lysosome, LB: lamellar body, MCII: MHC II compartment, MVB: multi-vesicular body, NCEH: neutral cholesteryl ester hydrolase, NP-C: Niemann-Pick type C (disease). NPC1: Nieman-Pick C1 protein, PC: phosphatidylcholine, P450scc: cytochrome P450 side chain cleavage complex, PMP: peroxisomal membrane protein, SPM: sphingomyelin, StAR: steroidogenic acute regulatory protein. TG: triglyceride, UC: unesterified (free) cholesterol, VAC: voltage activated channel, F-: famcsyl isoprenoid, P-: palmitate.

Lipid Transport Mechanisms of Neurons and Macrophages low levels of lipid asymmetry, often referred as ‘symmetric phospholipid bilayers’, as they are equipped with a set of bidirectional, ultrarapid and non-energyrequiring phospholipid translocases which promptly distribute newly synthesized phospholipids (22). Although ER is a major site for de novo cholesterol synthesis (in addition to the peroxisomes) ER-membranes paradoxically have a low cholesterol content, indicating that cholesterol does not persist in the ER-membranes but is rapidly released (reviewed in 3). The mechanisms for cholesterol sorting include scaffold proteins, such as caveolins or other molecular carriers that recognize and bind cholesterol, Golgi-mediated vesicular and other pathways (discussed later). Cholesterol synthesized via the 3-hydroxy-3methylglutaryl-coenzyme A (HMG-CoA) reductase dependent pathway is imported into the ER lumen through an as yet unidentified transport machinery. On the cytosolic side of the ER, particularly in macrophages (M␾s) and steroidogenic cells, free cholesterol is esterified by action of acylCoA:cholesterol acyltransferase-1 (ACAT1) and deposited in lipid droplets. Recent data indicate that ACAT1 is also enriched in the proximity of the trans-Golgi network (TGN), indicative of additional esterification at the cargo loading step in the vesicular export machinery from the TGN (23). According to earlier studies of Brown and Goldstein (24), the pool size and pattern of fatty acids and their CoAactivated forms in M␾s may critically determine cholesterol synthesis and esterification in these cells, and ACAT1 preferentially esterifies oleyl-CoA but not linoleyl-CoA (24–26 and references therein). This finding intimately links fatty acid metabolism to cholesterol esterification and targeting. While M␾s are specialized scavenging cells to ingest and store excess esterified cholesterol, neuronal cells contain virtually no cholesteryl esters (and triglycerides), indicating that synthesis, uptake and removal of cholesterol in neurons are tightly regulated (7,27 and references therein). In addition to its esterification, lumenal cholesterol can be further processed and incorporated into newly formed lipoproteins. Although the major sites for lipoprotein secretion are the liver and intestine, recent studies suggest that ‘nascent’ lipoproteins can be synthesized in cells distinct from these tissues. In a recent paper caveolin-1 was detected in lipoprotein particles secreted by exocrine pancreatic cell lines in vitro (28). Thus, synthesis and secretion of lipoproteins may be a more general process than expected previously. Golgi Membranes. Cholesterol content of Golgi membranes exhibits great variability. In hepatocytes,

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neuroendocrine and other ‘secretory’ cells Golgi membranes have a particularly low cholesterol:phospholipid ratio, indicating that cholesterol is rapidly removed and sorted from this compartment (2,3). In other cells however, the cholesterol content of TGN may reach the level of plasma membranes (3 and references therein). Plasma Membranes. In contrast to the ER, plasma membranes are highly asymmetric. Over the past years certain major players have been implicated for the maintenance of the phospholipid asymmetry in the plasma membrane (reviewed in 29): (i) Aminophospholipid translocase(s), inward-directed P-type ATPase pumps specific for phosphatidylserine (PS) and phosphatidylethanolamine (PE), are involved in the enrichment of the cytoplasmic leaflet in aminophospholipids. Moreover, aminophospholipid translocase also exhibits an inward translocase activity for phosphatidylcholine (PC)-analogues (30). Interestingly, the yeast aminophospholipid translocase homologue Dsr2p has been reported to act as a Golgi translocase (30–31). (ii) Floppase(s) are outward-directed pumps transporting both choline- and aminophospholipids, and (iii) scramblases facilitate bidirectional translocation across the plasma membrane bilayer for all phospholipid classes (29). Members of the phospholipid scramblase (PLSCR) family are thought to mediate Ca⫹⫹-induced phospholipid movements in activated, injured or apoptotic cells (32). Distribution of PLSCRs shows great differences, as PLSCR1 and PLSCR3 are widely expressed in certain human tissues but not in brain, PLSCR2 seems to be strictly confined for testis, and PLSCR4 is absent from lymphocytes but strongly expressed in many other tissues including brain (32). Although the substrate specificity of these translocases for phospholipids is well established, their role in transbilayer cholesterol movement is less clear. Particular interest has recently been focused on the superfamily of ATP-binding cassette (ABC) transporters that mediate unidirectional membrane translocation of a multitude of specific substrates by binding and hydrolysis of ATP. The ABC lipid transporters for cholesterol, phospholipids, retinoids, very long chain fatty acids, bile acids, leukotrienes, dietary sterols, etc. represent a distinct subset within this gene family (Table II), and they are extensively implicated in the development of certain human diseases (reviewed in 21 and references therein). ABCB1 (MDR1) may represent an exception in this subgroup, as it transports a broad spectrum of lipids and lipid soluble compounds, including sterol(s) and glucosylceramide (30,33 and references therein). A very recent paper demonstrating that overexpression of MDR1 increases cholesterol import

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Schmitz and Orsó

Table II. List of Disease-Associated ABC Lipid transporters. The Designation for ABC Transporters Is According to the Suggestions of the Human Gene Nomenclature Committee. The Conventional (Previous) Designations Are also Indicated in Brackets. (Adapted from Ref. 21.) Regularly Updated Website of Human ABC Transporters (with Links): http://www.med.rug.ml/mdl/humanabc.htm ABC transporter ABCA1 (ABC1) ABCA4 (ABCR)

ABCB4 (MDR3) ABCB11 (BSEP) ABCC2 (MRP2) ABCD1 (ALD) ABCD2 (ALDR) ABCD3 (PMP70) ABCG5 ABCG8

Substrate cholesterol? phospholipid(s)? retinoids N-retinylidene-PE

phosphatidylcholine bile acids glutathione conjugates, leukotriene C4 very long chain fatty acids? fatty acids or fatty acid-CoAs? fatty acids or fatty acid-CoAs? dietary sterol? dietary sterol?

Linked disease

OMIM accession

Familial HDL deficiency, e.g. Tangler disease Stargardt disease Cone-rod dystrophy Age-related macular dystrophy type 2 Retinitis pigmentosa type 19 Progressive familial intra-hepatic cholestasis type 3 Progressive familial intra-hepatic cholestasis type 2 Dubin-Johnson syndrome X chromosome-linked adrenoleukodystrophy Autosome-linked adrenoleukodystrophy Zellweger syndrome type 2? Sitosterolemia Sitosterolemia

#205400 #604091 #601691, #248200 #604116 #153800 #601718 #171060 #601847 #237500 #300100 #601081 #170995(?) #210250 #210250

into IEC-18 epithelial cells provides evidence for the involvement of ABC transporters in cellular cholesterol uptake too (34). Alternatively, the role of MDR1 in cholesterol uptake may be indirect, as the transport mediated by MDR1 is usually outward directed. In addition to its major impact in lipid translocation, a role for MDR1 in the secretion of A␤ amyloid peptide has been suggested by Lam and colleagues (35). Sterol sensitive ABC transporters comprise a further subgroup of the ABC family, partially in overlap with the ABC lipid transporters (36), that are intimately involved in cellular lipid transport processes.

Segregation of Membrane Microdomains Compartmentalized membrane (micro)domains have been investigated extensively in plasma membranes as these structures provide dynamic surface receptor patterns and concentrate signaling molecules. Although less evidenced, other cellular membranes may also develop segregated microdomains that contribute in molecular sorting (6). Microdomain organization in the plasma membrane is very complex and even ‘raft’ microdomains, that are dynamic lateral clusters of cholesterol and glycosphingolipids and mostly confined to the outer plasma membrane leaflet, represent no homogeneous entities. Rafts are often isolated as detergentinsoluble glycolipid membrane complexes (DIGs) after solubilization of cells in non-ionic detergents at low temperature. Using different detergents, content and characteristics of these isolated rafts may vary (6). Indeed, in a very recent report heterogeneity of raft mi-

crodomains has been shown, as the pentaspan plasma membrane protein prominin was localized in cholesterol-based Lubrol-rafts at the distal parts of microvilli in MDCK cells, distinct from cholesterol-sphingolipidbased and alkaline phosphatase containing Triton-rafts in the plane apical plasma membrane (8). Rafts are segregated in the TGN and it is tempting to speculate that different raft subclasses are assembled in distinct vesicles and sorted to specific plasma membrane (sub)domains (8,9). Indeed, segregation of sphingomyelin and cholesterol enriched raft-vesicles from COPI-coated vesicles upon their assembly was demonstrated by Brügger and co-workers (37). A further interesting finding that microvillar localization of Lubrol-rafts and /or prominin displays sensitivity to membrane cholesterol content, since the cholesterol clustering compound saponin induced fusion of small Lubrol-rafts into large rafts, while cholesterol depletion by using methyl-␤-cyclodextrin caused dissociation of the large type of Lubrol-rafts into the small type, which was no longer retained in microvilli (8). The established functional association between a cholesterol-based lipid microdomain and formation of microvilli may have wide implications: in this context cholesterol enriched membrane domains promote membrane ruffling and present a distinct subset of domain associated surface antigens involved in cell adhesion and signaling. This may represent a further, more direct level of cell regulation by sterol(s), in addition to the regulation via sterol-modulated gene expression (9 and references therein). In a recent work the in vitro spreading of human M0 derived M␾s, formation of filopodia and expression of a set of surface molecules

Lipid Transport Mechanisms of Neurons and Macrophages involved in adhesion, differentiation and scavenging showed a correlation with cellular cholesterol loading and/or depletion through a mechanism that involves CDC42, a member of the Rho-family GTPases (38), similar to the formation of sterol-regulated Lubrolrafts and microvilli of MDCK cells (8). Neurons are highly polarized cells and neuronal plasma membranes can be divided into somatodendritic and apical macrodomains, corresponding to basolateral and apical macrodomains of polarized epithel, respectively. These macrodomains contain numerous microdomains equipped with distinct sets of proteins. Raft microdomains have been implicated in targeting of certain axonal membrane proteins in fully mature hippocampal neurons (39). Moreover upregulated sphingomyelin (SPM) synthesis facilitates axonal polarization (40). By contrast, immature hippocampal neurons (stage 3 cells) are unable to form lipid-protein rafts efficiently, suggesting that raft microdomains might have minor impact in the initiation of neuronal polarization. In the latter event the enforced actin dynamics and increased bulk flow of membrane constituents into the axon may play a leading role (41 and references therein). It has to be mentioned however, that axonal shrinkage has been observed in stage 3 cultured hippocampal neurons upon disruption of Golgi trafficking by brefeldin A (42). Taken together, vesicular mechanisms and polarized membrane traffic, distinct of raft assembly may contribute to the establishment of neuronal polarity (reviewed in 41). Similar to the process of domain segregation in spreaded M␾s, signaling events leading to neuronal polarization involve the Rho-family of GTPases (Rho, Rac and CDC42) (43). In addition Notch signaling has also been implicated, particularly in the control of dendritic development (44). Caveolae represent a distinct subset of microdomains, containing caveolin(s), their characteristic hairpin-like, palmitoylated, cholesterol binding scaffold proteins (reviewed in 5). While raft domains occur in all mammalian cells, caveolae and expression of caveolins seem to be not ubiquitious (5–6). Cells lacking caveolae (lymphocytes, some neurons) are able to mediate signaling through rafts (6). Although rafts and caveolae bear similar characteristics in respect of their lipid composition (4,5), they also exhibit significant differences. Both phospholipase D (PLD) isoforms are present in detergent insoluble glycolipidrich fractions (45–46), but only PLD2 is concentrated in caveolin-rich membranes (47). The functional role of caveolae is still unclear: they have been implicated in endocytosis, potocytosis, transcytosis, muscle development, efflux of cellular

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free cholesterol, cell cycle progression and certain signal transduction events (reviewed in 48–49 and 5–6). Presence of caveolae in neuronal cells and M␾s has long been debated. In a recent comprehensive paper Mikol et al. (50) provided clear evidences on the expression of caveolin-1 in Schwann cells, several Schwann cell-derived cell lines and in rat sciatic nerves, furthermore caveolae have been observed in Freund-adjuvant elicited rat peritoneal M␾s (51). Caveolin-1 was found in both soluble (e.g. cytoplasmic) and membrane associated (e.g. caveolar compartment) forms in sciatic nerves and Schwann cells, which is consistent with previous findings on nonneuronal cells that cholesterol-laden caveolin(s) can be transported to the plasma membrane either in a cytosolic chaperone heterocomplex consisting of heat shock protein and cyclophilin in addition to caveolin, or alternatively can be assembled in the Golgi complex involving vesicular mechanisms (3,5 and references therein). Caveolin-1 may regulate cholesterol trafficking during myelination and remyelination since its expression which is upregulated in myelinating Schwann cells, decreases after axotomy as Schwann cells revert to a premyelinating phenotype (50). Caveolins as sensors and modulators of membrane cholesterol levels were recently linked to Alzheimer disease (AD). This implication is based on the following findings: (i) Overexpression of recombinant caveolin-1 (49) or caveolin-3 (52) leads to an enhanced ␣-secretase mediated proteolysis of amyloid precursor protein (APP), which is a direct source of A␤ amyloid peptide, and the latter protein is accumulated in the senile plaques of brains in AD patients. (ii) In the brain APP and A␤ are associated with rafts/ caveolae, as both peptides have been isolated from DIGs, and depletion of cellular cholesterol inhibits A␤ formation (53,54). (iii) Caveolin-3, which was thought to be restricted to muscle cells, is expressed in astroglial cells and it directly interacts with APP and presenilins. Moreover, caveolin-3 is upregulated in astrocytes of AD patients, and this finding may provide a link to the disease pathogenesis (52). Membrane microdomains thus serve a ‘domain platform’ for molecular interactions and cellular signaling.

Cholesterol and Phospholipid Input (Endocytosis) Mammalian cells are able to synthesize free cholesterol de novo from acetate precursors although most do this at a low rate, and internalization of cholesterol

1050 from the extracellular milieu is the predominant mechanism to obtain cholesterol under quiescent conditions (reviewed in 3). Cells in rapid growth and proliferation (embryonic cells or the developing nervous system, for example) are not capable of covering their needs of cholesterol completely from external sources and considerable cholesterol biosynthesis occurs. Thus, phenotypic alterations resulted from genetic defects of cholesterol synthesis mainly involve dysmorphogenesis of multiple organs during embryonic development (55). The clinically most relevant disorders of this group are the monogenetic defects in postsqualene cholesterol biosynthesis, like X-chromosomal linked dominant chondrodysplasia punctata (CDPX2, enzymatic defect of sterol ⌬8-⌬7-isomerase/EBP, OMIM #302960), CHILD syndrome (defect of sterol ⌬8-⌬7isomerase/EBP too, OMIM #308050), desmosterolosis (deficiency of ⌬24-sterolreductase, OMIM #602398) and Smith-Lemli-Opitz syndrome (SLOS, defect of ⌬7-sterolreductase, OMIM #270400) (extensively reviewed in 55–57). Uptake of Cholesterol by Neuronal Cells—the LDL Receptor Supergene Family. The concept of receptor mediated endocytosis as a major mechanism for cellular cholesterol acquisition, emerged from studies of cells (mainly fibroblasts and hepatocytes) from familial hypercholesterolemia patients, led to discovery of the LDL receptor (LDLR) (25) and, later the transcriptional regulation of cellular cholesterol level by a set of molecules with a ‘sterol sensing domain’ (reviewed in 58). The receptor mediated endocytosis of LDL triggers certain events which allow cells to control their intracellular cholesterol content. The free cholesterol, liberated by the lysosomal hydrolysis of LDL cholesteryl esters upon activity of the lysosomal acid lipase (LAL), mediates a series of feedback control mechanisms that protect cells from cholesterol overaccumulation, including a decrease of LDL receptors on the cell surface, suppression of the key enzymes of cholesterol biosynthesis (HMG-CoA synthase and HMGCoA reductase), as well as activation of ACAT1 promoting storage of excess cholesterol in the form of cholesteryl ester lipid droplets (59 and references therein). The LDLR gene defines a supergene family of receptors that, in a concerted action with other cell surface proteins such as cubilin (with megalin), urokinase receptor uPAR (with LRP), Na⫹/H⫹ exchanger NHE3 (with megalin) or others, mediate endocytosis leading to lysosomal degradation of a broad spectrum of macromolecular ligands, including apoE containing

Schmitz and Orsó lipoproteins (59–60). The most prominent members of the LDLR family in mammals are: LDLR, LDLRrelated protein (LRP, CD91), megalin, LRP-3, very low density lipoprotein receptor (VLDLR), LR-3, SorLA-1 and apoE receptor 2 (ApoER2), the latter two receptors seem to be expressed predominantly in the brain (60 and references therein). Lessons from studies with knockout mice have suggested, that besides their well established function in endocytosis, members of the LDLR family are intimately involved in biosynthetic processing of other proteins like sphingolipid activator protein (SAP) or APP, as well as in reproduction, embryonic development and renal absorption and homeostatic regulation of low molecular weight, megalin-binding proteins, including apolipoprotein A-I (apoA-I), insulin, prolactin, retinol-binding protein and others (reviewed in 60). In addition to de novo cholesterol biosynthesis, uptake of lipoprotein derived cholesterol by neuronal cells as well as expression of certain lipoprotein receptors, mainly members of the LDLR family (LDLR, LRP, VLDLR, SorLA-1 and apoER2), by the central nervous system (CNS) and/or the peripheral nervous system (PNS) are also documented (61,62). Expression pattern of lipoprotein receptors in brain areas shows great variability, and the receptor densities even between axon and cell body within the same neuron may differ considerably. While theoretically all classes of circulating lipoproteins can reach peripheral nerve endings or varicose axon terminals, presence of lipoproteins in the cerebrospinal fluid and in the proximity of CNS neurons is more restricted. ApoB-containing lipoproteins (LDL, VLDL, etc.) are absent in CNS, instead, the size of CNS lipoprotein particles is usually very small (approximate diameter 5–15 nm) and they contain apo(lipo)proteins like apoE, apoA-I, apoD and apoJ, rather than apoB (reviewed in 27). Vance et al. (7) have provided evidence supporting a lipoprotein derived cholesterol but not phospholipid acquisition by neuronal cells. They observed severely impaired axonal growth in pravastatin (HMGCoA reductase inhibitor and blocker of endogeneous cholesterol synthesis) treated cultured rat sympathetic neurons. Axonal elongation was completely restored upon administration of exogeneous cholesterol, or serum lipoprotein, or LDL, or HDL to distal axons. Interestingly only LDLs but not HDLs were able to reverse the impaired neuronal growth upon administration of these lipoproteins to the cell bodies. It indicates that receptors involved in LDL uptake are expressed on both perikarya and distal axons, whereas ‘HDL

Lipid Transport Mechanisms of Neurons and Macrophages receptors’ are confined to the distal axons, at least in cultured rat sympathetic neurons derived from superior cervical ganglia (7,62). In the above discussed experiments pravastatin was applied in order to block endogeneous neuronal cholesterol synthesis. Currently the HMG-CoA reductase inhibitors (‘statins’) are widely used as first prescribed therapeutic drugs for reducing the pool size of atherogenic lipoproteins in several forms of hypercholesterolemia (reviewed in 63). On the other hand, peripheral neuropathy has been reported in individual cases after a prolonged statin therapy (64–65), thus it is tempting to speculate that HMG-CoA reductase inhibitors may influence axonal membrane biogenesis. ApoE has been implicated as cholesterol/lipid acceptor involved in scavenging and delivering lipids to the axons and myelin for nerve regeneration. According to Mahley and colleagues, M␾s rapidly migrate to the site of nerve injury and they dramatically increase their synthesis and secretion of apoE (66). The released apoE molecules then take up cholesterol and other lipids from the damaged myelin membranes and/or degenerating axons, pack them into lipoprotein particles and these locally formed lipoproteins serve alternative cholesterol supply for membrane biogenesis upon axonal regeneration, in addition to cholesterol synthesis (66,7 and references therein). In contrast to previous expectations, normal peripheral nerve regeneration has been reported in apoE deficient mice (67,68). An exciting recent concept is that apoE influences neuronal growth through receptor mediated signaling events (7,59). In this scenario reelin, the extracellular matrix secretory product of Cajal-Retzius cells and major regulator of neuronal migration and rearrangement in the developing cerebral cortex, directly binds to the extracellular domain of apoER2 (and VLDLR and cadherin-related neuronal receptors) expressed on the cell surface of migrating neurons, leading to the formation of a multicomponent signaling complex. Upon ligand binding the intracellular NPxY consensus domain of apoER2, involved in receptor targeting to coated pits, may activate Cdk5 via interaction with disabled-1 adaptor protein, furthermore, the PID-domain of differentially spliced apoER2 triggers activation of downstream components such as JNK-interacting protein(s) JIP-1 and /or JIP-2, and c-Jun NH2-terminal kinase (JNK), and c-Jun (69–70,59). Other differential splice variants of apoER2 have been implicated in the clearance of ␣2-macroglobulin / proteinase complexes from the local microenvironment of neurons and /or cerebrospinal fluid. This aspect may further link apoE and/or apoER2 to the pathogenesis of

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AD, since ␣2-macroglobulins have been demonstrated in neuritic plaques of AD patients (71), and more recently Hughes and co-workers (72) have detected a direct molecular association between ␣2-macroglobulin and A␤ peptide by using a yeast two-hybrid screening approach. Thus, the initially more restricted function for the members of LDLR family as endocytosiscompetent receptors has to be extended, as these receptors serve a multitude of distinct events even in neuronal cells (59 and references therein). Wide Variety of Mechanisms for Lipid Acquisition in Macrophages. In contrast to neurons, MOs/M␾s display a variety of distinct mechanisms by which these cells acquire extracellular cholesterol (and other lipids), and some of these pathways may lead to accumulation of large amount of cholesterol and foam cell formation, hallmarks for early events of atherosclerosis. In general, expression of members of the LDLR family in M␾s is generally poor, instead, M␾s exhibit a set of scavenger receptors. These surface receptors have initially been defined as binding sites on M␾s that mediate the uptake and degradation of acetylated LDL (acLDL; or other, modified LDL like oxidized LDL /oxLDL/) and produce massive intracellular cholesterol deposition (73–75). Currently, it is known that the first described receptor belongs to a large supergene family of scavenger receptors. The mechanisms for uptake and lysosomal degradation of modified lipoproteins mediated by scavenger receptors (e.g. class A scavenger receptors) are principally not different from those of LDL receptors, although some important considerations should be taken into account (76 and references therein). (i) Modified lipoproteins do not represent single well-defined entities but rather show a variety of oxidized and /or acetylated and /or enzymatically (or by other factor) modified particles of varying content. The broad spectrum of modified lipoprotein components underlines the weak specificity of scavenger receptors in ligand binding, as most scavenger receptors are multifunctional, recognizing wide variety of ligands. (ii) In contrast to native LDL, modified lipoproteins taken up by scavenger receptors are less rapidly and incompletely degraded in the lysosomal compartment. Unesterified cholesterol and oxysterols however, are able to leave the lysosomes and they locate in all cell membranes. (iii) In contrast to LDL receptor mediated cholesterol internalization, sterol uptake mediated by scavenger receptors is not under negative feedback control, thus scavenger receptor mediated events may really contribute to excessive cholesterol accumulation in M␾s and foam cell formation.

1052 On the basis of recent studies, loading of human MOs with enzymatically modified LDL according to Bhakdi et al. (77), but not with acLDL or oxLDL, leads to a cellular phenotype of lipid laden M␾s, closely resembling foam cells and marked upregulation of Fc receptors (CD16a, CD32, CD64), but only slight increase in the expression of CD36 and scavenger receptor BI (SR-BI) or CLA-1. Enzymatic modification of lipoproteins by inflammatory hydrolases occurs very likely in vivo, in ‘response to retention’ and subsequent interaction with secretory cell products via paracrine routes, as suggested by Kovanen (78). In this scenario enzymatic modification may induce limited proteolysis, cross-linking and aggregation of LDL and such particles may prefer Fc receptor mediated (via “pseudo-opsonization”) and subsequent phagocytic or pinocytic mechanisms upon entering the cells rather than scavenger receptor-mediated pathways (79). Aggregated LDLs and chylomicron remnants can be taken up by a cytochalasin D sensitive phagocytic pathway (80,81). According to Tabas, large ␤-VLDL particles first enter peripheral surface-connected tubuli of M␾s before they undergo lysosomal degradation (82–83). This mechanism seems to be clearly distinct of clathrincoated pit-linked vesicular events leading to a rapid cholesterol esterification (26). Patocytosis represent an actin-dependent characteristic uptake mechanism for aggregated lipoproteins, cholesterol-phospholipid liposomes, microcrystalline cholesterol and other hydrophobic compounds first observed by Kruth et al. (84) in human MO-derived M␾s. Patocytosis is not associated with membrane fission, instead materials enter a surface connected labyrinthic system of M␾s in which they can persist. Alternatively, the acquired macromolecules or particles can be resecreted or partially exposed to lysosomes. The lysosomal degradation of patocytosed LDL aggregates is usually poor (26 and references therein). The selective uptake of HDL cholesteryl esters defines a high capacity, hormone-inducible sterol delivery process by which the core cholesteryl ester is taken into the cell (reviewed in 85–87). This pathway is fundamentally different from the LDL receptor pathway in that binding of HDL to the cell surface is not associated with endocytosis and lysosomal degradation of the lipoprotein particle (86 and references therein). Instead, after cholesteryl esters are transferred to cells, cholesterol-depleted HDL is released from the cell surface. This selective cholesteryl ester uptake appears to be essential for HDL-cholesterol delivery to the liver as a final step of ‘reverse cholesterol

Schmitz and Orsó transport’ or to steroidogenic tissues (mainly of rodents) both in vitro and in vivo, but less evidenced for M␾s. This mechanism also processes LDL and intermediate density lipoprotein (IDL) particles (86,88). The description of the scavenger receptor class B type I (SR-BI) as ‘HDL receptor’ (89) provided the first candidate receptor for the selective process. The detailed molecular mechanism of the selective HDL cholesteryl ester uptake pathway is not yet clear and various hypotheses are postulated (reviewed in 86–87). According to a concept preferred by Krieger and colleagues, upon HDL binding to caveolar SR-BI, HDL cholesteryl esters are rapidly and reversibly incorporated into the plasma membrane facilitating the collision of the HDL particle with the plasma membrane, which is followed by irreversible internalization of cholesterol into intracellular compartments (90 –91, 87). Another model was presented by Williams et al., in which SR-BI forms a hydrophobic channel to permit HDL exposure to the plasma membrane, and cholesteryl ester molecules can diffuse along the concentration gradient from HDL to the plasma membrane (92). This latter hypothesis is supported by the finding that activation energy for SR-BI mediated cholesteryl ester uptake is quite low, indicating that cholesteryl ester uptake occurs via a non-aqueous pathway. Since the plasma membrane bilayer can hold only a very low amount of cholesteryl esters (especially in raft/ caveolar microdomains), ongoing selective cholesteryl ester uptake requires either hydrolysis of cholesteryl esters or their rapid transfer to intracellular sites, such as lipid droplets (86). A further hypothesis proposes, that cholesteryl esters are rapidly converted into free cholesterol within the membrane during the interaction of HDL with caveolae. Free cholesterol is then “flopped” from the outer to the inner leaflet probably by a not yet identified “sterol floppase”, and finally transported from the inner leaflet into the cell interior. In this scenario SR-BI co-associates with these putative sterol floppase and mediates capture and binding of HDL particles to the receptor complex. The involvement of a putative sterol floppase is supported by the following evidences: (i) cholesterol but not cholesteryl esters are present in caveolar membrane domains and cholesterol loading leads to caveolar internalization (93), (ii) there is evidence for neutral cholesteryl ester hydrolase (NCEH) activity in the plasma membrane (94), (iii) SR-BI mediates transfer of more free cholesterol than cholesteryl esters to cells from either HDL or LDL (85), (iv) various tissue associated lipases (hepatic lipase and others) and plasma lipid transfer proteins

Lipid Transport Mechanisms of Neurons and Macrophages (cholesteryl ester transfer protein, CETP at least) may remodel lipid components of HDL and thus modulate the ability of SR-BI to mediate selective HDL cholesterol uptake (95–96). Another member of the class B scavenger receptors CD36, which is also localized in caveolar/raft domains and can mediate high affinity HDL binding, mediates poor HDL lipid transfer into the cells (97), but it has recently been identified as fatty acid and anionic phospholipid transporter (98,99 and references therein).

Cholesterol and Phospholipid Output (Secretion) Mammalian cells are equipped with different mechanisms in order to avoid toxicity of free cholesterol. In addition to the previously discussed intracellular storage of cholesterol in lipid droplets, multiple pathways exist for removal of cellular cholesterol. The mobilization of cellular cholesterol to the plasma membrane and efflux to extracellular acceptors, such as HDL (mainly HDL3 subclass) or its major protein constituent apoA-I, is an important ubiquitious mechanism of extrahepatic cells involved in net removal and maintenance of cellular cholesterol. Upon cellular efflux, HDL3 acquires free cholesterol, followed by lecithin:cholesterol acyltransferase (LCAT)-generated esterification in HDL, which increases the capacity of HDL3 to accept further cholesterol molecules. It is the initial step in ‘reverse cholesterol transport’, which transfers cholesterol from peripheral cells to the liver. Enzymatic interactions of HDL can alter the contents of the particles and, thus influence the capability of cholesterol acquisition, as suggested for phospholipid transfer protein (PLTP, 100). Cholesterol efflux involves at least two independent pathways, ‘diffusional efflux’ and ‘binding- and translocation dependent efflux’, and the latter process operates through a direct binding of apoA-I/HDL to the cell membrane and causes the transfer of intracellular cholesterol to the plasma membrane and, subsequently to the lipid acceptor (reviewed extensively in 101–103). Despite of certain detailed investigations the precise molecular mechanism how cellular lipid efflux occurs is largely unknown, but ABCA1 and SRBI have been suggested as major players, at least in the apoA-I mediated efflux. Tangier disease (TD) fibroblasts, showing impaired cellular cholesterol and phospholipid efflux are mutant for ABCA1 (104 –107), furthermore overexpression of ABCA1 leads to an enhanced apoA-I mediated efflux of cholesterol and PC

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in RAW 264.7 cells (108). The ABCA1-dependent cholesterol and PC efflux to apoA-I (in human vascular cells) occurs very likely in a two-step process, in which apoA-I acquires PC from the plasma membrane prior to cholesterol desorption from caveolae (109), as PC-containing apoA-I being a better cholesterol acceptor than lipid free apoA-I. Based on a recent model provided by Oram (110) and Schmitz and Langmann (111), ABCA1 may function as a direct plasma membrane transporter which translocates cholesterol and phospholipid, delivered by vesicular pathways and, incorporated into the inner plasma membrane leaflet. Similar to the pore-structure considered for MDR1 (112), ABCA1 forms an aqueous chamber upon apoA-I binding that protrudes from the cell and pumps the cholesterol and phospholipid molecules out for removal. This hypothetical model implies that ABCA1 is an active pump for cholesterol (and phospholipids), similar to several other members of the ABC-superfamily. Very recently Remaley and colleagues (113) have shown that ABCA1-mediated cellular lipid efflux is not resticted to apoA-I, but rather is a more general phenomenon for other exchangeable apo(lipo)proteins containing amphipathic helical domains, such as apoA-II, apoA-IV, apoC-I, apoC-II, apoC-III and apoE. Presence of ABCA1 mRNA has been detected in human brain, predominantly in the putamen and in the occipital lobe, although its expression is one order of magnitude lower than in M␾s, which are one of the highest ABCA1-containing cell types (114). ApoE has been suggested to promote cholesterol and PC efflux from cultured neurons as astrocytes through a mechanism that likely results apoE-containing ‘nascent neuronal HDL particles’ (115). Efflux for both classes of lipids was blocked by heparinase and lactoferrin, indicating that heparane sulfate proteoglycans and/or LRP may interact with apoE upon efflux. Only cholesterol but not PC efflux showed sensitivity to brefeldin A or monensin, and inhibition of protein kinase C (PKC) (115), suggesting that the molecular mechanisms for lipid efflux between neuronal and extraneuronal cells may differ. The other candidate protein that is likely involved in cellular lipid efflux is SR-BI, as it can mediate both the influx (‘selective uptake’) and efflux of cholesterol from cells to lipoproteins and other acceptors (85, 116–117). It has recently been shown that there is a bidirectional exchange of free cholesterol between cells and HDL (and LDL) lipoproteins that is facilitated by SR-BI and, that the direction of cholesterol flux is not concentration dependent (85). The further

1054 interesting feature of SR-BI is that, similar to ABCA1, efflux to HDL seems to be dependent on the phospholipid content of the acceptor (117). This indicates that the capability of apoA-I (via the C-terminal domain) to bind phospholipid is required for efficient cholesterol efflux as was suggested by earlier reports (118), or alternatively a cholesterol-phospholipid co-transport may occur upon efflux. The major sites for cholesterol efflux are very likely the caveolar/raft domains of the plasma membrane, where most SR-BI receptors and probably ABCA1 locate and these domains are enriched in cholesterol and (glyco)sphingolipids. An analogous cascade mechanism to the above mentioned signaling for apoER2 may exist for cellular lipid efflux, since considerable evidences have been adduced that the (putative) efflux mediating ‘HDL receptor’ may act as a signaling protein. In this concept binding of HDL and /or apoA-I to the cell surface is accompanied with activation and translocation of protein kinase C (PKC; preferentially ␣, ␦, ⑀ and ␨ subclasses) to the plasma membrane, through the phosphoinositol pathway (119–121). Surface binding of HDL3 leads to certain protein phosphorylation, including ERK1 and ERK2 mitogen-activated protein kinases (122) and hydrolysis of PC by phospholipases C and D (PLC and PLD, respectively) (123–125). Promoted cholesterol efflux has also been linked to enhanced protein kinase A (PKA) activity (126). Drobnik et al. (127) reported growth abnormalities and G2-arrested cell cycle related to impaired Golgi function and sphingolipid signaling in ABCA1 deficient TD fibroblasts. By contrast, in a very recent report Nofer and co-workers (128) have observed normal cell proliferation, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2) turnover and Ca⫹⫹ mobilization in TD fibroblasts in response to HDL, while the cholesterol efflux was significantly reduced. They have concluded, that abnormal PI-PLC activation does not account for a defective cholesterol translocation and efflux in TD cells as HDL-associated lysosphingolipid induced PI-PLC activation was paralleled with mitogenesis but not cholesterol efflux (128). The current concept is that the above mentioned signaling mechanisms may contribute to lipid translocation from various intracellular pools to the cell membrane and present them for efflux by desorption (126). Besides ABCA1 and/or SR-BI mediated mechanisms, extrahepatic cells, particularly M␾s contain a set of further machineries for cellular lipid removal. Earlier studies have shown that HDLs may enter M␾s as holoparticles, take up cholesterol through interaction with lipid droplets and finally undergo for resecretion that resembles to retroendocytosis (129,130).

Schmitz and Orsó It is consistent with previous findings that NCEHmediated cholesteryl ester hydrolysis but not cholesterol acceptor(s) correlate with sterol removal (131). Macrophages and neuronal cells are both capable of synthesizing and secreting amphipathic helical apoE molecules into the extracellular space that may self-associate into multimers in the aqueous cellular microenvironment, and acquire/integrate lipids from extracellular leaflets of plasma membranes. In this scenario apoE-containing nascent HDLs can be formed via autocrine mechanisms. It can not be excluded however, that apoE molecules acquire phospholipids and cholesterol prior to their release along the intracellular secretory pathway, thus small nascent lipoprotein holoparticles are secreted (132,133). A further indirect evidence for a ‘brain specific’lipoprotein traffic has been provided by findings that the major lipoprotein remodeling enzymes (phospholipid transfer protein /PLTP/, CETP etc.) are widely expressed in the brain. In addition to the ovary, thymus, placenta and heart, PLTP mRNA expression has also been detected in brain (134), moreover CETP activity was found in conditioned medium from human neuroblastoma and neuroglioma cells, as well as sheep choroid plexus (135). According to Yamada and co-workers (136) expression of CETP is confined for astroglial cells, and CETP-positive astrocytes may play a role in the tissue repair in AD brain. Oxidative mechanisms represent additional facilities for cholesterol removal from extrahepatic tissues. Sterol 27-hydroxylase, a widely expressed species of the ancient mitochondrial cytochrome P450 family, mediates a high capacity conversion of cholesterol into both 27-hydroxycholesterol and 3␤hydroxy-5-cholestenoic acid, which can be effluxed to molecular acceptors from the cells and transported to the liver, further oxidized in hepatocytes and finally secreted into the bile (137,138 and references therein). These polar oxysterols are formed within the mitochondrion followed mitochondrial cholesterol uptake and transferred to the plasma membrane by various pathways, partially by oxysterol binding protein OSBP-mediated mechanisms. The sterol 27-hydroxylase system is particularly important for removal of intracellular cholesterol in M␾s exposed to low level of lipoproteins, as HDL serves acceptor mainly for 27hydroxycholesterol but major part of 3␤-hydroxy-5cholestenoic acid associates to albumin in the circulation (reviewed in 138). The gene for sterol 27-hydroxylase is mutated in cerebrotendinous xanthomatosis (CTX, OMIM #213700), characterized by neuronal and xanthomal

Lipid Transport Mechanisms of Neurons and Macrophages accumulation of cholesterol and cholestanol, cataracta and early atherosclerosis “despite” of low (or normal) levels of plasma cholesterol. CTX patients show reduced formation of bile acids which is accompanied with compensatory increase of endogenous cholesterol synthesis and cholesterol 7␣-hydroxylation (the microsomal /CYP7A1/ and mitochondrial/CYP7B1/ cholesterol 7␣-hydroxylases are both involved) (139) and, thus may explain the low LDL levels in plasma (138). Recently a brain specific hydroxylase (24S-hydroxylase) has been described which converts cholesterol into a blood-brain permeable 24S-hydroxycholesterol that shows a significant flux from brain to the liver. This flux is likely to be of importance for neuronal cholesterol homeostasis (138,140). Extrahepatic formed polar oxysterols account for approximately 5% of total bile acid formation, thus they contribute to the total sterol homeostasis of the body (138 and references therein). Oxysterol binding proteins (OSBP itself and the OSBP-related proteins /ORPs/) have been implicated as major players in cholesterol esterification, translocation and cellular sterol balance (141). Stably transfected cells overexpressing OSBP were shown to display a marked decrease in cholesteryl ester synthesis which is paralleled with an increase in cholesterol synthesis (142). In addition, forced OSBP expression resulted in the upregulation of mRNA levels of a set of sterol-regulated genes, including HMG-CoA reductase, HMG-CoA synthase and the LDL receptor (142). The known interaction of OSBP with cellular membranes is attributed to its N-terminal pleckstrin homology domain, which likely associates with PIP 2 and / or related phosphoinositide(s) on membranes of the Golgi complex (142–143). In addition, OSBP regulates the interaction of PC or PI with the PI-transfer protein in a sterol ependent manner (143) and thereby modulates CDP-choline cytidyltransferase, the key enzyme of PC biosynthesis. The C-terminal moiety of OSBP accounts for oxysterol binding and in case it is removed, the truncated protein localizes to the Golgi apparatus spontaneously (144). OSBP contains a socalled GAP-domain (145) that likely interacts with the Rho-family member CDC42 and thereby functions as a modulator of vesicular trafficking through the Golgi complex. Golgi disruption by brefeldin A reduces the action of oxysterols on cholesterol synthesis (146), moreover Kes1, the OSBP homologue in Saccharomyces has been linked to the biogenesis of Golgiderived vesicles (147). The Osh1p, a further OSBP homologue, also contains a pleckstrin homology domain which is sufficient to target fusion proteins to mammalian Golgi membranes (143).

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While OSBP is ubiquitiously expressed in cells with predominance in kidney and liver tissues, ORPs show higher tissue specificity and the extent of their expression levels is different (148 and references therein). In neuronal cells ORP-1, ORP-2, ORP-4 and ORP-6 mRNAs are most abundantly expressed (148). The ORP-1 mRNA has been detected at high levels in the cortical areas of human brain and displays sterolregulated expression in cultured human neuroblastoma cells (148). In summary, current evidences support the concept that OSBPs play multiple roles in organelle lipid composition, cellular sterol and phospholipid balance and membrane traffic.

Vesicular Transport and Sorting Vesicular transport is one of the major processes involved in cellular trafficking of certain molecular classes. In this process, membranous carrier structures bud and pinch off a donor compartment and fuse with the recipient one, thus delivering their content and vesicle membrane components to the target organelle. In general, steps of vesicular transport consist of the same stages irrespective of donor and acceptor membranes, including (i) sorting of proteins and lipids and formation of transport vesicles, (ii) movement of the vesicles along cytoskeletal filaments, (iii) recognition of the target organelle and fusion of the vesicles with the acceptor membrane/compartment. Deficiencies in these steps lead to dysfunction of the vesicular transport machinery (extensively reviewed in 149). Previous research approaches have focused on establishing protein components and their function in membrane traffic and vesicle formation. More recently the lipid-protein interactions have extensively been implicated in vesicle biogenesis, fission and fusion. The major lipid binding proteins are listed in Table III. The signaling phosphoinositide molecules (notably PIP3, PIP2, PIP and IP3) formed in the donor membranes play critical roles in lipid-based trageting mechanisms and in the membrane interactions with cytosolic proteins involved in budding and pinching-off (19–20). Their down-regulator synaptojanin, which exerts poly-phosphoinositide phosphatase activity, has been linked to uncoating of synaptic vesicles upon their fission from the donor membrane. It has also been demonstrated that formation of Golgi-derived COPI-coated vesicles critically requires fatty acyl-CoA (150), furthermore that the fatty acyl-CoA molecules are involved in the fission step (151). Synaptophysin, one of the major cholesterol binding membrane constituent of neuronal

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Schmitz and Orsó Table III. List of the Major Lipid Binding Transporter Proteins Implicated in Membrane Traffic and Their Associated Disorders

Major players in membrane lipid traffic/vesicular transport 1. Plasma membrane associated translocases/transporters aminophospholipid translocases scramblases floppases ABC transporters (many of them; see Table 2.) caveolin(s) 2. Endo-lysosomal transporters/docking proteins NPC1 protein (Niemann-Pick disease C protein 1) HE-1 protein (Niemann-Pick disease C protein 2) lysosomal acid lipase (LAL) acidic sphingomyelinase ceramidase ␣-galactosidase A palmitoyl-protein thioesterase 1 (PPT1) oxysterol binding protein (OSBP) OSBP-related proteins (ORPs) OCRL-1 3. Endoplasmic reticulum associated molecules HMG-CoA reductase ACAT2 DGAT2 microsomal transfer proteins (MTPs) 4. Golgi and secretory vesicle associated molecules endophilins (lysophosphatidic acid acyltransferases) BARS-50 (CtBP) synaptojanin (poly-phosphoinositide phosphatase) synaptophysin phospholipase D 5. Lipid droplet associated transporters ACAT1 DGAT1 neutral cholesteryl ester hydrolase (NCEH) hormone sensitive lipase (HSL) 6. Mitochondrial transporters steroidogenic acute regulatory protein (StAR) MLN64(?) peripheral benzodiazepine receptor (PBR) 7. Peroxisomal translocases/transporters peroxisomal ABC transporters (see Table 2.) PEX-1 protein PHYH (phytanoyl-COA hydroxylase)

Associated human genetic deficiency

? (Scott syndrome?) ? See (partially) Table 2. ?, Caveolin-3 is deficient in autosomal dominant limb-girdle muscular dystrophy Niemann-Pick disease type C, form 1 Niemann-Pick disease type C, form 2 Wolman disease and Cholesteryl ester storage disease Niemann-Pick disease A and B (NPA, NPB) Farber disease Fabry disease Infantile neuronal ceroid lipofuscinosis (CLN1) ? ? Oculocerebrorenal syndrome (Loewe type) (Deficiency is embryolethal) ? ? ? ? ? ? ? (No obvious phenotype in knockout mice) ? ? ? ? ? Congenital lipoid adrenal hyperplasia ? (Deficiency is probably embryolethal) See (partially) Table 2. Infantile Refsum disease Refsum disease (with pipecolicacidemia)

synaptic vesicles, has recently been implicated in the generation of synaptic-like microvesicles (SLMVs) in PC12 neuroendocrine cells (152). Current data favour the suggestion that the interaction of synaptophysin with cholesterol substantially alter/promote membrane curvature and thereby support the segregation of SLMVs from the plasma membrane. The cytoplasmic lysophosphatidic acid (LPA) acyltransferase BARS-50 (brefeldin A-induced ADP-ribosylated substrate of 50 kD) mediates the fission of Golgi membrane tubules to Golgi-derived vesicles in an ADP-ribosylation dep-

endent manner (153). Similar to BARS-50, a further protein with LPA acyltransferase activity, named endophilin A1, seems to be essential for synaptic vesicle endocytosis (154) through an interaction with dynamin and/or amphiphysin (both required for vesicle formation). Endophilin A1 defines a small protein family, currently consisting of four members in mammals (endophilins A1–A3 and endophilin B), although endophilin is also present in other species including Drosophila, C. elegans, S. cerevisiae indicating an evolutionary conservation with the endophilin family

Lipid Transport Mechanisms of Neurons and Macrophages (155). While endophilin A2 is ubiquitiously expressed in many tissues, endophilin A1 seems to be neuron specific (155 and references therein). The consequence of the LPA acyltransferase activity of endophilin A1 is the conversion of LPA and arachidonoyl-CoA (ARA-CoA) to phosphatidic acid (PA). According Huttner and Schmidt (155) an opposite membrane insertion of a cone-shaped, negative curvature inducer lipid Ara-CoA and a wedge-shaped LPA results a cylindrical PA that promotes ‘negative membrane curvature’. By contrast, insertion of a negative cone-shaped LPA promotes ‘positive membrane curvature’. Furthermore by analogy to the G-protein cycle, endophilin A1 likely exists in two states, a substrate-bound (LPA/Ara) state which supports the ‘positive membrane curvature’, e.g. does not promote budding and fission, and a product-bound (PA) state, which contributes to the ‘negative membrane curvature’, e.g. promotes membrane invagination and pinching-off. In this scenario the shape of membrane lipids is a key determinant of membrane curvature (extensively reviewed in 155). A recent study of Kobayashi et al. (156) emphasizes that lyso-bisphosphatidic acid (LBPA), which is enriched in the endo-lysosomal compartment, is critically involved in the Niemann-Pick disease C protein 1 (NPC1)-mediated egress of cholesterol from the acidic (sub)compartment. Thus distinct classes of lipids, such as lysophospholipids or hexagonal phase-preferring lipids (i.e. PE) may promote positive or negative membrane curvature, repectively (156–157). In case of sphingolipids the head group specificity seems to determine their trafficking by modulating membrane invaginations and vesicle formation (157). This established link between membrane lipid modifications and vesicle formation and/or membrane fission suggests further implications. It is tempting to speculate for example, that molecular mechanisms working in the opposite direction to membrane fission may promote vesicular fusion with acceptor membrane(s).

Non-Vesicular Mechanisms Cellular compartmentalization requires targetspecific transport mechanisms among intracellular organelles. Little is known at present about cholesterol transport in the cytoplasm but certain phospholipid transfer proteins (PLTPs) have been identified with the capability of transferring lipids from donor to acceptor membranes and thus implicated not only in

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vesicular transport but also in non-specific sterol movement in the cytoplasm (reviewed in 158). Three major classes of soluble lipid transfer proteins have been investigated in details: (i) The monospecific PLTPs (e.g. PC transfer protein) exhibit an exclusive specificity for one phospholipid class, thus they have no impact in sterol transfer. (ii) The oligospecific PLTPs recognize a restricted set of phospholipid species as transfer substrates, thus their role as efficient sterol transporter seems to be unlikely. The PI/PC transfer proteins contain only one phospholipid binding site per protein monomer and they perform the high affinity binding of two dissimilar phospholipids (PI and PC) in a mutually exclusive binding reaction (159). It has to be mentioned however, that PI serves primary ligand for the mammalian transfer protein(s) as the rate of transfer for PI about 20-fold greater than for PC (159). Another interesting feature of these oligospecific PLTPs is a very high level of primary sequence conservation across species boundaries, moreover they are extensively involved at multiple sites of the secretory pathway as well as in signaling (reviewed in 160). (iii) The non-specific transfer proteins are capable of mobilizing sterols, glycolipids and most phospholipid species. The best studied member of this protein class is the sterol carrier protein-2 (SCP-2) which promotes exchange of a wide variety of sterols and all classes of phospholipids between membranes. In addition, SCP-2 increases the synthesis and esterification of cholesterol as well as bile acid synthesis in various in vitro assays (see in 3,161). Overexpression of SCP-2 in hepatocytic cell lines results enhanced cholesterol cycling and altered cellular cholesterol pool (162). SCP-2 defines an ancient, small SCP-2 gene family of four members (SCP-2, SCP-x, D-bifunctional enzyme, UNC-24/ hSLP-1) involved in certain peroxisomal functions (161 and references therein). Their common feature is the presence of an SCP-2like domain at the C-terminus. Particularly low levels of SCP-2 have been detected in cells affected by Zellweger syndrome, a primary peroxisomal biogenesis disorder, without mutations in the SCP-2 gene. At present no human disease has been linked to genetic SCP-2 deficiency (161). Moreover, mice with targeted inactivation of Scp-2 show normal cholesterol metabolism, instead they are incapable of catabolizing methyl-brached fatty acylCoAs (163). Taken together, the current data favor the concept that SCP2 functions as fatty acylCoA transfer molecule involved in peroxisomal fatty acid metabolism rather than cytosolic sterol carrier (Table III). The role of

1058 SCP-2 in cytoplasmic cholesterol transport seems to be an indirect or secondary phenomenon (161). There is also considerable evidence that a significant part of cholesterol is rapidly transported from the ER to the plasma membrane, first to caveolae/raft microdomains and then to the non-caveolar regions (reviewed in 3). This transport process is unaltered by compounds disrupting the cytoskeleton (colchicine, cytochalasin, etc.) or the Golgi-complex (monensin, brefeldin A) but significantly inhibited by depletion of the cellular ATP-pool. A cytosolic chaperone heterocomplex consisting of caveolin, heat shock protein and cyclophilin (in a dynamic assembly with steroid hormone receptors, such as progesterone receptor) may

Schmitz and Orsó mediate this transport step (3,157,164), thus nonvesicular mechanisms are extensively involved in intracellular movement of sterols. According to a further concept certain lipid classes, including cholesterol and the major phospholipids can be transported through the cellular membrane continuum. An excellent experimental demonstration of this membrane continuity has been provided by Scow and Blanchette-Mackie (165; reviewed in 166) in cells of brown and white adipose tissue, showing that fatty acids (FAs) and monoacylglycerols are translocated via lateral movement along interfacial membrane continuum to mitochondria (Figure 2).

Fig. 2. Route proposed for transport of fatty acids from capillary lumen to adipocytes by lateral movement in an interfacial continuum of cell membranes. The interfacial continuum (represented by a broad stippled line) is composed of the external leaflets of the plasma membrane of endothelium (1), the membrane of transcellular channels in endothelium (2), the plasma membrane of adipocytes (3), the membrane of surfaceconnected channels (4), the membrane of endoplasmic reticulum (5), and the outer mitochondrial membrane in adipocytes (6). The external leaflets of the endothelial cell (1) and adipocyte (3) are continuous at the contact sites. The interfacial continuum would include the chylomicron surface film when the surface film is fused temporarily with the external leaflet of the endothelial plasma membrane (1). Fatty acids formed by lipoprotein lipase (site of lipolysis) enter the continuum in capillary lumen, move in the continuum to the endoplasmic reticulum of the adipocyte, leave the continuum after re-esterification (site of esterification) to triacylglycerol (TG) and then accumulate as lipid droplet between leaflets of the reticulum. Fatty acids formed by action of tissue lipase on intracellular TGs would re-enter the continuum at the site of lipolysis, move along the continuum to mitochondria, and leave the continuum after activation of fatty acids in the outer mitochondrial membrane for transfer to the inner mitochondrial membrane (7). Note that the cellular projections (spikes) of the endothelial cell penetrate the basement membrane (10). Cytoplasmic leaflets of membranes are represented by broad white lines. Crista (8) and inner mitochondrial matrix (9) are also indicated. (Adapted from ref. 165., with the permission of E. J. Blanchette-Mackie.)

Lipid Transport Mechanisms of Neurons and Macrophages In this model, site of lipolysis (by endotheliumassociated lipoprotein lipase), or site(s) of esterification and lipolysis (in association with lipid droplets), or site of mitochondrial acyl activation in brown or white adipocytes are ‘traffic junctions’ equipped with specific ‘gatekeeper’ proteins that mediate transfer and/or regulation (Figure 2). Lamellar bodies are specialized subcellular organelles (0.1–2.5 ␮m) that have been detected by electron microscopy in various cell types under normal and pathological conditions. They have been referred to be specifically regulated forms for storage and secretion of certain lipids, including sterols, phospholipids, fatty acids and small amount of proteins (extensively reviewed in 167). The best characterized lamellar bodies are produced by pneumocyte type II cells as secretory forms of the lung surfactant system. Further examples for physiological lamellar body formation are the Odland bodies of the skin as constituents of the waterproof “sealing layers” of epithelial cells, the hydrophobic protective lining of the gastric mucosa, the lamellar phosholipids as major sterol carriers in bile, and others. Accumulation of lamellar bodies have been implicated in various diseases such as infant and adult respiratory distress syndromes, development of fatty streaks and atherosclerotic plaques, inflammatory bowel diseases, psoriasis, ichtyosis, certain lysosomal storage diseases, etc. (167). Regarding to the alternative transport of membrane soluble molecules through the interfacial membrane continuum, compounds in lamellar form can accumulate under any circumstances when gatekeepers are impaired, via overcrowding the membrane compartment direct upstream of the defected gatekeeper (168). In this scenario the precise subcellular localization and composition of the first accumulated lamellar material may indicate the nature of damage. Thus as an example it is obvious, that lysosomal SPM accumulates in fingerprint-lamellar bodies in Niemann-Pick type A (NPA) disease, since acid sphingomyelinase as lysosomal gatekeeper is deficient in this inborn error. Macrophage-Neuron Interactions in Traffic Disease Development There are several conditions in the pathogenesis of vesicular traffic disorders in which macrophageneuron interactions may play a role in disease development and/or progression. Some selected examples are presented here. Familial HDL Deficiency: Tangier Disease (TD). Tangier disease (OMIM #205400) is the ABCA1-

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linked, autosomal recessive inherited HDL deficiency characterized by an impaired apoA-1 mediated cellular efflux of cholesterol and phospholipids due to a cellular defect in a Golgi to plasma membrane directed cholesterol transport (see Cholesterol and Phospholipid Output (Secretion) section). The major symptoms of TD including hyperplastic, orange tonsils or ‘pharyngeal plaques’, splenomegaly and lymphadenopathy are attributed as consequences of the excessive accumulation of cholesteryl esters in cells of the reticuloendothelial system (110 and references therein). Peripheral neuropathy is one of the most frequent clinical signs of TD, especially in advanced forms of the disease (169,170). The neuropathy may be presented either as an asymptomatic or transient or relapsing multifocal, demyelinating mono- or polyneuropathy, or as a progressive syringomyelia-like form. Morphologically the first form is characterized by de- and remyelination of peripheral nerve(s) with mild histiocytic infiltration, and vacuolar lipid inclusions in Schwann cells. The latter form is accompanied with axonal degeneration of small unmyelinated and myelinated fibers and small dorsal ganglia, thus explaining the preferential loss of thermo- and nociception. Lipid inclusions are present in various neuronal cells, Schwann cells, endoneurial fibroblasts, cells in the vasa nervorum, etc. (169,170). At cellular level certain other abnormalities of intracellular lipid metabolism have been reported. In contrast to normal MOs/M␾s, these cells in TD individuals degrade internalized HDL in unusual lysosomes (129,171), a process that is promoted by cholesterol loading with acLDL (172). Characteristic ultrastructural findings in various cell types from TD patients include an abnormal morphology of the Golgi apparatus and the presence of unusual translucent and dense lysosomes. The cisternae of the TGN are dilated and markedly hyperplastic, and the altered dense lysosomes appear to degrade Golgi-material (172). The defect in intracellular lipid trafficking is associated with an abnormal ceramide-backbone and glycerophospholipid turnover (173), which is probably related to the significant reduction in the in vitro growth rate and an increase in their cell surface area of fibroblasts from TD patients (127). The abnormal lipid deposition in Schwann cells is generally implicated to be responsible for the peripheral neuropathy associated with ABCA1 deficiency (110 and references therein). It can not be ruled out however, that the impaired Golgi to plasma membrane directed vesicular transport involves primarily the neurons, thus a mild neuronal disturbance contributes

1060 to the TD neuropathology. It is consistent with recent preliminary findings that Abca1 deficient mice (DBA/ 1J/C57BI/6 knockouts) develop slow reactivity and slight developmental deficiencies (Orsó E. et al., unpublished data). By contrast, Christiansen-Weber et al. (174) have failed to observe any significant neural defect by using tail-flick test and hot-plate test in another Abca1 knockout murine model (sv129/C57BI/6 knockouts). Niemann-Pick Type C (NPC) Disease. The NPC disease (OMIM #257220) is an autosomal recessive inherited unique error of cellular lipid traffic, clearly distinct from NPA and Niemann-Pick type B (NPB) diseases, which are allelic variants and results of lysosomal acid sphingomyelinase deficiency (175). Although the clinical presentation of NPC disease is heterogeneous, most patients exhibit visceral storage of cholesterol, SPM and glycosphingolipids and progressive degeneration in the central nervous system (reviewed in 175). Neuronal storage is accompanied with a variety of cellular inclusions as well as occasional presence of neurofibrillary tangles, meganeurites or axonal spheroids. Sea blue histiocytes and foamy M␾s are observed in several tissues although such cells are often found in many other so-called ‘storage diseases’, thus their presence in NPC disease is likely unspecific. The most prominent cellular finding of NPC fibroblasts is the accumulation of LDL-derived, unesterified cholesterol in a unique, late endosomal subcompartment (NPC-compartment) distinct from lysosomes or endosomes containing adaptins, cathepsin-D or mannose 6-phosphate receptors (176,177). The cellular defect is also accompanied with an altered down regulation of HMG-CoA reductase and LDL receptors. In addition, cholesterol accumulates in the TGN and its re-location to and from the plasma membrane is substantially delayed (177,178). The NPC-compartment co-localizes with Rab7 and Rab9, members of the Rabfamily of small molecular weight GTPases (177). Rab7 is known as a regulator of vesicular endosomal traffic to lysosomes (179), while Rab9 is a regulator of endosomal traffic to the TGN (180). These findings clearly indicate that cholesterol egress from the specific NPCcompartment is mediated by vesicular transport and, NPC1 seems to be one of the regulators of this transport (reviewed in 177,178). Interestingly, caveolin(s) are upregulated in NPC disease (181–182). Whether the increased caveolin expression represents a compensatory mechanism for the delayed post-lysosomal cholesterol exit via the NPC-compartment, or both NPC1 and caveolin(s) are present in the same vesicular compartment is not yet clear.

Schmitz and Orsó The NPC1 gene encodes a multispan transmembrane glycoprotein with sterol sensing domain, similar to HMG-CoA reductase, SCAP (sterol response element binding protein cleavage activating protein) and the morphogen receptor for ‘hedgehog’ named ‘patched’ (183), which is mutated in 85–90% of patients suffering with NPC disease (184). Using EST database search for human NPC1 homologues a novel gene, termed NPC1-like 1 (NPC1L1) has recently been identified (185 and references therein). The predicted NPC1L1 protein shares 42% amino acid identity and 51% similarity to NPC1, as it contains the conserved sterol sensing domain and the NPC1 domain. These latter findings indicate that NPC1 and NPC1L1 might constitute a new family of related proteins involved in subcellular lipid traffic (185 and references therein). The two proteins however, are different with respect to their putative intracellular targeting signals, as NPC1 contains an LLNF endosomal/ lysosomal targeting motif, whereas NPC1L1 possesses a characteristic plasma membrane to TGN, YQRL motif (185). Furthermore, the two genes are probably regulated by different manner, since analysis of the putative promoter region of NPC1L1 identified the presence of a sterol regulatory element (SRE) and a consensus sequence for a YingYang-1 (YY1) factor (185). By contrast, these elements are absent from the NPC1 putative promoter region (186). Currently, three major concepts have been presented to a proposed function of NPC1 (176,185 and references therein): (i) NPC1 may function as a direct cholesterol transporter by collecting of cholesterol from the NPC compartmental membrane system and transporting the lipid to the TGN. (ii) NPC1 may act as a docking/fusion protein, promoting cholesterol filled vesicles to dock and fuse with recycling endosomes for subsequent delivery to the TGN. (iii) NPC1 is a pump that drives the movement of cholesterol (and other lipids) from the endosome/lysosome to the TGN. In this speculative model NPC1, similar to prokaryotic molecular pumps that use a proton-motive gradient as the energy source for mobilizing their substrates, forms a channel within the membrane. In this scenario NPC1, NPC1L1 and probably Patched would serve the mammalian counterparts of these prokaryotic transporters. It is also tempting to speculate that prokaryotic transporters would play housekeeping functions for specific substrates in higher eukaryotes (reviewed in 185,178). Although considerable efforts have been invested to elucidate the NPC1 function and the pathomechanism of NPC disease, there are several unresolved questions. It is still unclear why paradoxically, GM2-

Lipid Transport Mechanisms of Neurons and Macrophages gangliosides are the major storage products in NPC brain and not cholesterol. Furthermore, it has recently been shown that in the monkey brain NPC1 protein is localized exclusively in astrocytes but not in neurons (187), thus neurodegeneration in NPC seems to be rather a consequence of demyelinization than primary neuronal loss. In a more recent approach the relative contribution of ganglioside accumulation in the neuropathogenesis of NPC was investigated (188). Liu and co-workers (188) bred NPC model mice with mice carrying a targeted mutation in GalNAc-T, the gene encoding the ␤-1,4-N-acetyl-galactosaminyltransferase responsible for the GM2-ganglioside synthesis. Surprisingly, the double knockout mice did not exhibit accumulation of GM2-gangliosides or GA1 or GA2 glycolipids in their brain. Detailed histological analysis revealed a significant reduction of the neuronal storage pathology characteristic for NPC, but similar visceral pathology in the double mutants. On the other hand, despite of strongly reduced ganglioside accumulation in the brain, the clinical phenotype in the double knockouts did not improve, thus other, not yet characterized major players, distinct from GM2gangliosides are responsible for the NPC-related neuropathology (188). Shortly before completing the present manuscript mutations in a novel gene termed HE-1 have causatively been linked to NPC disease (189). In a minor group of NPC patients (NPC2 individuals), not deficient for NPC1 gene but clinically indistinguished from NPC1 mutants, absence of the lysosomal protein HE-1 was found. Accumulation of LDL-derived cholesterol in NPC2 fibroblasts has been reduced by in vitro administration of recombinant HE-1 (189). Although the molecular mechanism of HE-1 action is largely unknown, it is tempting to speculate that HE-1 may work as a part of the endo-lysosomal lipid ‘output system’. Alzheimer Disease (AD) and Atherosclerosis. Alzheimer disease is a neurodegenerative dementia syndrome associated with characteristic histopathologic findings in brain tissues: (i) intraneuronal deposition (mainly in cerebral neurons) of microtubule-associated Tau proteins in fibrillar form leading to intracellular tangles and, (ii) extracellular cerebral accumulation of the secretory proteolytic product of APP in ␤-pleated sheet fibrils (A␤ amyloid peptide) forming senile plaques. Mutations in the genes encoding APP or two other brain specific, ER and cis-Golgi localized intracellular proteins, presenilin-1 (PS1) and presenilin-2 (PS2), with yet unknown function, have been linked to some but not all early-onset forms of familial AD (190

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and references therein). According to current hypotheses, these mutations lead to an abnormal APP processing, by promoting the formation of fibrillogenic and neurotoxic fragments of APP, namely the 42- and 43amino-acid-long A␤42 and A␤43 peptides, respectively (190). The A␤42 and A␤43 peptides are processed in the ER and Golgi compartments and caveolae have been implicated in their secretion (see chapter 3). In contrast, the 40-amino-acid-long A␤40 peptide, which is nonamyloidogenic and represents the ‘normal’ and most abundant proteolytic product of APP, is processed in the endo-lysosomal acidic compartment under the regulatory control of bleomycin hydrolase (190). According to current concepts, generation of A␤ fragments comprises a set of strictly regulated sequential proteolytic processes of APP by ␤- and ␥-secretases (191). In a recent paper Yu et al. (192) have reported a novel transmembrane glycoprotein named nicastrin which interacts with presenilins and the carboxy-terminal tub’ of APP, and likely regulates ␥-secretase. Interestingly nicastrin was found to be required for Notch processing, too. By using a knockout approach in C. elegans Yu and co-workers observed a phenotype of nicastrin-null nematode offsprings that was indistinguished from knockouts, in which genes involved in Notch signaling were inactivated (192). In this scenario presenilins and nicastrin are likely to be functional components of a multimeric protein complex required for the intramembranous proteolysis of distinct proteins such as APP and Notch/GLP-1 (193). The familial forms of AD account for only 5–8% of the cases. Most individuals suffering with either familial or sporadic AD become affected after the age of 65, thus the disease is considered as late-onset AD. Numerous epidemiological studies have shown a striking association between a common genetic polymorphism of the apoE gene and the late-onset of AD, especially the occurence of the ␧4 allele of apoE seems to have a major impact in the pathogenesis of AD (reviewed in 190,194). ApoE, first described as a 34 kD agrinine-rich protein, is a key regulator of plasma lipid levels by affecting all classes of lipoproteins, modulating their receptor-mediated clearance and lipolytic processing and production of very low density lipoproteins of hepatic origin (195 and references therein). ApoE has also been implicated extensively in neuronal repair as well as in the maintenance of the brain lipid homeostasis (see chapter ‘Uptake of cholesterol by neuronal cells—the LDL receptor, supergene family’). In human, the structural gene locus for apoE is highly polymorphic, three common alleles (designated

1062 as ␧2, ␧3 and ␧4) exist encoding three major isoforms E2, E3 and E4, respectively (reviewed in 195–196). This polymorphism leads to six different phenotypes which can be distinguished by isoelectric focusing or immunoblotting: three homozygous (E2/E2, E3/E3 and E4/E4) and three heterozygous (E2/E3, E2/E4 and E3/E4) forms. The isoforms are distinct from each other in a single amino acid substitution. While the most common E3 isoform contains Cys at the residue 112 and an Arg at the residue 158, the E2 isoform contains two cysteines and the E4 isoform two arginines at each residue, respectively. As a result, apoE4 bears one more positive charge compared to apoE3, and the E2 isoform has one less. Regarding that the receptorbinding site of apoE is located between residue 136 and 158, this charge difference is likely responsible for the different binding affinities of apoE isoforms to members of the LDLR family. The Cys112Arg substitution in apoE4 apperas to reduce disulfide bond(s) of this isoform with other sulfhydryl-containing proteins (196). Further single amino acid substitutions within the 136–150 residue region, containing several Arg and Lys residues critical for receptor binding, may also modify other protein-protein interactions since it has long been known that clusters of Arg act as efficient ER-retention signals and hydrophobic amino acids, particularly Leu residues, usually promote Argclustering (197). There are several hypotheses how apoE isoforms participate (promote or protect) in AD-pathogenesis. One of the most plausible concepts is that apoE polymorphisms influence the production, distribution or clearance of A␤ (190 and references therein). This hypothesis is supported by the evidences that A␤ directly interacts with apoE, furthermore that lipid-depleted apoE4 binds A␤ with higher avidity than delipidated apoE3. Moreover, apoE and A␤ are likely taken up from the extracellular space by receptors LDLR family (particularly by LRP) and these two substrates may compete for clearance through the receptors (190,196). It is also hypothesized that apoE4containing particles internalized by neuronal cells may induce lysosomal accumulation of APP-fragments leading to neuronal death (190,196). Alternatively, apoE4-containing lipoproteins might be trapped extracellularly by A␤ deposits, mobilizing soluble A␤ peptides and thus consequently enlarge amyloid plaques (190,196). These above mentioned mechanisms may operate at different stages of AD-pathogenesis and suggest a chaperone-like function for the apoE particles that promotes cerebral deposition of A␤. This concept is supported by the (indirect) evidences that

Schmitz and Orsó overexpression of mutant (e.g. amyloidogenic) APP in mice with intact apoE leads to development of histopathologic alterations in brain that are concordant with AD (198). By contrast, expression of the same transgene on apoE-deficient background results dramatically reduced extracellular A␤ deposits in murine brain (198). Besides to a proposed modulatory role for apoE in APP processing, there are convincing evidences that this lipoprotein also participates in the formation of neurofibrillary tangles. It has been demonstrated that apoE3 binds to Tau protein better than apoE4, indicating that apoE3 may sequester Tau, thus reducing its capability of self-association and hyperphosphorylation (190,199 and references therein). Taken together, apoE polymorphisms seem to be involved in AD-development at certain cellular levels, the precise mechanism(s) however, how these lipoprotein isoforms exert their action is still largely unclear and further research is required. In addition to AD-pathogenesis, a distinct set of evidences implicate that apoE polymorphisms are also intimaltely involved in the development of atherosclerosis (195,200). This concept is suggested by the findings that (i) MOs and vascular smooth muscle cells produce significant amounts of apoE and both cell types are critically involved in the development of vascular lesions during atherogenesis (reviewed in 195). (ii) In vitro differentiation of human mononuclear phagocytes (MNPs) derived from healthy blood donors homozygous for the E3/E3 or E4 /E4 phenotype was analyzed upon induction with macrophage colonystimulating factor (M-CSF). Expression of CD16a, suggesting Fc receptor-dependent phagocytic activity, was significantly higher in apoE4/E4 MOs than in apoE3/E3 cells, indicative that this single polymorphism of apoE may affect monocyte differentiation (201). Expansion of a monocyte subset (MNP3), characterized as CD16⫹/CD14low and enhanced expression of CD11a, CD11b and CD49d integrins, has been shown to correlate with increased plasma levels of ‘atherogenic’ lipids/lipoproteins (e.g. elevated total cholesterol and triglycerides, decreased HDLcholesterol) (202,203) as well as inflammation (204, 205) and acute phase reaction (206,207). (iii) In addition to the expanded MNP3 subset in apoE4/E4 cells during in vitro differentiation, a significantly decreased intracellular apoE concentration and a reduced amount of secreted apoE were found in apoE4/E4 MOs, as compared to apoE3/E3 MOs (201). Interestingly, apoC-I and CETP showed an increased intracellular expression of apoE4/E4 MOs in comparison to apoE3/E3 cells (Kapinsky M., unpublished data).

Lipid Transport Mechanisms of Neurons and Macrophages These data suggest that apoE polymorphisms affect the intracellular lipid metabolism in MOs/M␾s and thus may influence the foam cell formation upon atherogenesis (208).

CONCLUSION During the past ten years considerable evidences have accumulated that neuronal cells, in contrast to previous concepts, exhibit an extensive cholesterol and phospholipid metabolism. The present study focuses on the major protein players that establish cellular distribution of cholesterol and phospholipids. Evidences are provided that neuronal cells and monocytes/ macrophages are equipped with comparable intracellular lipid trafficking mechanisms. Selected examples are presented that trafficking dysfunctions lead to disease development or contribute to the pathogenesis of diseases.

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