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Journal of Lipid Research, Vol. 45, 1375-1397, August 2004
Copyright © 2004 by American Society for Biochemistry and Molecular Biology
Thematic Review |
Department of Internal Medicine, University of Texas Southwestern Medical School, Dallas, TX 75390-8887
Published, JLR Papers in Press, May 16, 2004. DOI 10.1194/jlr.R400004-JLR200
1 To whom correspondence should be addressed. e-mail: john.dietschy{at}utsouthwestern.edu
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ABSTRACT |
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23 mg/g). Furthermore, even though the CNS accounts for only 2.1% of body weight, it contains 23% of the sterol present in the whole body pool. In all animals, most growth and differentiation of the CNS occurs in the first few weeks or years after birth, and the cholesterol required for this growth apparently comes exclusively from de novo synthesis. Currently, there is no evidence for the net transfer of sterol from the blood into the brain or spinal cord. In adults, the rate of synthesis exceeds the need for new structural sterol, so that net movement of cholesterol out of the CNS must take place. At least two pathways are used for this excretory process, one of which involves the formation of 24(S)-hydroxycholesterol. Whether or not changes in the plasma cholesterol concentration alter sterol metabolism in the CNS or whether such changes affect cognitive function in the brain or the incidence of dementia remain uncertain at this time.
Abbreviations: ABC, ATP binding cassette; apoE, apolipoprotein E; CNS, central nervous system; kcal, kilocalories; LDL-C, cholesterol carried in low density lipoprotein; LDLR, LDL receptor; LRP, LDLR-related protein; LXRß, liver X receptor-ß; NPC1, Niemann-Pick C1; NPC2, Niemann-Pick C2; NPC1L1, Niemann-Pick C1-like 1; PNS, peripheral nervous system; SR-BI, scavenger receptor class B type I; SREBP, sterol-regulatory element binding protein; TC, total cholesterol
Supplementary key words Alzheimer's disease • amyloid precursor protein • oxysterols • dementia • amyloid ß peptide • blood-brain barrier • apolipoprotein E • apolipoprotein A-I • lipoprotein transporters • ATP binding cassette transporters
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INTRODUCTION |
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During the last 40 years, as the role of lipoproteins in the development of atherosclerosis was being defined, there was renewed interest in the possibility that the level of circulating cholesterol also affected, in some manner, the function of the CNS. It was postulated, for example, that the concentration of cholesterol circulating in the plasma of the newborn infant might affect brain development and even intelligence. In the adult, it was suggested that low levels of circulating cholesterol might be responsible for depression and violent, or even suicidal, behavior (6–8). Still more recently, the incidence of dementia has been related directly to the level of circulating cholesterol (9–13).
This possibility that the differentiation and function of the CNS was in some way influenced by the concentration of cholesterol circulating in low density (LDL-C) or high density lipoproteins in the plasma received further support from recent developments in our understanding of the molecular events involved in transmembrane cholesterol movement. It was recognized, for example, that the brain contained or expressed a number of different apolipoproteins, e.g., apolipoprotein E (apoE) and apoA-I; lipoprotein receptors, e.g., the low density lipoprotein receptor (LDLR) and scavenger receptor class B type I (SR-BI); and members of the ATP binding cassette (ABC) family of transporters, e.g., ABCA1 and ABCG1 (14–19). Because several of these proteins have well-established roles in cholesterol metabolism in many organs elsewhere in the body, it was assumed that they played similar roles in the CNS. However, direct experimental evidence that this is the case remains elusive. This review, therefore, was undertaken to summarize our current knowledge of the processes that result in the accumulation of cholesterol in the CNS and that are responsible for the net movement of sterol among cells of the brain and between the brain and circulating lipoproteins in plasma.
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NET MOVEMENT OF CHOLESTEROL ACROSS THE PLASMA MEMBRANE AND THROUGH THE WHOLE ANIMAL |
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Major proteins involved in cellular cholesterol transport
As illustrated in Fig. 1
, in the prototypic cell most cholesterol is unesterified and is located in either the outer (red) or inner (green) leaflet of the plasma membrane. Only a small portion of the pool of unesterified cholesterol in most tissues is located in intracellular structures such as the endoplasmic reticulum, Golgi apparatus, or nucleus. Nevertheless, this small, metabolically active pool of sterol plays a critical role in regulating the content of cholesterol in the cell. Cholesterol accounts for 20–25% of the lipid molecules in the plasma membrane of most cells, whereas various phospholipids, sphingomyelin, and glycolipids make up the remainder. The distribution of these different lipids within the leaflets of the plasma membrane, however, is not uniform. The glycolipids and, to a lesser extent, sphingomyelin are found predominantly in the outer leaflet, and the various phospholipids are predominantly located in the inner leaflet. In many cell membranes, portions of these leaflets are organized into microdomains that are enriched with cholesterol, sphingomyelin, and glycolipids and therefore are resistant to solubilization in detergents (20–22). Although not shown in Fig. 1, embedded within the plasma membranes are a variety of transporters and signaling molecules as well as various aquaporins (23). These latter proteins are responsible for the observation that plasma membranes, in general, are highly permeable to water but relatively impermeable to ions and protons. Obviously, the concentration of unesterified cholesterol in these leaflets must be tightly regulated because this molecule plays an important role in determining the fluidity and permeability characteristics of the membrane as well as the function of both the transporters and signaling proteins.
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Cholesterol is continuously shed from the outer leaflet of the plasma membrane, and much of this movement may simply be driven by chemical gradients between the leaflet and lipoprotein receptors in the plasma (37, 38). However, at least three other mechanisms have also been described that bring about net excretion of cholesterol out of the cell. These include the transporters ABCG5/8 (5) and ABCA1 (6) and various enzymes capable of hydroxylating cholesterol (7). ABCG5/8 is made up of two half transporters that can move unesterified cholesterol from the cell membrane or cytosol to the external environment (39). Similarly, ABCA1 also apparently transports unesterified cholesterol out of the plasma membrane in certain cells (37, 40). In an entirely different mechanism, many cells contain enzymes capable of hydroxylating the cholesterol molecule in the 7
, 24, 25, and 27 positions, and these hydroxylated sterol molecules, in turn, can diffuse out of the cell across the plasma membrane [reviewed in ref. (41)]. Although not shown in Fig. 1, a few types of cells, such as hepatocytes and enterocytes, can also incorporate cholesteryl esters, along with triacylglycerol, into nascent lipoprotein particles and export these packaged lipids to the surrounding aqueous phase.
Cholesterol transport in the whole animal
As illustrated in Fig. 2
, these various transport mechanisms are expressed in specific organs in such a way that there is essentially net unidirectional movement of cholesterol from the peripheral organs to the intestinal lumen. Apparently, every cell in the periphery, including the CNS, can synthesize cholesterol and move it up to the leaflets of the plasma membrane. Because the concentration of sterol in these leaflets remains constant, cholesterol must be continuously removed and becomes associated with circulating apoA-I. In cells such as macrophages, this desorption process may be under the control of ABCA1, but this seems not to be the case with most other cell types (40). Within the plasma space, the HDL particle enlarges as cholesteryl ester is formed under the influence of LCAT. Depending upon the species, a portion of this cholesteryl ester may then be transferred by cholesteryl ester transfer protein to apoB-containing lipoproteins and taken up into the liver by means of the LDLR and the clathrin-coated pit pathway. Alternatively, the cholesteryl ester may be selectively transported by SR-BI directly into the hepatocyte. In either case, the cholesteryl ester is hydrolyzed and the unesterified cholesterol is mixed with other pools of sterol that come from de novo synthesis or from the intestine (42). After a portion of this pool is metabolized to bile acid, these sterols are transported across the canalicular membrane of the hepatocyte by a complex process that requires three separate transport proteins. These three proteins bring about the movement of phospholipid (ABCB4), bile acid (ABCB11), and cholesterol (ABCG5/8) into the bile and, ultimately, into the intestinal lumen. Although both the cholesterol and bile acid molecules are partially reabsorbed and recycled back to the liver through an enterohepatic circulation, ultimately there is net excretion of both of these molecules from the body as fecal neutral and acidic sterols. It should be emphasized that the rate-limiting step in this unidirectional transport system appears to reside within the individual cells of the peripheral organs. Functional deletion of ABCA1, apoA-I, LCAT, SR-BI, or LDLR does not significantly alter the overall flux of cholesterol from these peripheral tissues to the intestinal lumen (43, 44).
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Many of these same proteins involved in the movement of cholesterol throughout the body are also expressed in the CNS. These include many members of the LDLR family of receptors as well as SR-BI (15, 46). The apolipoproteins apoE, apoA-I, apoA-IV, apoD, and apoJ have also been detected in cerebrospinal fluid, and various members of the ABC family of transporters are expressed in specific cells of the CNS (16, 47). The crucial question, then, is which if any of these proteins is involved in the net movement of cholesterol across the blood-brain barrier into the CNS, between individual cells within the brain and from the CNS back into the plasma.
Concentration of cholesterol in various organs in the steady state
This flow of cholesterol from the sites of synthesis in the cells of the peripheral organs to the liver and intestine is so tightly regulated that in the steady state, the concentration of sterol in cell membranes is kept remarkably constant at a level that is characteristic of each particular tissue. As illustrated in Fig. 3
, the pool of cholesterol in the whole animal is 2,200 mg/kg body weight. This is true for essentially all species from the mouse to the primate and indicates that the average concentration of cholesterol in the whole animal is 2.2 mg/g fresh tissue (48). However, as also shown in this figure, this steady-state concentration varies markedly among the different organs.
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Turnover of cholesterol in various organs and in the whole animal in the steady state
Thus, in essentially all tissues there is a continuous flow of cholesterol from the endoplasmic reticulum to the cell membrane, and from this plasma membrane to the liver and intestine. During this process, the concentration of sterol in the plasma membranes of each organ (Fig. 3) and the size of the pool of cholesterol in the whole animal remain essentially constant. However, the rate of movement of sterol through these pathways, and therefore the rate of plasma membrane cholesterol turnover, is very different in animals with different basal metabolic rates. As shown in Fig. 4
, for example, the basal metabolic rate in the mouse is 170 kilocalories (kcal)/day/kg, and the flow of cholesterol from all peripheral organs to the liver is greater than 100 mg/day/kg (43, 58). In contrast, the basal metabolic rate in the human is only 25 kcal/day/kg, and the flow of sterol from the periphery is only 10 mg/day/kg. Because the size of the steady-state pool of sterol is nearly the same in these various species, these data indicate that 8% of the cholesterol in plasma membranes of the mouse is replaced each day and that only 0.7% of that found in the membranes of humans is turned over (43, 59).
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CONCENTRATION OF CHOLESTEROL IN THE PLASMA AND DISORDERS OF THE CNS |
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40 mg/dl to 25 mg/dl and the LDL-C decreases from 25 mg/dl to only 10 mg/dl over the course of fetal development (62). Similar declines are seen in both the pig and human fetuses, with TC and LDL-C concentrations reaching 55 and 30 mg/dl, respectively, at birth in humans (63, 65). With the onset of suckling, however, the plasma sterol levels rapidly increase 2- to 4-fold in all species, including sheep, pig, and human. The breast-fed human newborn, for example, ingests 18 mg cholesterol/day/kg body weight under circumstances in which the infant is synthesizing 25 mg/day/kg (dietary cholesterol intake is 72% of endogenous synthesis) (66, 67). Because of this relatively large dietary cholesterol (and triacylglycerol) load, the TC promptly increases to 170 mg/dl, the LDL-C increases to 90 mg/dl, and there is suppression of endogenous sterol synthesis (67, 68). However, as also shown in Fig. 5C, if the infant is placed on a low-cholesterol synthetic formula, dietary cholesterol intake is reduced to only 2 mg/day/kg (8% of endogenous synthesis), the TC and LDL-C levels increase to only 95 and 35 mg/dl, respectively, and there is much less suppression of endogenous sterol synthesis (67, 69, 70). Thus, all species show a marked increase in the plasma cholesterol concentration during the suckling period, although, in the human, this physiological hypercholesterolemia can be partly abrogated by feeding a low-cholesterol formula diet (Fig. 5C).
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135 and 70 mg/dl, respectively, in adults of all ages, and these groups have virtually no coronary artery disease (71, 73–78). In contrast, humans who are weaned onto the usual Western diets containing 300–500 mg cholesterol/day (43–71% of endogenous synthesis) and in whom >40% of calories come from triacylglycerol typically have TC and LDL-C levels of >200 and >110 mg/dl, respectively, and these populations have a very high incidence of coronary artery disease (79–81). These differences in plasma lipid levels in the adults are not attributable to genetic differences in these groups of people; rather, they are primarily the result of differences in intake of dietary lipids. When people of Amerindian genetic background, for example, are placed on Western diets, their TC and LDL-C values promptly increase and become indistinguishable from those values found in people of Caucasian or African genetic backgrounds eating the same Western diets (78, 82). Thus, there are wide fluctuations in the TC and LDL-C concentrations at different times during the development of the animal or human. Furthermore, during the suckling and adult periods, additional variation in the circulating lipid levels can be induced by either dietary or pharmacological manipulations. Assuming that cholesterol metabolism in the CNS is in some manner affected by the concentration of sterol in the blood, some investigators have raised the possibility that these manipulations might have a detrimental effect on brain function. Certainly, children with the Smith-Lemli-Opitz syndrome who cannot convert 7-dehydrocholesterol to cholesterol in the blood or CNS have profound abnormalities in brain development and function (83). However, more subtle effects of fluctuations in circulating cholesterol levels on brain function have been postulated over the years in at least four areas. First, because the physiological hypercholesterolemia seen during suckling (Fig. 5) coincides with the period of major growth and myelination in the brain, it was suggested that formula feeding or limitations on dietary cholesterol intake might be deleterious to the differentiation of the CNS or even to the level of intelligence (84, 85). Second, in several studies in adults, aggressive, suicidal, or even criminal behavior was reported to be more common in individuals with low circulating cholesterol levels (6–8, 86–88). It was postulated that these low plasma levels might lead to a loss of cholesterol from membranes in the brain and, hence, to a reduction in the number of serotonin receptors (87, 89). Indeed, the concentration of serotonin in the blood was found to be lower in men with persistently low plasma cholesterol levels (90). Third, a number of epidemiological studies suggested that the relationship between all-cause mortality rates and the plasma cholesterol concentration was U-shaped. That is, mortality rates were increased in people with high cholesterol levels, presumably because of coronary artery disease and strokes; however, these rates were also increased in individuals with very low plasma cholesterol concentrations, for reasons that were not immediately obvious but that might be related to emotional changes (91–94). Finally, it was suggested that there was a direct correlation between the plasma cholesterol level and the incidence of dementias, including Alzheimer's disease (10, 95–97). Furthermore, treatment of older individuals with pharmaceutical agents that decreased the plasma cholesterol concentration appeared to be associated with a lower incidence of such dementias (11, 12).
It should be emphasized that many of these associations have not been confirmed by more recent and rigorous epidemiological studies. Nevertheless, these observations raise the possibility that there is a relationship between the plasma level of cholesterol carried in LDL, HDL, or other lipoproteins and the growth, myelination, and function of the CNS. If this were true, these lipoproteins might make important contributions to the cholesterol pools within the brain and spinal cord and possibly affect, directly or indirectly, the processing of proteins such as serotonin receptors or amyloid precursor protein (98).
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CHOLESTEROL METABOLISM AND EVOLUTION OF THE BRAIN |
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The problem of nerve conduction velocity
Although the permeability characteristics of the cholesterol/phospholipid bilayer allow potential differences to be maintained across the plasma membranes of virtually all cells, only neurons (and muscle cells) propagate electrical signals over long distances. The velocity of movement of this electrical signal down the axon is inversely proportional to both the electrical resistance encountered in the axon (ra) and the capacitance of the plasma membrane surrounding the axon (cm) (100). Thus, conduction velocity in nerves could be greatly increased by reducing the term racm. One approach to this problem was to reduce axonal resistance by evolving neurons with axons of very large diameter (0.5 mm), as did some cephalopods like the giant squid (100, 101). However, such a solution could not be used in the more complex brains of higher vertebrates. The alternative approach was to greatly reduce the capacitance of each neuron by increasing the thickness of the cholesterol/phospholipid membranes surrounding each axon.
This was accomplished by evolving two new types of cells, the oligodendrocyte in the CNS and the Schwann cell in the peripheral nervous system (PNS) (101). As shown diagrammatically in Fig. 6
, the oligodendrocyte is an extraordinary cell that synthesizes vast sheets of plasma membrane that extend outward from the cell body. These sheets are surrounded by narrow loops or channels of persistent cytosol. Each of these sheets first wraps around a section of adjacent axon 1 mm in length, between nodes of Ranvier. Thus, within the CNS, each oligodendrocyte may contribute plasma membranes to 10–15 different neurons, whereas in the PNS, each Schwann cell interacts with only a single neuron (102). The second step in the formation of this myelin coat involves the removal of most of the aqueous phase surrounding the tightly wrapped plasma membranes. This process involves specific proteins (e.g., myelin basic protein) that bring together in tight apposition the inner leaflets of the bilayers and, similarly, pull together the outer leaflets of adjacent plasma membranes (103). In this manner, nearly all of the aqueous electrolyte solution in both the extracellular and cytosolic compartments is "squeezed" out of the mature myelin and its capacitance is correspondingly reduced. Although more primitive forms of poorly packed myelin are found in lower phyla like Annelida and Crustacea, true compact myelin is seen only in Gnathostome vertebrates (104). Thus, the evolutionary adaptation of the cholesterol-rich plasma membrane to form compact myelin made it possible to "wire" the complex brain with numerous relatively small-diameter, low-capacitance axons that manifested very high conduction velocities.
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73% of the weight of the liver but only 40% of isolated myelin (105, 106). The concentration of unesterified cholesterol equals only 3 mg/g in the hepatocyte (Fig. 3) but may reach 40 mg/g in the spinal cord. Although this sterol accounts for a similar proportion (17–22%) of the total lipids found in plasma membranes of both the liver and the CNS, membranes in the brain contain relatively more glycolipids (28% vs. 7%) and less sphingomyelin (8% vs. 19%) than do plasma membranes of other organs like the liver (107). The fact that the cholesterol molecule exists in this relatively anhydrous, hydrophobic environment raises the issue of how such sterol is replaced during the normal turnover of cholesterol that takes place in all such plasma membranes.
The problem of brain size
Although the evolution of myelin largely solved the problem of action potential conduction velocity, this insulatory layer on axons did add additional volume to the CNS, particularly in the large brains of primates, where there was massive expansion of the neocortex. Over a very large range of brain sizes, the volume of the neocortex increased from only 16% of the volume of the whole brain in small animals like Soricomorpha to 74% in Hominoidea. In contrast, the relative volume of the cerebellum remained constant at 13% of whole brain volume, regardless of the absolute size of the brain (108, 109). As the size of the neocortex progressively evolved, so also did the volume occupied by myelinated nerve fibers. The quantitative nature of this relationship is shown in Fig. 7
, where the volume of white matter is plotted against the volume of gray matter in 59 different species (110). As is apparent, there is a remarkably tight relationship between these two variables in all animals from the pigmy shrew to the elephant. These data support the view that as the neocortex increased in size and complexity, there was necessarily a disproportionate increase in the volume of brain devoted to "wiring." Thus, in Fig. 7, the volume of the white matter approximately increases as the 4/3 power of the volume of the gray matter (99, 110).
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0.5 and 1,400 g, respectively. Although the total body cholesterol pool in these two species is essentially the same, 2,150 and 2,200 mg/kg, respectively, the proportion of this pool residing in the CNS is significantly greater in the human (490 mg/kg, 23% of the total body pool) than in the mouse (330 mg/kg, 15% of the total body pool) (53, 59). Thus, because of the disproportionate increase in the volume of white matter required to support the greatly expanded neocortex in the human, there is a correspondingly disproportionate fraction of the whole-body pool of unesterified cholesterol in the CNS.
The problem of the female pelvis
Although solving the problem of nerve conduction velocity, this disproportionate increase in brain size created another challenge that ultimately dictated when brain growth and myelination could take place during fetal and neonatal development. Obviously, the cross-sectional area of the female pelvis limited the degree of development of the CNS before birth by limiting the size of the head that could pass through the birth canal. Various species have dealt with this problem in essentially two ways.
In Fig. 8
, the degree of myelination at different times during perinatal development is approximated by the mean concentration of cholesterol in the whole brain. Ungulates, represented in Fig. 8A by the sheep, as a group begin myelinating the brain during late fetal development. Although these hooved animals are not noted for their intellectual capacity, this early myelination allows the newborn to be immediately mobile so that it can be led away from the birthing site before attracting the attention of local predators. The guinea pig represents another species that myelinates the CNS in the few weeks before birth so that the newborn pup is also quite mature and fully mobile (111). In contrast, many species, represented by the mouse and hamster in Fig. 8B, have essentially no myelination in the brain and are nearly helpless at birth. Thus, although the concentration of cholesterol in the brain of the sheep is 12 mg/g at birth, it is only 3.5 mg/g in mice and hamsters. This value is similar to the concentration of sterol found in most other organs at birth in these species. However, in the 3 weeks after birth, cholesterol rapidly accumulates in the CNS. The human, also born essentially helpless, follows a similar pattern of myelination, although a number of years, rather than weeks, are required for the average concentration of cholesterol in the brain to increase from 6 mg/g at birth to 23 mg/g found in the young adult (Fig. 8C).
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The problem of excess energy expenditure
A final problem with the evolution of the CNS is explaining how the vertebrate, in general, and the encephalized primate, in particular, could afford the massive expenditure of energy required to support a large brain. In the newborn human, for example, 60% of basal energy expenditure goes to support ion transport, neurotransmitter synthesis, and other metabolic processes in the CNS (99). In the adult, the organ-specific basal metabolic rate of the brain (230 kcal/day/kg) is nine times higher than the average basal metabolic rate (25 kcal/day/kg) for the whole body (60). The gastrointestinal tract has a similarly high rate of organ-specific basal metabolic energy expenditure (250 kcal/day/kg).
For nearly all animals, the rate of basal energy expenditure increases as the 3/4 power of body weight (58). Surprisingly, humans follow this same relationship despite their disproportionately large CNS (60). An explanation for this apparent paradox recently has been presented as the "expensive-tissue hypothesis." This hypothesis suggests that the massive expansion of the neocortex in humans came at the expense of the gastrointestinal tract. The left column in Fig. 9 shows the weights of various organs that would be expected in a 65 kg human based upon projections from the relative weights of these same organs in various nonhuman primates. Such a hypothetical human should have a relatively small brain but a very large gastrointestinal tract to cope with foods of poor nutritional value and digestibility. However, the situation in actual humans, shown in the right column, reveals that the brain is more than three times larger and, importantly, the intestine is only half as large as projected from these calculations. Thus, it has been suggested that primitive humans with enlarging brains became more efficient hunter/gatherers and so gained greater access to more nutritious and easily digestible foods (e.g., fruits, nuts, and meat) and so no longer needed the large gastrointestinal tract. Humans had, in effect, conserved energy by greatly reducing the size of the intestine and then shifted this basal energy expenditure to support a much larger brain (60). Whether or not this hypothesis is correct, the CNS still has a very high basal metabolic rate, so that another paradox remains. Why is the rate of cholesterol turnover in the membranes of the brain apparently much slower than the rate of turnover in the plasma membranes of other organs? This question remains to be answered.
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BRAIN GROWTH AND CHOLESTEROL POOLS IN THE CNS |
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0.28 to 0.52 g (16.3 g/kg body weight), and the content of cholesterol in the whole brain increases from 1.5 to 10.6 mg (330 mg/kg). As is also apparent in Fig. 10B, the rate of increase in this sterol pool is greatest during the first 3 weeks after birth, after which the rate of accumulation markedly decreases as the animal matures.
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360 g in the newborn to 1,400 g (21.5 g/kg) in the adult (Fig. 10C), during which time the content of cholesterol increases from 2,700 to 32,200 mg (490 mg/kg) (114). Thus, the size of the pool of cholesterol in the brain per kilogram of body weight in the mouse varies greatly between 1 week old (290 mg/kg) and 3 week old (670 mg/kg) pups but becomes constant in the adult animal at 330 mg/kg, so that in the 26 week old mouse this pool represents only 15% of the whole-body pool. In the human, however, this pool decreases slightly from 770 mg/kg in the newborn infant to 490 mg/kg in the adult, in which it occupies 23% of the whole-body sterol pool.
In the adult CNS, this pool of unesterified cholesterol must be distributed among at least three different compartments. These include the cholesterol present in the membranes of myelin and the sterol present in the plasma membranes of neurons and glial cells. Although data are very limited, it is possible to approximate the amount of cholesterol present in each of these compartments. In the rat and mouse, it is reported that 70% and 80% of brain cholesterol, respectively, is in myelin (49, 106, 115). This would suggest that in the adult mouse the pool of cholesterol in myelin equals 260 mg/kg, whereas the remaining 70 mg/kg presumably resides in the membranes of cellular elements. Approximately 10% of these cells are probably neurons and the remainder are different types of glial cells and vascular elements. If these figures are appropriate for the mouse, the pool of cholesterol in neurons must equal only 7 mg/kg, whereas the remaining 63 mg/kg resides predominantly in the glial cell compartment (59). The metabolism and turnover of cholesterol in each of these three major cellular compartments is almost certainly very different.
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CHOLESTEROL MOVEMENT INTO AND OUT OF THE CNS |
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Characterization of the blood-brain barrier
Any cholesterol that enters or leaves the CNS must do so by crossing the blood-brain barrier that, anatomically, is made up primarily of the unique endothelial cells found in the capillary network within the brain and spinal cord. The inner or basal membranes of these cells are also intimately associated with the foot processes of adjacent astrocytes (14). In general, in different regions of the body, capillaries have different permeability characteristics that are determined by whether or not they manifest fenestrations, tightly adherent junctional complexes, or transcellular vesicular movement (116). Capillaries of the brain apparently have essentially no fenestrae and show little bulk phase vesicular transport. Furthermore, adjacent endothelial cells are tightly adherent, so that there is also little or no paracellular molecular diffusion (117, 118). Thus, the endothelial cells of these capillaries form a very high resistance barrier where the movement of molecules, including cholesterol, between the plasma and the CNS must largely take place across the two parallel plasma membranes covering each surface of the capillary endothelial cell (14).
As with other plasma membranes, these membranes of the endothelial cells are freely permeable to water and, in addition, contain a number of different transport proteins, e.g., glucose and amino acid transporters and P-glycoprotein (ABCB1) (14, 119). In addition, these bilayers are permeable to a number of more hydrophobic molecules, including sterols, that diffuse across the blood-brain barrier down existing activity gradients (120). The maximal rate of such movement is dictated by the product of the maximal activity that a particular molecule can achieve in the blood or extracellular fluid of the brain and its passive permeability coefficient (120–122). Importantly, when a sterol like cholesterol is hydroxylated, there is an increase in the maximal aqueous activity that can be achieved but a reduction in the passive permeability coefficient. However, the increase in solubility is proportionately greater than the reduction in the permeability coefficient, so that the net effect of hydroxylation is to greatly increase the maximal rate of passive diffusion of the molecule across the blood-brain barrier.
From these considerations, it is very unlikely that cholesterol carried in lipoproteins can reach the CNS either through fenestrations in the capillary membranes or through paracellular diffusion. However, three other pathways are at least theoretically available to promote net cholesterol movement in either direction across the blood-brain barrier. First, it is possible that the plasma membranes of the endothelial cells contain functional lipoprotein transporters such as the LDLR or SR-BI or transporters directed at unesterified cholesterol like ABCA1. Second, although vesicular transport appears to be minimal in the brain, it is still possible that a small amount of bulk-phase endocytic transcellular movement could take place in the endothelial cells. Third, it is also possible that unesterified cholesterol or, more likely, hydroxylated cholesterol could diffuse passively across the blood-brain barrier down existing activity gradients.
Cholesterol movement from the plasma into the CNS
There is now a variety of data from both in vitro and in vivo studies that can be used to identify which, if any, of these transport pathways function to promote net cholesterol uptake into the CNS. Certainly, the observation that the suckling animal routinely develops high levels of circulating lipoproteins (Fig. 5) during the time when major sterol accretion in the brain is taking place (Fig. 8) raises the possibility that lipoproteins might cross the blood-brain barrier. Consistent with this possibility, cells of the CNS are known to express the liver X receptor-ß (LXRß) (and, to a lesser degree, LXR) nuclear receptor that can regulate transporters such as ABCA1 (16, 17, 123). Furthermore, the endothelial cells making up the blood-brain barrier express mRNAs for LDLR, SR-BI, and ABCA1 (15, 124). Under in vitro conditions, these proteins have been shown to promote the movement of cholesterol across these endothelial cells, suggesting that these transporters might be involved in either the net uptake of LDL-C into the CNS or the net excretion of sterol from the brain (15, 124).
Four other groups of studies, however, have examined this question of whether there is net cholesterol uptake into the CNS under in vivo conditions. First, the clearance of lipoprotein cholesterol into the brain has been measured in sheep, rabbit, and mouse using 125I-labeled homologous LDL. In both the adult mouse and rabbit, net clearance of these particles into the CNS was undetectable (<0.5 µl/h/g) under circumstances in which the liver and adrenal gland in these same species took up LDL at rates varying from 100 to 1,000 µl/h/g (125, 126). Furthermore, this very low clearance rate of <0.5 µl/h/g was the same when the LDL particle was methylated to block interaction with the LDLR and in animals genetically lacking the LDLR (125, 126). Similar studies were also undertaken during the fetal and suckling periods in the sheep, as shown in Fig. 11
. Between 90 days before birth and 18 days after birth, the concentration of cholesterol in all regions of the CNS increased dramatically, reaching 7 mg/g in the frontal and parietal cortex and 40 mg/g in the spinal cord. However, despite these high rates of sterol accretion in the brain and spinal cord, LDL clearance was still <0.5 µl/h/g, a value not different from 0. This was true in every region of the CNS and at times before the closure of the blood-brain barrier, even though there was active uptake of LDL in the liver and adrenal gland of these same animals (113). Identical results have been found in the mouse using HDL labeled with [14C]cholesteryl ester. Finally, although mRNA for the LDLR is found in the brain, the level of expression does not change in the newborn mouse or rabbit when circulating hypercholesterolemia is present and maximal accretion rates of brain cholesterol are found (127, 128). Thus, these various observations provide no direct experimental evidence for the uptake of lipoprotein cholesterol across the blood-brain barrier into the CNS at any time during late fetal or postnatal development.
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A third set of experiments has explored the possibility that small amounts of bulk-phase endocytic transcellular movement of plasma lipoproteins take place across the endothelial cells of the blood-brain barrier. In mice fed oleic acid and cholesterol, the plasma TC was increased from 228 to 1,629 mg/dl. Even at a minimal bulk flow clearance rate of 0.5 µl/h/g, this 7-fold increase in lipoprotein cholesterol concentration should have delivered a significant amount of cholesterol into the CNS. However, under these conditions, there also was no change in the concentration or rate of synthesis of cholesterol in any region of the CNS, including the cerebrum, cerebellum, midbrain, brain stem, and spinal cord (53). Thus, these studies again failed to demonstrate any net cholesterol movement from the plasma into the CNS.
Finally, there is a series of observations using either unlabeled or isotopically labeled sterols to examine the permeability characteristics of the blood-brain barrier. Unfortunately, such studies do not specifically measure net sterol transport into the brain because bidirectional molecular exchange could also result in the appearance of these respective sterols in the CNS. Nevertheless, the results of these experiments are useful. In one study, rats were treated with an inhibitor of cholesterol synthesis so that most organs, including the CNS, contained predominantly 7-dehydrocholesterol. When these animals were fed exogenous cholesterol, the content of this sterol in the plasma and liver promptly increased, but there was no change in the level of cholesterol in the brain (130). This result was similar to observations in both mouse and human with loss of function of the ABCG5/8 transporter that various sterols of plant origin accumulate in relatively high concentrations in the plasma and liver, but only trace quantities of these sterols could be detected in the CNS (45, 131). In another experiment, rat pups were fed artificial milk diets intragastrically between 5 and 16 days of age. When the cholesterol concentration in this artificial milk was increased 6-fold, the concentration of cholesterol in the liver also increased 6-fold, but it remained unchanged in the brain. Furthermore, the deuterated cholesterol present in the milk came to label 70% of the sterols in the plasma and liver but virtually none of the cholesterol in the brain (132). In baboons administered [14C]cholesterol intravenously, virtually all organs became heavily labeled after 70–85 days. The exception was the brain, which acquired virtually no radioactive cholesterol (133). In a similar experiment in humans, no radioactive sterol was found in the brain 6 days after the intravenous administration of [14C]cholesterol. As in the baboon, this latter study indicated that the CNS was not part of the miscible pool of cholesterol that was present in nearly all other organs of the body (134).
Taken together, these many observations in different species represent a compelling body of evidence that there is no net, or even bidirectional, movement of cholesterol from plasma lipoproteins across the endothelial cells of the blood-brain barrier to the cells of the CNS. This seems to be the case in the mature animal, in which sterol turnover is very slow, as well as in the developing fetus and suckling newborn, in which the rates of sterol acquisition are greatest. These findings, therefore, raise the question of whether there can be a causal relationship between the concentration of plasma cholesterol and abnormalities such as depression, violent behavior, and dementia.
Cholesterol synthesis in the CNS
If there is no movement of lipoprotein cholesterol from the plasma into the CNS under circumstances in which massive expansion of the pools of sterol in the brain and spinal cord is taking place, then de novo synthesis must account for all cholesterol accretion and turnover in these tissues. However, these rates of synthesis are grossly underestimated when substrates such as [14C]acetate, [14C]octanoate, or [14C]glucose are used to make the measurements either in vitro or in vivo. This large error comes from the failure of these substrates to readily penetrate the blood-brain barrier and, equally important, from uncertainties about the specific activity of the precursor pool of [14C]acetyl-CoA generated from t
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INTRODUCTION
