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- Publisher
- American Society for Biochemistry and Molecular Biology
- Copyright
- Copyright © 2007 Elsevier Inc.
- ISSN
- 0022-2275
- eISSN
- 1539-7262
- DOI
- 10.1194/jlr.m700125-jlr200
- Publisher site
- See Article on Publisher Site
Abstract
Receptor-mediated and bulk-phase endocytosis cause macrophage and cholesterol accumulation in Niemann-Pick C disease † 1, * * * * Benny Liu, Chonglun Xie, James A. Richardson, Stephen D. Turley, and John M. Dietschy Departments of Internal Medicine* and Pathology, University of Texas Southwestern Medical School, Dallas, TX 75390-9151 Abstract These studies explored the roles of receptor- hypercholesterolemia and Niemann-Pick type C (NPC) dis- mediated and bulk-phase endocytosis as well as macrophage ease. The major lipoproteins in the plasma expressing apo- infiltration in the accumulation of cholesterol in the mouse lipoprotein E (apoE) are chylomicron remnants (CMrs) and with Niemann-Pick type C (NPC) disease. Uptake of LDL- very low density lipoprotein remnants (VLDLrs), whereas cholesterol varied from 514 mg/day in the liver to zero in the major fraction expressing apoB-100 is LDL (1, 2). As the central nervous system. In animals lacking LDL re- illustrated in Fig. 1, to reach the surface of the parenchy- ceptors, liver uptake remained about the same (411 mg/day), mal cell, these lipoproteins must traverse the barrier repre- but more cholesterol was taken up in extrahepatic organs. This uptake was unaffected by the reductive methylation of sented by the endothelial lining of the capillaries. Some of LDL and consistent with bulk-phase endocytosis. All tissues these capillaries, like those in the central nervous system, accumulated cholesterol in mice lacking NPC1 function, are impermeable to these particles (3, 4). Other capillaries, but this accumulation was decreased in adrenal, unchanged like those in the liver, have large fenestrations of 50–90 nm in liver, and increased in organs like spleen and lung when in diameter that are freely permeable to lipoproteins (5). LDL receptor function was also deleted. Over 56 days, the These apoE- and apoB-100-containing particles that spleen and lung accumulated amounts of cholesterol greater reach the pericellular space compete for binding to low than predicted, and these organs were heavily infiltrated with density lipoprotein receptors (LDLRs) on the plasma macrophages. This accumulation of both cholesterol and membrane of the various cells (Fig. 1, step A) (6, 7). These macrophages was increased by deleting LDL receptor func- tion. These observations indicate that both receptor- ligand/receptor complexes then concentrate and cluster mediated and bulk-phase endocytosis of lipoproteins, as into coated pits (step B) and, along with a small volume well as macrophage infiltration, contribute to the choles- of bulk pericellular fluid, are endocytosed into the cell terol accumulation seen in NPC disease. These macro- (step C). A vacuolar ATPase translocates protons into phages may also play a role in parenchymal cell death in this these vesicles, acidifying the contents of the endosome syndrome.—Liu, B., C. Xie, J. A. Richardson, S. D. Turley, (step D), which leads to unfolding of the LDLR, release of and J. M. Dietschy. Receptor-mediated and bulk-phase endo- the lipoprotein particle, and hydrolysis of the cholesteryl cytosis cause macrophage and cholesterol accumulation in ester by lysosomal acid lipase (8). The unesterified cho- Niemann-Pick C disease. J. Lipid Res. 2007. 48: 1710–1723. lesterol formed by this reaction in the late endosomal/ lysosomal compartment (step E) is solubilized by NPC2 Supplementary key words hepatic dysfunction & lung failure & low density lipoprotein receptor & lysosomal cholesterol & apoptosis & protein (9, 10) and then transported into the cytosol neurodegeneration & lipoprotein clearance by NPC1 protein (step F) (11–14). This cholesterol, along with newly synthesized sterol (step H), forms a metaboli- cally available pool of cholesterol (step G) that is used for Receptor-mediated endocytosis of lipoproteins through a variety of purposes by the cell. The size of this meta- clathrin-coated pits plays a major role in the cellular up- bolically active pool is sensed and tightly regulated by take of cholesterol from the plasma. Although the general mechanisms in the endoplasmic reticulum and nucleus outline of this clathrin-coated pit pathway is understood, that can alter the level of LDLR activity in the cell mem- there are several major quantitative details that are still poorly defined but that may be important in understand- ing the pathophysiology of such diverse diseases as familial Abbreviations: apoE, apolipoprotein E; CMr, chylomicron rem- nant; LDL-TC, total cholesterol carried in LDL; LDLR, low density lipoprotein receptor; NPC, Niemann-Pick type C; VLDLr, very low Manuscript received 13 March 2007 and in revised form 19 April 2007. density lipoprotein remnant. To whom correspondence should be addressed. Published, JLR Papers in Press, May 2, 2007. DOI 10.1194/jlr.M700125-JLR200 e-mail: [email protected] Copyright D 2007 by the American Society for Biochemistry and Molecular Biology, Inc. 1710 Journal of Lipid Research Volume 48, 2007 This article is available online at http://www.jlr.org This is an Open Access article under the CC BY license. Fig. 1. Diagrammatic representation of the processes of receptor-mediated and bulk-phase endocytosis of lipoprotein particles into the cells of the body. Plasma lipoproteins such as LDL carrying predominantly cholesteryl ester must diffuse from the plasma space across the endothelial barrier into the pericellular fluid. The resistance of this endothelial barrier to diffusion of the lipoprotein particles can be described by a reflection coefficient in which a value of 0 represents no resistance and a value of 1.0 represents infinite resistance. The lipoprotein particles may then enter the endocytic vesicle of a clathrin-coated pit after binding to an LDL receptor (steps A, B) or in solution in the pericellular bulk solution (step C). After various steps, designated D–G, the unesterified cholesterol that is formed is transported from the late endosomal/lysosomal compartment into a metabolically accessible pool of sterol in the cytosolic com- partment. The net transport of the lipoprotein particles, therefore, takes place by two independent pro- cesses that are either receptor-dependent or receptor-independent, bulk-phase uptake. In both cases, the lipoproteins are fed into the late endosomal/lysosomal compartment for processing. NPC1, Niemann-Pick type C1. brane, the rate of sterol synthesis, and the rate of sterol LDLR (familial hypercholesterolemia), for example, lead export (15, 16). In this manner, the absolute concentra- to marked increases of the plasma cholesterol concentra- tion of cholesterol in every cell is kept remarkably constant tion but essentially no detectable change in the cellular over the life of an animal or human, and the various apoE- level of sterol or the rate of cholesterol synthesis (17, 18). and apoB-100-containing lipoproteins are efficiently re- In contrast, a mutation in lysosomal acid lipase (Wolman moved from the plasma. disease) causes a major increase in the contents of cho- This whole system, however, breaks down in the face of lesteryl ester and triacylglycerol, presumably in the late mutations that inactivate one of the proteins mediating endosomal/lysosomal compartment, in the cells of many the three major steps in this sequence. Mutations of the organs, yet the plasma cholesterol concentration remains Receptor-mediated and bulk-phase lipoprotein uptake 1711 2/2 2/2 the activity of both of these proteins (ldlr /npc1 ) (17, 21, relatively unchanged (19, 20). Similarly, mutations inac- 25). These animals were housed in plastic colony cages in rooms tivating NPC1 or NPC2 (NPC disease) lead to massive with alternating 12 h periods of light and dark. All animals were accumulation of unesterified cholesterol, but not triacyl- fed ad libitum a low-cholesterol (0.02%, w/w) rodent pellet diet glycerol, in the late endosomal/lysosomal compartment of (No. 7001; Harlan Teklad, Madison, WI) until they were studied the cells in all tissues but only a marginal increase of the at ?7–9 weeks of age. All experiments were carried out during circulating cholesterol concentration in the plasma (21). the fed state, ?1–2 h before the end of the dark cycle, and the These observations highlight a number of uncertainties experimental groups contained equal numbers of male and concerning this whole pathway of receptor-mediated female animals. All experimental protocols were approved by the Institutional Animal Care and Use Committee of the University endocytosis. If, for example, the LDLR is responsible for of Texas Southwestern Medical Center. the uptake of lipoproteins such as LDL, what process ac- counts for the clearance of these particles when the LDLR Isolation and radiolabeling of LDL and albumin is nonfunctional, and why is there little evidence of altered 2/2 Mouse LDL was harvested from both male and female ldlr cellular cholesterol homeostasis in this syndrome? Fur- mice that had been maintained on a low-cholesterol diet, whereas thermore, what characteristics of this putative second ovine and human LDL was obtained from the plasma of normo- transport process could account for the fact that it clears cholesterolemic sheep and humans, respectively. In all cases, the more cholesterol associated with LDL (LDL-TC) from the LDL fraction was isolated by preparative ultracentrifugation in the plasma each day than normally is cleared when the LDLR density range of 1.020–1.055 g/ml. These LDL preparations, along is functional (22)? Additionally, if the accumulation of with mouse albumin (Sigma, St. Louis, MO), were then radio- 125 131 unesterified cholesterol in cells with mutational inactiva- labeled with either [ I]tyramine cellobiose or I (17, 23, 26, 27). The apoE-containing HDL contaminating some of these LDL tion of NPC1 comes from lipoprotein sterol bound to the fractions was removed by passing the lipoprotein solution over a LDLR, why doesn’t deletion of the function of this re- heparin-Sepharose 6b column (28). In one case, a portion of the ceptor markedly decrease the rate of sterol accumula- human LDL was reductively methylated to block its interaction with tion in the NPC syndrome? Such a manipulation actually the LDLR (29). After extensive dialysis, these radiolabeled prepa- decreases the cholesterol content in a few tissues but in- rations were passed through a 0.45 mm Millex-HA filter. creases it in most others (23, 24). Finally, is there a differ- ent role in monocytes and macrophages for lipoprotein Measurement of clearance rates for LDL and albumin clearance through receptor-mediated endocytosis or other Mice were anesthetized and a catheter was inserted into a jug- mechanisms in NPC disease? ular vein. After awakening, each animal was given a bolus of To explore these questions and further understand the [ I]tyramine cellobiose-labeled LDL or albumin followed by a pathogenesis of NPC disease, the current studies exam- continuous infusion of the same preparation for 4 h. The rate of ined five specific areas of lipoprotein transport. The first this continuous infusion was adjusted for the turnover rate of each protein to maintain a constant specific activity in the plasma set of experiments quantitated the rates of clearance of over the entire infusion (30). Ten minutes before the termina- LDL-TC in mice, both in the presence and absence of the tion of this continuous infusion, a bolus of I-labeled LDL or LDLR. A second group of studies explored whether there albumin was administered to each animal to determine the vol- was residual LDLR binding activity in animals supposedly ume of distribution of that particular preparation at time 0. The lacking LDLRs. A third set of experiments measured the animals were then exsanguinated at 4 h, and all major organs rates of bulk-phase endocytosis and determined whether were removed. The residual carcass, containing principally mus- this process, as well as receptor-mediated uptake, delivered cle, bone, and marrow, was homogenized. Tissue and plasma sam- 125 131 cholesterol into the late endosomal/lysosomal compart- ples were assayed for their content of Iand I. After correcting for the initial volume of distribution of the various radiolabeled ment. A fourth group of experiments compared actual proteins, the rates of clearance of each of these probes in the rates of cholesterol accumulation in the tissues of animals different organs were determined and expressed as microliters of lacking NPC1 function with the theoretical rates of lipo- plasma cleared of the particular molecule each hour per gram protein cholesterol uptake by both receptor-mediated and wet weight of tissue (ml/h/g). When these values were multiplied bulk-phase endocytosis. Finally, a histological survey of the by the organ weight, the clearance rates per whole organ were various organs in these animals was undertaken to deter- obtained (ml/h/organ or ml/day/organ). In the case of the LDL mine whether there were significant differences in macro- preparations, when these values were multiplied by the concen- phage recruitment and cholesterol accumulation in mice tration of LDL-TC in the plasma, the absolute values for the lacking functional NPC1 activity compared with those micrograms of cholesterol taken up each day into each organ were obtained (mg/day/organ). The sum of the clearance rates lacking functional LDLR activity. in all organs gave the whole animal clearance rates, and these values, along with the plasma LDL-TC concentration, could be used to calculate the milligrams of LDL-TC turned over each day by the whole animal per kilogram of body weight (mg/day/kg). MATERIALS AND METHODS Animals and diets Measurement of plasma and tissue cholesterol concentrations and liver function tests These studies were undertaken using three different groups of genetically modified mice in addition to appropriate control The plasma total cholesterol concentration was measured 1/1 1/1 animals (designated ldlr /npc1 ). These groups included enzymatically (kit 1127771; Boehringer Mannheim, Indianapolis, 2/2 1/1 animals lacking LDLR activity (ldlr /npc1 ), mice lacking IN). All tissue samples were removed from the animals at the end 1/1 2/2 functional NPC1 activity (ldlr /npc1 ), and animals lacking of the experiments and extracted. The unesterified and esterified 1712 Journal of Lipid Research Volume 48, 2007 fractions were separated, and the cholesterol in each fraction was of homologous mouse LDL by the organs of the control 1/1 1/1 then quantitated by gas-liquid chromatography (31–33). These ldlr /npc1 animals were first measured. As anticipated values are presented as amounts of sterol in each whole organ per from previously published work in the rat, hamster, rabbit, kilogram of body weight (mg/organ/kg). Plasma liver function and cynomolgus monkey (31, 34, 35), the rates of clear- tests were measured by a commercial laboratory. ance per gram of tissue were very high (.250 ml/hr/g) in adrenal and liver (Fig. 2A), but in all of the remaining Histological examination of various tissues organs they were very low (,30 ml/hr/g). Notably, no At the termination of the experiments, various tissues were uptake was detected in the skin or central nervous system. removed, fixed in 10% buffered formalin, and embedded in When organ weights (Fig. 2B) and the plasma LDL-TC con- paraffin. These blocks were then sectioned (5 mm thick) and centration were taken into consideration, the uptake of stained with hematoxylin and eosin. cholesterol carried in LDL overwhelmingly took place in the liver. Thus, whereas the whole animal cleared 656 mg Calculations of LDL-TC per day (Fig. 2C), the liver accounted for The data from all experiments are presented as means 6 SEM 514 mg/day of this uptake. Of the other organs, only the for the number of animals indicated. Where necessary, differ- residual carcass (44 mg/day) and small bowel (35 mg/day) ences between mean values were tested for statistical significance had notable, although much lower, rates of lipoprotein (P , 0.05) using a two-tailed, unpaired Student’s t-test (Graph- Pad Software, Inc., San Diego, CA). cholesterol uptake. As the residual carcass in this experi- ment consisted primarily of muscle, bone, and marrow, and because muscle itself takes up virtually no LDL-TC, this finding implied that the cells of the marrow actively RESULTS used LDL-TC. Assuming that the plasma volume in these To sort out the various processes that result in the tis- mice equaled 60.4 ml/kg body weight (36), the pool of sue uptake of circulating LDL-TC, the rates of clearance LDL-TC in these animals equaled 4.23 mg/kg, the rate of 1/1 1/1 Fig. 2. Total cholesterol carried in LDL (LDL-TC) clearance into the major tissues of the ldlr /npc1 mouse. These animals were infused with [ I]tyramine cellobiose-labeled homologous mouse LDL, and rates of lipoprotein uptake were quantitated in the whole animal and in every major organ. A: Rates of uptake expressed as clearance values in microliters of plasma entirely cleared of its LDL-TC content by a given tissue per hour per gram wet weight. B: Mean weights of the whole animal and each major tissue. C: When these clearance values are multiplied by the concentration of LDL-TC in the plasma and by the respective organ weights, the data shown are obtained in micrograms of LDL-TC taken up each day into each organ. D: These same uptake rates normalized to a constant animal body weight of 1 kg. In this experiment, carcass refers to what tissues remain after the other organs are removed. All data represent means 6 SEM for measurements made in 11 mice. Receptor-mediated and bulk-phase lipoprotein uptake 1713 LDL-TC turnover was 23 mg/day/kg (Fig. 2D), and the rate of LDL-TC turnover was increased to 41 mg/day/kg fractional catabolic rate equaled 5.4 pools/day. (Fig. 3D), and the fractional catabolic rate was decreased to These measurements were next repeated in mice in 0.67 pools/day. which all LDLR activity had presumably been inactivated From these two sets of data, it appeared that the clear- (Fig. 3). In these animals, the relative clearance of LDL-TC ance of LDL-TC by the various organs could be separated per gram of tissue was reduced markedly in the adrenal into receptor-mediated and receptor-independent (mea- 2/2 1/1 and liver but to a much lesser degree in the other organs sured in the ldlr /npc1 mice) components. The rela- 2/2 (Fig. 3A). Although the organ weights in these ldlr / tive importance of the receptor-independent component 1/1 npc1 mice were similar to those of the control animals varied markedly in the different tissues, being very low in (Fig. 3B), the circulating plasma LDL-TC concentration the adrenal (1.8% of total clearance) and liver (6.1%) but was 14-fold higher. As a consequence, the relative amounts much higher in organs like spleen (58%), small bowel of LDL-TC taken up in each tissue varied greatly from the (35%), lung (63%), and residual carcass (64%). Never- values found in the control animals. The mass of LDL-TC theless, it was still conceivable that in some manner this 2/2 1/1 cleared by the whole ldlr /npc1 animal, for example, apparent receptor-independent component of clearance 2/2 was significantly greater (1,109 mg/day) than that in the was the result of residual LDLR activity in the ldlr / 1/1 control mice (656 mg/day) (Fig. 3C), whereas uptake in npc1 animals. To further explore this important issue, the liver (411 vs. 514 mg/day) was nearly the same. In many studies were next undertaken to identify LDL preparations other organs, such as the small bowel (150 vs. 35 mg/day), that could not interact with the mouse LDLR. 1/1 spleen (29 vs. 3 mg/day), lung (26 vs. 3 mg/day), and re- As seen in Fig. 4A, C, E, G, various tissues of the ldlr / 1/1 sidual carcass (392 vs. 44 mg/day), the amount of LDL-TC npc1 mice cleared ovine LDL from the plasma at taken up was actually markedly increased in the ani- higher rates but cleared human LDL at lower rates than 1/1 mals lacking LDLR activity. Thus, in contrast to the ldlr / these organs cleared homologous mouse LDL. Thus, both 1/1 npc1 mice, the extrahepatic organs became the pre- of these heterologous LDL preparations still interacted dominant site for the clearance of LDL-TC. The pool of with the LDLR of the mice, albeit at different rates. How- LDL-TC in these mice was increased to 61.2 mg/kg, the ever, when the human LDL was reductively methylated, 2/2 1/1 Fig. 3. LDL-TC clearance into the major tissues of the ldlr /npc1 mouse. As described in the legend to Fig. 2, these animals were infused with radiolabeled homologous mouse LDL and rates of lipoprotein uptake were quantitated in the whole animal and in every major organ. As in Fig. 2, these uptake rates are represented as clearance values in A, and the weight of each organ is given in B. C illustrates the absolute amount of LDL-TC taken up each day by each organ, and D illustrates the amount of LDL-TC taken up each day into each organ when the animal weight is normalized to 1 kg. All values represent means 6 SEM for measurements made in 10 animals. 1714 Journal of Lipid Research Volume 48, 2007 Fig. 4. Rates of clearance of various heterologous and derivatized preparations of LDL. To find an LDL prepa- ration that bound poorly, or not at all, to the mouse low density lipoprotein receptor (LDLR), LDLs of mouse, sheep, and human origin were radiolabeled 2/2 1/1 1/1 and infused into both the ldlr /npc1 and ldlr / 1/1 npc1 animals. Similar studies were carried out with human LDL that had been reductively methylated to block its interaction with the LDLR. Uptake rates were 2/2 1/1 also measured in the ldlr /npc1 animals for mouse albumin. Clearance rates for these various prepara- tions are shown for the adrenal (A, B) and liver (C, D), two organs with very high proportions of receptor- mediated transport, and for the spleen (E, F) and small bowel (G, H), two organs with very low rates of receptor-mediated uptake. Each value represents the mean 6 SEM for measurements made in 7–14 animals. In all four organs, there were no significant differences in clearance values of mouse LDL, methylated human 2/2 1/1 LDL, or mouse albumin in the ldlr /npc1 animals (B, D, F, H). clearance in the adrenal decreased from 274 to 9 ml/hr/g whole animal cleared 1.70 ml of bulk fluid/h/g (Fig. 3A), (Fig. 4A) and that in the liver decreased from 111 to which equals a clearance rate of 40.8 ml/day/kg body 6 ml/hr/g (Fig. 4C). In contrast, this reductive methyla- weight. Because the plasma volume in these animals tion only decreased the clearance of the human LDL equaled 60.4 ml/kg, this finding implied that nearly two- particle from 24 to 16 ml/hr/g in the spleen (Fig. 4E) and thirds of the plasma volume was endocytosed every 24 h. from 17 to 7 ml/hr/g in the small bowel (Fig. 4G), two Furthermore, the fact that the control animals cleared ho- organs with only a very small apparent component of mologous mouse LDL at a rate of 14.1 ml/hr/g (Fig. 2A), or receptor-mediated uptake. Most importantly, and in con- 338 ml/day/kg, dramatically demonstrated how the bind- trast to these findings in the control animals, the rates ing of LDL to the LDLR and the subsequent concentration of clearance of homologous mouse LDL and reductively and clustering of these complexes into coated pits (Fig. 1A, methylated human LDL were virtually identical in all four B) increased by .8-fold the clearance of LDL in the normal 2/2 1/1 organs of the ldlr /npc1 mice (Fig. 4B, D, F, H). mouse (338 40.8 ml/day/kg). Furthermore, a second indifferent protein, mouse albu- Although these conclusions were based upon measure- min, also was cleared in these organs at the same rates as ments of LDL clearance using either LDL that was not a was the mouse LDL. Thus, these findings strongly ligand for the mouse LDLR or, alternatively, animals that supported the conclusion that there was no detectable lacked LDLR activity, a second test of this conclusion was 2/2 1/1 LDLR activity remaining in the ldlr /npc1 mice and possible. The total amount of LDL-TC taken up by both that the LDL clearance that was observed in these animals receptor-mediated and bulk-phase endocytosis in any or- represented bulk-phase endocytosis. gan equals the product of the clearance rate in that organ The magnitude of this process could be calculated from and the concentration of LDL-TC in the plasma (Fig. 2C). the rates of clearance of methylated human LDL in either In any organ like adrenal that relies predominantly on 1/1 1/1 2/2 1/1 the ldlr /npc1 or ldlr /npc1 animals or from the receptor-mediated LDL-TC uptake (98.2% of total), dele- 2/2 1/1 tion of LDLR activity should decrease the amount of cho- uptake of homologous mouse LDL in the ldlr /npc1 animals. In this latter group of mice, for example, the lesterol reaching the tissue. In contrast, in organs like Receptor-mediated and bulk-phase lipoprotein uptake 1715 spleen and lung that rely much less on receptor-mediated creased significantly, mirroring the relative increases in uptake (?40% of total), deletion of LDLR function should LDL-TC uptake measured directly in these same tissues greatly increase LDL-TC uptake (Fig. 3C). These predicted (Figs. 2C, 3C). Similarly, the decline in the content of differences in the mass uptake of cholesterol through both cholesterol in the adrenal (Fig. 5G) and the lack of change receptor-mediated and bulk-phase endocytosis could be in the liver (Fig. 5H) also reflected the fact that LDL-TC measured directly in the various tissues of animals lacking uptake was reduced in this endocrine organ but was essen- NPC1 function, because, in this circumstance, sterol that is tially unchanged in the liver (Figs. 2C, 3C). endocytosed by either mechanism presumably becomes Two other points concerning this study should be em- trapped in the late endosomal/lysosomal compartment phasized. First, we have previously shown that liver dys- (Fig. 1). Thus, based on the differences in the rates of function in NPC disease is essentially a linear function of LDL-TC uptake in the various tissues after deletion of LDL the amount of cholesterol trapped in the liver (37). In this function (Fig. 3C vs. Fig. 2C), the amount of unesterified study, deletion of LDLR activity did not further change the cholesterol trapped in the adrenal should be reduced, that content of sterol in the liver (Fig. 5H) and did not fur- in the liver should be relatively unchanged, and that in ther alter the plasma transaminase levels (Fig. 5D, E). Sec- extrahepatic organs like lung, spleen, and small bowel ond, in the presence of the NPC1 mutation, nearly all of should be increased. the cholesterol in the tissues is unesterified (21). The excep- As seen in Fig. 5, in the absence of NPC1 function in the tion is the adrenal, which takes up large amounts of choles- 1/1 2/2 ldlr /npc1 mice, the plasma cholesterol concentra- teryl ester carried in HDL by a mechanism that bypasses the tion (Fig. 5A), various liver function tests (Fig. 5C–E), and block in transport out of the endosomal/lysosomal com- the levels of unesterified cholesterol were all significantly partment (24). In the adrenal, where the concentration of 1/1 increased compared with those in the control ldlr / unesterified cholesterol decreased significantly with inter- 1/1 npc1 mice. This increased content of cholesterol in the ruption of LDLR function (Fig. 5G), the concentration of whole animal (Fig. 5F) and various organs (Fig. 5G– J) cholesteryl ester was normal (data not shown). As these two sets of data confirmed the validity of presumably reflected the amount of sterol being taken up by both receptor-mediated and bulk-phase endocytosis the measurements quantitating clearance rates for both through the clathrin-coated pit pathway in these animals receptor-mediated and bulk-phase endocytosis of lipo- with normal LDLR function. However, when the receptor- protein cholesterol, it was next possible to explore the mediated component of this uptake process was elimi- quantitative nature of the unesterified cholesterol seques- nated and the plasma cholesterol concentration increased tration that takes place in every tissue when NPC1 protein 2/2 2/2 markedly, as in the ldlr /npc1 mice (Fig. 5A), the is mutationally inactivated. In Fig. 6A, the absolute amount of excess cholesterol that accumulated in the organs of the relative content of unesterified cholesterol in the whole 1/1 2/2 animal (Fig. 5F), spleen (Fig. 5I), and lung (Fig. 5J) in- ldlr /npc1 mouse by 56 days of age is shown by the Fig. 5. Effect of deleting LDLR activity on the accumulation of unesterified cholesterol in various tissues of the animals lacking NPC1 1/1 1/1 1/1 2/2 2/2 2/2 function. In this study, three groups of animals genotyped as ldlr /npc1 , ldlr /npc1 , and ldlr /npc1 were maintained on the low-cholesterol diet and euthanized at 56 days of age, when measurements of plasma total cholesterol concentration (A), whole body weight (B), and various liver function tests (C–E) were made. In addition, the content of unesterified cholesterol was also determined in the whole mouse (F) as well as in the adrenal (G), liver (H), spleen (I), and lung ( J). These values are presented as milligrams of sterol present in each organ per kilogram of body weight. Each value represents the mean 6 SEM for measurements made in 8–11 animals. * P , 0.05 1/1 1/1 - 1/1 2/2 compared with the control ldlr /npc1 animals; P , 0.05 compared with the ldlr /npc1 animals. AP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase. 1716 Journal of Lipid Research Volume 48, 2007 1/1 2/2 Fig. 6. Theoretical and actual rates of cholesterol accumulation in the tissues of the ldlr /npc1 mouse. 1/1 1/1 1/1 2/2 Both ldlr /npc1 and ldlr /npc1 animals were maintained on the low-cholesterol diet and eutha- nized at 56 days of age. The mass of unesterified cholesterol present in the whole animal and in various organs was determined and expressed as milligrams per kilogram of body weight. This mass of sterol found 1/1 2/2 in the ldlr /npc1 mouse minus the mass found in the control animal represented the amount of excess cholesterol that accumulated in the mutant animal over 56 days as a result of the NPC1 mutation. These values are shown as closed bars in A. The open bars represent the theoretical amount of cholesterol that should have accumulated over this period based on rates of lipoprotein cholesterol uptake. The theoretical value shown for the whole mouse represents the sum of all of the LDL-TC cleared (2,856 mg/kg) and the dietary cholesterol absorbed (896 mg/kg) during this period, whereas the value for the liver equals the sum of that portion of the LDL-TC pool taken up (2,240 mg/kg) and the dietary cholesterol absorbed (896 mg/kg). As indicated, these two values are underestimates of the theoretical value to the extent that clearance rates for the very low density lipoprotein remnant (VLDLr) and biliary cholesterol absorbed through the intestine were not available. The theoretical rates for the other organs were calculated solely from their rates of LDL-TC clearance (see Fig. 2), assuming that chylomicron remnant and VLDLr were not take up by these organs. In B, the actual observed amounts of cholesterol accumulated in the tissues were divided by the respective theoretical amounts that should be present based upon these rates of lipo- protein clearance. In this experiment, carcass refers to what tissues remained after the other organs were removed. The actual rates of accumulation were derived from two groups of animals, each containing 10 animals each. closed bars and is expressed as milligrams of sterol per trapped in these tissues during the 56 day period. These kilogram of body weight. At this age, the whole mutant theoretical values were calculated using the clearance data mouse had accumulated an excess of 3,100 mg/kg that had just been determined (see legend). cholesterol compared with the control animal, whereas As is apparent, in the whole mouse the actual accumu- 1,102 mg/kg of this excess was found in the liver. Lesser lation of sterol was close to the minimal theoretical value amounts accumulated in organs like small bowel, spleen, (3,100 vs. 3,752 mg/kg), whereas accumulation in the liver lung, and carcass (presumably largely in marrow cells). For was much lower than predicted (1,102 vs. 3,136 mg/kg). In comparison, the open bars show the amount of lipo- contrast, although the extrahepatic organs accumulated protein cholesterol that should have been taken up by much less sterol than the liver over this period, in many both receptor-mediated and bulk-phase endocytosis and organs this accumulation was greater than predicted from Receptor-mediated and bulk-phase lipoprotein uptake 1717 the rates of LDL-TC uptake. This is emphasized in Fig. 6B, repeated in animals also lacking LDLR function, accu- in which actual sterol accumulation over 56 days was mulation in the liver was essentially unchanged (1,260 vs. shown to be 3- to 6-fold greater than predicted in organs 1,207 mg/kg), whereas accumulation in tissues like the like spleen, lung, and carcass. Conceivably, this difference spleen (95 vs. 65 mg/kg) and lung (149 vs. 109 mg/kg) in the accumulation of cholesterol in some extrahepatic was increased. Thus, in the presence of the NPC1 muta- organs, compared with the liver, reflected an unexpected tion, there was exaggerated cholesterol accumulation in a shift in the clearance of lipoproteins like the CMr to the number of extrahepatic organs, and bulk-phase endocy- periphery in the mutant animals. This was not the case, tosis played an important role in this accumulation. 1/1 2/2 however, because treatment of the ldlr /npc1 mouse These findings raised the question of whether there with ezetimibe to block cholesterol absorption decreased were morphological alterations in some organs of the 1/1 2/2 sterol accumulation in the liver from 1,207 to 742 mg/kg ldlr /npc1 mouse that could account for this exces- but had essentially no effect on accumulation in the pe- sive and unpredicted accumulation of cholesterol. As illus- ripheral organs. Furthermore, when these studies were trated in Fig. 7, in several organs, like skeletal and cardiac 1/1 1/1 1/1 2/2 Fig. 7. Comparison of the histopathology of the major organs of the ldlr /npc1 and ldlr /npc1 mice. To examine the histological alterations that might have occurred in the presence of the NPC1 mutation (see Fig. 6), tissue sections were prepared and stained with hematoxylin and eosin. These pairs of tissue samples are arranged beginning with those manifesting no histopathology (A–H), those with modest changes (I–P), and, finally, those with marked abnormalities (Q–X). In J–X, the arrows point to histological abnormalities found in certain tissues and described in the text. Each panel is a repre- sentative section taken from the different animals, and all were photographed at the same magnification. Bar 5 40 mm. 1718 Journal of Lipid Research Volume 48, 2007 2/2 2/2 muscle, adrenal, and kidney, no histological changes were in the ldlr /npc1 mice was entirely through bulk- evident (Fig. 7A–H). The morphology of the small intes- phase endocytosis. tine also was essentially normal except for the presence of an occasional foamy macrophage in the villous core (Fig. 7J) and a vesicular transformation of the cytosol DISCUSSION in the autonomic neurons (Fig. 7L) that was similar to changes seen in the neurons and glial cells of the central Mutational inactivation of the transporter NPC1 leads to 1/1 2/2 nervous system in the ldlr /npc1 mouse (38). Both cellular dysfunction in virtually every tissue in the body. the testis (Fig. 7N) and ovary (Fig. 7P) also revealed similar Clinically, in the human, this cellular abnormality may histologic abnormalities. However, the most marked be expressed as severe liver disease, pulmonary failure, changes were seen in those organs that typically have chronic diarrhea, or progressive neurological disease large numbers of macrophages and are part of the reticu- (39–42). The biochemical hallmark of this syndrome is loendothelial system. Clusters of foamy macrophages were an age-related, progressive accumulation of unesterified scattered throughout the liver and interspersed with cholesterol in the cells of every organ (21, 33, 43). This swollen hepatocytes with vesicular cytoplasmic changes sequestered sterol is derived from various lipoproteins (Fig. 7R). Similar macrophage invasion was found in the taken up into the cells by both receptor-mediated and spleen (Fig. 7T) and mucosa-associated lymphoid collec- bulk-phase endocytosis through clathrin-coated pits and tions in Peyer’s patches (Fig. 7V). Most striking was the is manifest histologically as vesicular lipid accumulation lung, which showed marked accumulation of foamy mac- throughout the cytosolic compartment (38, 43, 44). In a rophages within alveoli and apparent reduction in air number of organs, including liver, lymphoid tissue, lung, spaces (Fig. 7X). Presumably, there were similar collec- and brain, there is also infiltration and activation of mac- tions of macrophages in the marrow, but this was not rophages (glia, in the case of the central nervous system), examined specifically. Thus, some of those same organs which may play a role in the death of cells in each of these with the greatest unpredicted rates of cholesterol accu- organs (Fig. 8) (33, 45, 46). Furthermore, when more mulation at 56 days of age (Fig. 6B) seemed to have the lipoprotein cholesterol is shifted to some of these extra- greatest degree of macrophage infiltration (Fig. 7). hepatic organs, there is apparently an increase in this in- In a final experiment, the degree to which these mor- filtration of macrophages. These studies raise important phological changes were dictated by the amount of LDL- issues with respect to the role of this cholesterol accumu- TC taken up and trapped within the late endosomal/ lation in parenchymal cells, as opposed to macrophage lysosomal compartment was explored. As established ear- infiltration, in initiating cell death and, hence, the devel- lier, deletion of LDLR function had little effect on LDL- opment of clinical symptoms in this genetic disorder. TC uptake in the liver (411 vs. 514 mg/day) but increased LDLRs are expressed in many tissues, including cells of uptake in the extrahepatic organs (698 vs. 142 mg/day) the central nervous system, and bind lipoproteins contain- in general and in the spleen and lung (Figs. 2, 3) in par- ing apoE with high affinity but those with apoB-100 with ticular. However, this shift in cholesterol uptake did not lesser affinity (2, 47–49). However, access to these recep- alter the morphology of these tissues. As shown in Fig. 8, tors is limited by the permeability characteristics of the there was no macrophage infiltration and no vesicular endothelial barriers in each organ (Fig. 1), a fact that transformation of the cytosol in the cells of the adrenal probably accounts for the observation that nearly all CMr (Fig. 8A, B), liver (Fig. 8E, F), spleen (Fig. 8I, J), or lung and VLDLr particles and 70–80% of LDL particles are 2/2 1/1 (Fig. 8M, N) in the ldlr /npc1 mice compared with cleared from the plasma by the liver, an organ with a 1/1 1/1 the control ldlr /npc1 animals. Similarly, in the adre- fenestrated sinusoidal capillary system (5). Only 20–30% nal, which primarily uses cholesterol derived from HDL of the plasma LDL-TC pool normally is cleared by the and not LDL-TC (24), tissue morphology also was essentially extrahepatic organs in most species, including humans, 1/1 2/2 2/2 unchanged in the ldlr /npc1 (Fig. 8C) and ldlr / although none is taken up into the central nervous system 2/2 npc1 (Fig. 8D) mice. However, in the animals with these (3, 22, 50). As this process of receptor-mediated endocy- same genotypes, there were striking differences in mor- tosis involves interaction of the LDL particles with a finite phology in those tissues that actively took up LDL-TC number of receptor sites on the liver, the rate of LDL-TC uptake manifests saturation kinetics with respect to the through receptor-mediated and bulk-phase endocytosis. In the absence of NPC1 function, there were clusters of concentration of this lipoprotein in the plasma (34). How- foamy macrophages scattered throughout the liver (Fig. 8G), ever, because the apoE-containing CMr and VLDLr parti- and the density of these collections was about the same cles bind more effectively, and so compete with the binding when LDLR function was also ablated (Fig. 8H). This of the LDL particles, the apparent Michaelis constant for infiltration of macrophages was more pronounced in the the uptake of LDL-TC is shifted to much higher values 1/1 2/2 spleen (Fig. 8K) and lung (Fig. 8O) of the ldlr /npc1 than would be true in the absence of such apoE-containing mice, and in contrast to the liver, the degree of infiltra- lipoproteins (7). As a consequence, the steady-state con- 2/2 2/2 tion was much more pronounced in the ldlr /npc1 centration of LDL-TC in the plasma is typically much animals (Fig. 8L, P). Thus, the severity of the macrophage higher than the steady-state levels of CMr and VLDLr. Apart from this receptor-mediated process, these same infiltration appeared to correlate with the magnitude of LDL-TC uptake in these organs,eventhoughthisuptake cells, including macrophages, typically engulf small vol- Receptor-mediated and bulk-phase lipoprotein uptake 1719 Fig. 8. Macrophage infiltration of various organs in the mouse lacking NPC1 function. Representative histological sections of four organs from control animals (A, E, I, M), animals lacking the LDLR (B, F, J, N), animals with the NPC1 mutation (C, G, K, O), and animals lacking both LDLR and NPC1 function (D, H, L, P) are shown. The arrows in panels G, H, K, L, O, and P point to clusters of pale-staining, foamy macrophages. All of these sections were photographed at the same magnification. Bar 5 100 mm. umes of bulk pericellular fluid during invagination of the LDL-TC into the cells. The magnitude of such uptake can endocytotic vesicles that typically are 80–200 nm in di- be measured using homologous LDL in mice lacking ameter (Fig. 1). Because LDL particles are present in this LDLRs, derivatized LDL that cannot bind to the LDLR, or pericellular fluid, this bulk-phase endocytosis represents a indifferent probe molecules such as albumin, inulin, or second, receptor-independent, process for the uptake of sucrose. In the current studies, three different methods 1720 Journal of Lipid Research Volume 48, 2007 were used, and all gave essentially the same values (Fig. 4B, of LDL-TC uptake was through bulk-phase endocytosis, in D, F, H). It should be noted that because the rate of uptake tissues like the spleen, lung, and carcass (i.e., marrow), by this process does not involve interaction with a finite nearly two-thirds of uptake was normally through bulk- 1/1 1/1 number of receptor sites, but is proportional to the con- phase endocytosis, even in the ldlr /npc1 mice. Fur- centration of LDL-TC in the pericellular fluid, the kinetics thermore, the amount of cholesterol sequestered in these 1/1 2/2 of this uptake process are linear and not saturable (34). As tissues in the ldlr /npc1 animals was three to six times expected, clearance of the LDL particles from the plasma greater (Fig. 6B) than predicted from the rate constants by this bulk-phase process was much lower in the whole for LDL-TC uptake measured in the control mice (Fig. 2C). mouse (46 ml/h; Fig. 3A) than it was in the animals in Like the liver, several of these organs were enlarged rela- which receptor-mediated endocytosis was the predomi- tive to body weight and heavily infiltrated with macro- nant transport mechanism (391 ml/h; Fig. 2A). phages (Fig. 8K,O) (45). This finding raised the possibility, However, because the rate of receptor-mediated LDL- therefore, that much of the unpredicted sequestration of TC uptake is saturable and that of bulk-phase endocytosis cholesterol found in these organs came about because of is a linear function of the plasma lipoprotein concentra- LDL-TC clearance by the infiltrating macrophages as well tion, more LDL-TC is actually taken up and degraded in as the parenchymal cells of the respective organs. As an the absence of LDLRs than in the presence of these aside, this same infiltration of lipid-laden macrophages is 2/2 1/1 receptors. In this study, for example, the ldlr /npc1 seen in the lungs of children with NPC disease and is mice took up 41 mg/day/kg (Fig. 3D) LDL-TC compared described pathologically as “lipoid pneumonitis” (41). with 23 mg/day/kg removed from the plasma by the con- That these macrophages play a role in NPC disease is 1/1 1/1 trol ldlr /npc1 animals (Fig. 2D). Similarly enhanced further supported by the observation that both the choles- LDL-TC turnover has been reported in the rabbit (105 vs. terol accumulation and cellular infiltration were made 19 mg/day/kg) and human (40 vs. 13 mg/day/kg) lacking worse when more lipoprotein cholesterol uptake was in- functional LDLRs (22, 35). In these situations, the VLDLr duced in these tissues. With deletion of LDLR func- particles are apparently removed more slowly from the tion, LDL-TC uptake remained about the same in the liver plasma by the liver so that a greater percentage of these (411 vs. 514 mg/day; Figs. 2,3), whereas uptake in the extra- particles is converted to LDL. As a result, although less hepatic organs increased (698 vs. 142 mg/day; Figs. 2, 3). cholesterol is returned to the liver as VLDLr, more is Under these circumstances, hepatic cholesterol content cleared from the plasma as LDL-TC. Thus, in familial (Fig. 5H), the degree of macrophage infiltration (Fig. 8H), hypercholesterolemia, although there is expansion of the and the liver function abnormalities (Fig. 5D, E) remained pool of cholesterol in the plasma, sterol levels and turn- unchanged. In these same animals, however, LDL-TC uptake was increased markedly in tissues like spleen (29 vs. over in the tissues are essentially normal, as is the histology of the major organs (Fig. 8B, F, J, N) (17, 18, 30). 3 mg/day), lung (26 vs. 3 mg/day), and carcass (392 vs. These studies reveal that both receptor-mediated and 44 mg/day) (Figs. 2, 3). Importantly, this enhanced uptake bulk-phase endocytosis contribute to the age-related ex- was associated with increased cholesterol accumulation pansion of the unesterified cholesterol pool in the late (Fig. 5I, J) and greater apparent macrophage infiltration endosomal/lysosomal compartment of the parenchymal (Fig. 8L, P). Thus, just as the severity of the liver disease cells in all tissues of the NPC animals. Because LDL-TC could be varied by altering the amount of cholesterol uptake from the plasma in the whole mouse (656 mg/day; reaching the hepatocytes through receptor-mediated Fig. 2C) takes place predominantly in the liver (514 mg/ uptake of the CMr, so also the severity of the histopathol- day; Fig. 2C), it is not surprising that this organ accounts ogy in other organs like lung and lymphoid tissue could be for a major portion of the progressive expansion of made worse by increasing cholesterol uptake through the whole-body cholesterol pool seen in NPC disease bulk-phase endocytosis of LDL-TC. (Fig. 5F, H) (21, 33). Furthermore, this expansion is These biochemical and histopathological findings in associated with an increase in relative liver size, infiltra- the liver and extrahepatic tissues are similar to those re- tion of this organ with foamy macrophages (Fig. 7R), ported in the central nervous system in NPC disease. vesicular lipid inclusions in swollen hepatocytes, evi- Although various members of the LDLR family are ex- dence of cellular death through apoptosis, and abnormal pressed in cells of the brain, the movement of cholesterol liver function tests (Fig. 5C–E) (33). The amount of from glial cells to neurons probably takes place through cholesterol sequestered in the liver of 56 day old mice sterol bound to apoE (49, 51–53). In the NPC mouse, can bevariedover a rangeof ?15–90 mg by manipula- there is accumulation of cholesterol in neurons and glial tion of the amount of cholesterol absorbed across the cells, activation of microglia, the central nervous system intestine and carried to the liver in the CMr (37). Im- equivalent of macrophages, and, ultimately, death of se- portantly, the severity of liver damage in such animals, lected populations of neurons and glia (38, 43, 45, 54, 55). as assessed by liver function tests, varies directly with However, neither varying the level of cholesterol absorp- the amount of cholesterol reaching the cells of this organ tion across the intestine, which alters the severity of the and becoming entrapped within the late endosomal/ liver disease, nor manipulation of the level of LDLR activ- lysosomal compartment. ity, which alters the severity of the histopathology in tissues like lung, has any effect on this neurodegeneration. 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Journal
Journal of Lipid Research – American Society for Biochemistry and Molecular Biology
Published: Aug 1, 2007
Keywords: hepatic dysfunction; lung failure; low density lipoprotein receptor; lysosomal cholesterol; apoptosis; neurodegeneration; lipoprotein clearance