Proc. Nati. Acad. Sci. USA Vol. 85, pp. 9821-9825, December 1988 Neurobiology Localization of mRNA for low density lipoprotein receptor and a cholesterol synthetic enzyme in rabbit nervous system by in situ hybridization (3-hydroxy-3-methylglutaryl-coenzyme A synthase/apoprotein E/myelination/Watanabe heritable-hyperlipidemic rabbit) LARRY W. SWANSON*, DONNA M. SIMMONS*, SANDRA L. HOFMANNt, JOSEPH L. GOLDSTEINt, AND MICHAEL S. BROWNt *The Salk Institute for Biological Studies and Howard Hughes Medical Institute, La Jolla, CA 92037; and tDepartments of Molecular Genetics and Internal Medicine, University of Texas Southwestern Medical Center, 5323 Harry Hines Boulevard, Dallas, TX 75235 Contributed by Joseph L. Goldstein, September 15, 1988

ABSTRACT The low density lipoprotein receptor and one macrophages. Thus, apoE and the LDL receptor play a role of its ligands, apoprotein E, are known to be synthesized in the in the healing of peripheral nerves. central nervous system. In the current study, we used in situ A role for apoE in the central nervous system (CNS) was hybridization to localize the receptor mRNA in selected neu- demonstrated by Boyles et al. (5), who showed by immuno- rons and glia throughout the nervous system of 9-day-old cytochemistry that astrocytes produce apoE. This finding rabbits. Particularly high levels were found in sensory ganglia, stimulated a search for LDL receptors in the brain. Hofmann sensory nuclei, and motor-related nuclei. The same regions et al. (7), using RNA blotting to measure LDL receptor contained high levels of mRNA for 3-hydroxy-3-methylglu- mRNA, and Pitas et al. (8), using immunocytochemistry, taryl-coenzyme A synthase, a regulated enzyme in cholesterol identified LDL receptors in rabbit, bovine, rat, and monkey biosynthesis. The distribution of low density lipoprotein recep- brain. The levels of mRNA and protein were higher in white tor mRNA was similar in mature and immature rabbits. The matter than in cortex. The mRNA was present at particularly data suggest that certain cells in the nervous system have high high levels in the medulla, pons, and spinal cord. The amount requirements for cholesterol, which they satisfy through cho- of mRNA did not decline when myelination of the CNS was lesterol synthesis and through receptor-mediated uptake of completed, indicating that the brain required LDL receptors cholesterol-carrying lipoproteins. The latter originate in astro- even in adult life (7). Pitas et al. (8) showed that LDL cytes which synthesize and secrete apoprotein E. These data receptors were present on astrocytes abutting on the arach- suggest that the nervous system of mammals contains an active noid space and in nonidentified cells deeper in the brain. They system for continuous redistribution and recycling of choles- also showed that cerebrospinal fluid contains relatively high terol that is physically distinct from the lipoprotein transport levels of apoE. These studies suggest a hitherto unsuspected system in plasma. system for cholesterol transport in the adult CNS. ApoE- containing lipoproteins secreted by astrocytes would be The low density lipoprotein (LDL) receptor binds cholester- taken up by other brain cells to supply them with cholesterol. ol-carrying plasma lipoproteins that contain apoprotein (apo) In addition to this system for cholesterol transport, the B-100 or apoE. Binding leads to cell uptake by receptor- rabbit brain contains surprisingly high levels of mRNA for mediated endocytosis, thereby supplying cholesterol to cells 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) syn- (1). The production of LDL receptors is driven by a cell's thase, an enzyme in the cholesterol biosynthetic pathway (7). cholesterol requirement. Cells that require large amounts of The level of mRNA declines only by 50-60% when myeli- cholesterol, such as those in the liver and adrenal gland and nation is complete, further suggesting that the brain has an those that grow in tissue culture, produce relatively large ongoing requirement for cholesterol that is fulfilled by LDL numbers of LDL receptors. Cells with low requirements, receptors and by endogenous cholesterol synthesis. such as resting lymphocytes, produce few receptors. When In the current studies, we have used the technique ofin situ the LDL receptor is genetically defective, as in patients with mRNA hybridization to identify the cells that contain mRNA familial hypercholesterolemia or in Watanabe heritable- for the LDL receptor and HMG-CoA synthase. The data hyperlipidemic (WHHL) rabbits, LDL is not taken up by indicate that both of these mRNAs are distributed widely in cells normally, and plasma LDL levels rise (2). neurons in the brain and in sensory ganglia. One of the ligands for the LDL receptor, apoB-100, is secreted almost exclusively by the liver (3). The other ligand, apoE, is secreted by hepatic and extrahepatic cells. One METHODS nonhepatic cell type shown to secrete apoE is the peritoneal Animals. New Zealand white rabbits (9- or 10-day-old and macrophage (4). When loaded with excess cholesterol, mac- adult males) were used for experiments as indicated. WHHL rophages secrete cholesterol-containing lipoproteins that rabbits were raised in Dallas. contain apoE, which targets the lipoproteins to LDL recep- In Situ Hybridization. RNA probe synthesis was carried out tors. ApoE-containing lipoproteins are also secreted by as described by Melton et al. (9) by using either SP6 or T7 macrophages that enter damaged peripheral nerves and polymerase and DNA fragments cloned into pGEM-3 or become engorged with cholesterol derived from degraded pGEM-4 (Promega). Templates for LDL receptor RNA myelin (5, 6). When the nerve regenerates, the neurons and synthesis were derived from a 2.4-kilobase Xmn I-Xho I Schwann cells produce LDL receptors and this allows them fragment corresponding to nucleotides 53-2475 of a cDNA to use the lipoprotein-bound cholesterol secreted from the Abbreviations: apo, apoprotein; 35S-cRNA, 35S-labeled complemen- The publication costs of this article were defrayed in part by page charge tary RNA; CNS, central nervous system; HMG-CoA, 3- payment. This article must therefore be hereby marked "advertisement" hydroxy-3-methylglutaryl-coenzyme A; LDL, low density lipopro- in accordance with 18 U.S.C. §1734 solely to indicate this fact. tein; WHHL rabbit, Watanabe heritable-hyperlipidemic rabbit. 9821 Downloaded by guest on September 29, 2021 9822 Neurobiology: Swanson et al. Proc. Natl. Acad. Sci. USA 85 (1988) for the rabbit LDL receptor (10) cloned into pGEM-3 and cell nuclei. Adjacent series of unhybridized sections were linearized with EcoRI (antisense probe) or cloned into also stained with thionin to identify neuronal size and shape. pGEM-4 and linearized with HindIII (sense-strand control). S1 Nuclease Analysis. LDL receptor mRNA from normal This fragment contains nucleotide sequences corresponding and WHHL rabbit tissues was analyzed as described (7). to nearly the entire coding region of the receptor cDNA. Templates for HMG-CoA synthase RNA synthesis were RESULTS AND DISCUSSION derived from a HindIll fragment of plasmid p53-312 (11) corresponding to nucleotides 29-1581 of the hamster HMG- Fig. 1 Upper shows autoradiograms of tissue sections from a CoA synthase cDNA. This fragment was cloned into pGEM- 9-day-old rabbit that were incubated with a 3"S-labeled 3 in both orientations (for antisense probe and sense-strand antisense RNA complementary to the LDL receptor mRNA. control) and linearized with EcoRI. As a positive control we first examined the adrenal gland, the Animals were anesthetized with phenobarbital sodium (50 richest known source of LDL receptors (1). Silver grains mg/ml; 0.2 ml for 9 day-old animals, i.p.; 2 ml for adults, i.v.) were present over the entire adrenal cortex, including the and perfused transcardially with aldehydes by using a "pH- zona glomerulosa, zona fasciculata, and zona reticularis. shift" method (12). Frozen sections (20,um thick) were cut on There was no hybridization to the medulla. Fig. 1 Upper also a sliding microtome, mounted on poly(L-lysine)-coated shows abundant hybridization ofthe 3"S-labeled complemen- slides, and air-dried under vacuum. Pretreatment, hybridiza- tary RNA (35S-cRNA) in two parts ofthe nervous system: the tion, and washing conditions were similar to those described trigeminal ganglion (Middle) and the hippocampus (Right). Control incubations with a 35S-labeled sense-strand probe (13, 14). Briefly, sections were treated with proteinase K (10 gave no signal in the adrenal or trigeminal ganglion (Lower). ,ug/ml, 37TC, 30 min) in adult tissues or Triton X-100 in Some background hybridization was observed in the hippo- neonatal tissues, acetylated, and dehydrated. After thorough campus, but it was much less intense than that obtained with drying, 70 ,ul of hybridization mixture containing 3"S-labeled the antisense probe. probe (107 cpm/ml, with 10 mM dithiothreitol) was spotted on Fig. 2 shows sections of the same three tissues incubated each slide, sealed under a cover glass, and incubated at 550C with the 35S-labeled antisense probe for HMG-CoA synthase for 16 hr. Slides were then rinsed, digested with ribonuclease mRNA. Again, the adrenal cortex showed abundant labeling, A (20 ug/ml, 37°C, 30 min), washed in 15 mM sodium whereas the medulla was negative. However, in marked chloride/1.5 mM sodium citrate at pH 7 for 30 min at 55°C, contrast to the results with the LDL receptor probe, the HMG- and dried. The sections were exposed to 8max Hyperfilm CoA synthase probe hybridized weakly to the zona glomeru- (Amersham) at 4°C for 4-6 days for preliminary analysis and losa in comparison with the other two zones. These studies to estimate liquid emulsion exposure time. The sections were suggest that the zona fasciculata and zona reticularis derive then delipidized with xylene, rinsed with absolute ethanol, cholesterol both from endogenous synthesis and from LDL air-dried, and coated with Kodak NTB2 liquid emulsion uptake, whereas the zona glomerulosa derives cholesterol diluted 1:1 with distilled water. After an exposure time of 2 primarily from LDL uptake. Glomerulosa cells require much weeks at 4°C, the slides were developed with Kodak D-19, less cholesterol than cells in the other two zones, since glomer- fixed, and stained through the emulsion with thionin. Be- ulosa cells produce relatively small amounts ofsteroid (15). The cause of the ribonuclease A treatment, thionin stained only trigeminal ganglion and the hippocampus showed extensive LDL RECEPTOR TRIGEMINAL GANGLION HIPPOCAMPUS I LL C/) z LU C/) I z I.4

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I FIG. 1. Dark-field photomicrographs showing hybridization of antisense- and sense-strand "S-cRNA probes for LDL receptor to tissue sections through the adrenal gland, trigeminal ganglion, and hippocampus of a 9-day-old rabbit. Note dense hybridization (silver grains appear white) to the entire thickness of the adrenal cortex (ZG, zona glomerulosa; ZF, zona fasciculata; ZR, zona reticularis), but not to the medulla (M). (x60.) In the trigeminal ganglion (and in dorsal root ganglia), many sensory ganglion cells are labeled, as are many small cells, presumably Schwann cells. (x200.) The pyramidal layer of fields CA3 and CA1 in the hippocampus are also heavily labeled, as are presumed glial cells in the corpus callosum (wide diagonal stripe in upper right corner). (x 15.) No specific label was observed in sections hybridized with sense-strand probes. Downloaded by guest on September 29, 2021 Neurobiology: Swanson et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9823 HMG CoA Synthase ADRENAL TRIGEMINAL GANGLION HIPPOCAMPUS

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FIG. 2. Dark-field photomicrographs showing hybridization with antisense- and sense-strand 35S-cRNA probes for HMG-CoA synthase in nearby sections of the same tissues illustrated in Fig. 1. Note that in the adrenal cortex, the zona fasciculata (ZF) shows dense hybridization and the zona reticularis (ZR) shows moderate hybridization, whereas hybridization in the zona glomerulosa (ZG) is near background levels. In contrast, the pattern of hybridization in the trigeminal ganglion and hippocampus is similar to that seen with probes to the LDL receptor (Fig. 1). Magnifications are the same as in Fig. 1.

hybridization with the HMG-CoA synthase cRNA probe, In Figs. 1-4 the sections were prepared from 9-day-old suggesting that cells in these regions synthesize cholesterol. rabbits, which are actively engaged in myelination (16). We Table 1 lists those areas of the CNS that contained also studied the LDL receptor in sections from adult rabbit relatively high levels of mRNA for the LDL receptor and brain and obtained the same general results. We have not yet HMG-CoA synthase in neurons, as judged by clusters of studied HMG-CoA synthase in sections from adult brain. silver grains over large pale nuclei (Fig. 3). Labeling for LDL To confirm that the visualized mRNA was derived from the receptor mRNA was very high in dorsal root ganglion LDL receptor gene, we performed an S1-nuclease analysis on sensory cells, the trigeminal ganglion, and the mesencephalic mRNA obtained from normal rabbits and from WHHL nucleus of the trigeminal nerve. LDL receptor mRNA was rabbits, which have a deletion of 12 base pairs in the coding also present in various motor-related and other neurons in region of the gene (10). A uniformly 32P-labeled cDNA probe widespread parts of the brain (Fig. 4) and in glia in many was allowed to hybridize to the various RNA samples, and regions ofthe nervous system. The distribution of mRNA for the mixture was then digested with the single-strand specific HMG-CoA synthase paralleled that for the LDL receptor S1 nuclease (Fig. 5). The products were subjected to elec- (Table 1). This finding suggests that cells in a single region trophoresis and autoradiography. Adrenal mRNA from nor- synthesize cholesterol and take it up from lipoproteins. It is mal animals protected the entire coding region of the probe not yet clear whether the same cells carry out both processes. from Si-nuclease digestion. Only the vector sequences at the In many areas of the CNS, hybridization signals for the 3' end were digested. Similar full-length protection was receptor and synthase were no higher than the levels ob- obtained with RNA from the , hippocam- served with sense-strand control probes. The most notable of pus, and trigeminal ganglion. The amounts of mRNA were these receptor and synthase-deficient areas were the cere- similar in the trigeminal ganglion in an adult animal and a bellum, striatum and lateral septum, isocortex, and lateral 10-day-old animal, as judged from the intensities of the part of the reticular formation. protected bands. Table 1. Neuron populations that contain mRNA for LDL receptor and HMG-CoA synthase in 9-day-old rabbits Sensory ganglion cells: dorsal root, trigeminal, and mesencephalic n. of trigeminal Somatic motor neurons: ventral horn (spinal cord), hypoglossal n., n. ambiguus, facial n., and trigeminal motor n. Preganglionic parasympathetic neurons: dorsal motor n. of vagus and Edinger-Westphal n. Motor-related cell groups: globus pallidus, subthalamic n., compact part of the , , red n., and lateral reticular n. Sensory relay nuclei: vestibular nuclei, external cuneate n., and superior colliculus and (all parts) Basal forebrain cholinergic regions: medial septal n., diagonal band n., substantia innominata, and magnocellular preoptic n. Amygdala (all parts) and bed n. of stria terminalis Cerebral cortex: piriform cortex and olfactory tubercle Reticular formation and related cell groups: large and medium-sized neurons throughout length of medial half of reticular formation, all , and n., nucleus. Downloaded by guest on September 29, 2021 9824 Neurobiology: Swanson et al. Proc. Natl. Acad. Sci. USA 85 (1988) A. Normal B. WHHL Adult 10 days Adult

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mRNA | i""""""3 :,G 3n.High-poe brih-il photo r'rp@#shwn hy- a, * ' 'm .4o**- [ P] cDNA probe I (646nt) _ Protected F 553nt Normal FragmentsProtetedLL 292nt 249nt WHHL FIG. 3. High-power bright-field photomicrograph showing hy- bridization of an antisense rabbit LDL receptor 35S-cRNA probe to FIG. 5. SI-nuclease analysis of LDL receptor mRNA from the dorsal lateral geniculate nucleus ofa 9-day-old rabbit. The section normal and WHHL rabbits of the indicated age. A uniformly was stained with thionin, but only nuclei are stained because the 32P-labeled probe corresponding to nucleotides 78-629 of the rabbit section was treated with ribonuclease A. Note clusters of silver LDL receptor cDNA (10) plus 93 nucleotides of cloning and phage grains over several large pale nuclei, which because of their size are M13 polylinker sequences was hybridized to 20 tkg of total RNA neurons. (x667.) isolated by the guanidinium isothiocyanate/CsCl method (17) from the indicated tissues and then digested with S1 nuclease. Digestions When the mRNA was obtained from WHHL rabbits, the were carried out with either 200 units (A) or 60 units (B) of S1 probe was cleaved by the S1 nuclease to yield fragments of nuclease (Bethesda Research Laboratories) at 37'C for 1 hr. Pro- 292 and 249 nucleotides (Fig. 5). Inasmuch as the site of this tected fragments were visualized by autoradiography after fraction- cleavage corresponds to the known site of the deletion in the ation on denaturing polyacrylamide gels. The gel was exposed to WHHL gene, this result demonstrates that the mRNA in the XAR-5 film for 10 hr at -70'C with an intensifying screen. Lengths CNS is produced by the authentic LDL receptor gene (10). ofthe protected fragments were estimated relative to standards from Msp I-digested pBR322 DNA. The slightly larger bands above the 292-nucleotide fragment derived from the WHHL allele is a conse- 1 quence of incomplete S1 digestion of poly(G) tails (generated in the cloning procedure) present at the 5' end of the probe. The design of the experiments and the expected results for the normal and WHHL mRNAs are indicated in the lower portion of the figure. nt, nucle- otides; sup. col., superior colliculus; hippo., hippocampus. The current results, when coupled with previous data (5- 8), imply that the brain has an active system for transporting and synthesizing cholesterol. This system appears to be physically distinct from the lipoprotein transport system in plasma. Cholesterol transport in the CNS originates in astrocytes, which synthesize and secrete apoE-containing lipoproteins. The cholesterol in these lipoproteins is most likely derived from the turnover of myelin and plasma membranes of neurons and glia. The apoE-containing lipo- proteins secreted by the astrocytes are targeted to cells that FIG. 4. Dark-field photomicrographs showing the pattern of express LDL receptors. As shown in the current studies and hybridization with an antisense rabbit LDL receptor 35S-cRNA in the studies of Pitas et al. (8), LDL receptor-bearing cells, probe to various parts of the 9-day-old rabbit brain. (A) Two cell including neurons and glia, are distributed widely in the CNS. groups in the dorsomedial medulla show dense hybridization: This transport mechanism would allow continuous recycling preganglionic parasympathetic neurons in the dorsal motor nucleus of cholesterol from one cell to another in the brain. It would of the vagus nerve (DMX) and motor neurons in the hypoglossal also allow cholesterol to be exported out of the brain through nucleus (XII). The area postrema (AP) and fourth ventricle (circular area) are in the midline. (x30.) (B) Neurons in the intermediate gray the movement of apoE-containing lipoproteins from cerebro- layer of the superior colliculus (right side of the brain) show spinal fluid to plasma (8). Cells in sensory ganglia may also particularly dense hybridization. (x 10.) (C) Neuronal cell groups in be able to use their LDL receptors to take up circulating the ventral that show clear hybridization include the red LDL. The capillary endothelia in these ganglia contain nucleus (RN), ventral tegmental area (VTA), and compact part ofthe fenestrae (18) that may permit the passage of plasma lipo- substantia nigra (SNc); considerably weaker hybridization is found proteins. in the reticular part of the substantia nigra (SNr) and in the own interpeduncular nucleus (IPN). (x40.) (D) This section through the Cells in the brain also have the ability to produce their basal forebrain shows dense hybridization to neurons in the medial cholesterol. In rabbits, the overall rate of cholesterol syn- septal nucleus (MS) and the contiguous nucleus of the diagonal band thesis, when averaged throughout the brain, is lower than the (DB). The ventral tips ofthe anterior horns ofthe lateral ventricle can rate in the adrenal gland, but higher than it is in kidney and be seen in the upper left and upper right. Frontal sections. (x 10.) skeletal muscle (19). The current studies raise the possibility Downloaded by guest on September 29, 2021 Neurobiology: Swanson et al. Proc. Natl. Acad. Sci. USA 85 (1988) 9825 that cholesterol synthesis may be much higher in certain 2. Goldstein, J. L. & Brown, M. S. (1983) in The Metabolic Basis regions of the brain. In general, the regions that show high ofInherited Disease, eds. Stanbury, J. B., Wyngaarden, J. B., levels of mRNA for HMG-CoA synthase also show high Fredrickson, D. S., Goldstein, J. L. & Brown, M. S. (Mc- levels of LDL receptor mRNA. These regions must have a Graw-Hill, New York), pp. 672-712. particularly high turnover of cholesterol that they satisfy 3. Mahley, R. W., Innerarity, T. L., Rall, S. C., Jr., & Weisgra- through endogenous synthesis and of ber, K. H. (1984) J. Lipid Res. 25, 1277-1294. uptake apoE- 4. Basu, S. K., Brown, M. S., Ho, Y. K., Havel, R. J. & Gold- containing lipoproteins. stein, J. L. (1981) Proc. Natl. Acad. Sci. USA 78, 7545-7549. The question arises as to whether the high requirement for 5. Boyles, J. K., Pitas, R. E., Wilson, E., Mahley, R. W. & cholesterol in certain regions ofthe brain is attributable solely Taylor, J. M. (1985) J. Clin. Invest. 76, 1501-1513. to the cholesterol required for structural purposes, or 6. Mahley, R. W. (1988) Science 240, 622-630. whether cholesterol might be converted into other products, 7. Hofmann, S. L., Russell, D. W., Goldstein, J. L. & Brown, such as steroid hormones, in these regions. LeGoascogne et M. S. (1987) Proc. Natl. Acad. Sci. USA 84, 6312-6316. al. (20) have shown that the cholesterol side-chain cleavage 8. Pitas, R. E., Boyles, J. K., Lee, S. H., Hui, D. & Weisgraber, enzyme is present in oligodendrocytes and other cells in the K. H. (1987) J. Biol. Chem. 262, 14352-14360. brain, particularly in the and in the olfactory 9. Melton, D. A., Krieg, P. A., Rebagliati, M. R., Maniatis, T., bulb. This enzyme produces pregnenolone. It is possible that Zinn, K. & Green, M. R. (1984) Nucleic Acids Res. 12, 7035- some of the LDL receptor activity and cholesterol biosyn- 7056. thetic activity in the nervous system is used to provide a 10. Yamamoto, T., Bishop, R. W., Brown, M. S., Goldstein, J. L. substrate & Russell, D. W. (1986) Science 232, 1230-1237. for this steroid synthesis. 11. Gil, G., Goldstein, J. L., Slaughter, C. A. & Brown, M. S. If the LDL receptor plays a physiologic role in neural (1986) J. Biol. Chem. 261, 3710-3716. tissues, backup systems must exist. Genetic defects in the 12. Gerfen, C. R. & Sawchenko, P. E. (1984) Brain Res. 290, 219- LDL receptor in humans or in WHHL rabbits do not cause 238. obvious malfunctions of the CNS (2). Brain cells that ordi- 13. Cox, K. H., DeLeon, D. V., Angerer, L. M. & Angerer, R. C. narily use LDL receptors may thus develop increased cho- (1984) Dev. Biol. 101, 485-502. lesterol synthesis when LDL receptors are deficient. Evi- 14. Simmons, D. M., Arriza, J. L. & Swanson, L. W. (1988) J. dence for such a backup was obtained in the adrenal gland of Histotech., in press. the WHHL rabbit, which has a 5-fold elevated rate of 15. Bondy, P. K. (1985) in Williams Textbook of Endocrinology, cholesterol synthesis (21). It will be important to determine eds. Wilson, J. D. & Foster, D. W. (Saunders, Philadelphia), whether cholesterol synthesis and levels of mRNA for pp. 816-833. 16. Dalal, K. B. & Einstein, E. R. (1969) Brain Res. 16, 441-451. cholesterol biosynthetic enzymes are elevated in certain cell 17. Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J. & Rutter, types of the CNS of WHHL rabbits. W. J. (1979) Biochemistry 18, 5294-5299. 18. Arvidson, B. (1979) Exp. Neurol. 63, 388-410. We thank David W. Russell for helpful advice. This research was 19. Spady, D. K. & Dietschy, J. M. (1983) J. Lipid Res. 24, 303- supported by Grants HL20948 and NS16686 from the National 315. Institutes of Health. S.L.H. is the recipient of the American Heart 20. LeGoascogne, C., Robel, P., Gouezou, M., Sananes, N., Association-Squibb Clinician Scientist Award 86-412. Baulieu, E.-E. & Waterman, M. (1987) Science 237, 1212-1215. 21. Dietschy, J. M., Kita, T., Suckling, K. E., Goldstein, J. L. & 1. Brown, M. S. & Goldstein, J. L. (1986) Science 232, 34-47. Brown, M. S. (1983) J. Lipid Res. 24, 469-480. Downloaded by guest on September 29, 2021