THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 263, No. 7. Issue of Marcb 5, pp. 3411-3417,1988 Printed in U.S.A.

Type C Niemann-Pick Disease LYSOSOMAL ACCUMULATION AND DEFECTIVE INTRACELLULAR MOBILIZATION OF LOW DENSITY LIPOPROTEIN CHOLESTEROL*

(Received for publication, September 4, 1987)

Jacob SokolS, E. Joan Blanchette-Mackies, Howard S . Kruthl, Nancy K. DwyerQ, Lynn M. AmendeQ, Jean D. Butler11 , Enid Robinson*, Shutish Patel**, Roscoe 0. Brady$, Marcella E. ComlyS, Marie T. Vanier$*, and Peter G . Pentchev*§Q From the $Developmental and Metabolic Neurology Branch, National Institute of Neurological and Communicative Disorders and Stroke, the §Endocrinology Section, Laboratory of CeUular and Developmental Biology, National Institute of Diabetes, Digestive and Kidney Diseases, the llLaboratory of Experimental Atherosclerosis, National Heart, Lung and Blood Institute, the I( Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Marylnnd 20892, the $$Laboratoire de Bwchimie, Institut National de la Sante et de la Recherche Medicale U 189, Faculte de Medecine Lyon-Sud, BP 12, F-69921 Oullins Cedex, Frame, and the **Neurological Service, Veterans Administration Medical Center, Newington, Connecticut 061 11

The intracellular accumulation of unesterified cho- lesterol was examined during 24 h of low density lip- oprotein (LDL) uptake in normal and Niemann-Pick C fibroblasts by fluorescence microscopy with filipin staining and immunocytochemistry. Perinuclear fluo- rescence derived from filipin-sterol complexes was ob- served in both normal and mutant cells by 2 h. This perinuclear cholesterol staining reached its peak in normal cells at 6 h. Subsequent development of fluo- rescence during the remaining 18 h of LDL incubation was primarily limited to the plasma membrane region of normal cells. In contrast, mutant cells developed a much more intense perinuclear fluorescence through- out the entire 24 h of LDL uptake with little enhance- ment of cholesterol fluorescence staining in the plasma membranes. Direct mass measurements confirmed that internalized LDL cholesterol more readily replenishes the plasma membrane cholesterol of normal than of mutant fibroblasts. Perinuclear filipin-cholesterol flu- orescence of both normal and mutant cells was co- localized with lysosomes by indirect immunocytochem- ical staining of lysosomal membrane protein.

Abnormal sequestration of LDL cholesterol in mu- tant cells within a metabolically latent pool is sup- ported by the finding that in vitro esterification of cellular cholesterol could be stimulated in mutant but not in normal cell homogenates by extensive disruption of the intracellular membranous structures of cells previously cultured with LDL.

Deficient translocation of exogenously derived cho- lesterol from lysosomes to other intracellular mem- brane sites may be responsible for the delayed homeo- static responses associated with LDL uptake by mutant Niemann-Pick Type C fibroblasts.

Type C Niemann-Pick disease is a human autosomal-reces- sive lipid storage disorder (1). Certain clinical, morphological, and biochemical similarities with type A and B Niemann- Pick diseases prompted the classification of this disorder as

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

5 s To whom correspondence should be addressed.

Type C Niemann-Pick disease (2). Although subsequent stud- ies showed both the type A and B disorders to be primary sphingomyelinase mutations (3-5), no consistent evidence of a similar primary lesion in sphingomyelin catabolism has been reported for Type C Niemann-Pick disease (6). To the con- trary, recent investigations have suggested that this disorder may, in fact, represent a primary lesion that disturbs critical balances in cholesterol metabolism (7-11).

Cellular cholesterol homeostasis involves a series of inte- grated responses that enable cells to maintain cholesterol levels within a critical range needed for optimal growth and development under environmental conditions that include both cholesterol excess and deprivation (12). Receptor-me- diated uptake and hydrolytic lysosomal processing of LDL’ in cultured fibroblasts derived from Niemann-Pick C patients are associated with cellular homeostatic responses that are uniformily delayed (11). Lipoprotein uptake by the mutant cells leads to an excessive intracellular accumulation and storage of cholesterol primarily as unesterified sterol (8). The inability of internalized cholesterol to initiate timely regula- tory responses in these mutant cells could have resulted from a primary lesion either in the initiation of a regulatory mes- sage commonly shared by all the affected responses or in the intracellular transport of cholesterol. The present data will document that a sterol transport error plays a major role in the cellular pathology of Niemann-Pick C disease.

EXPERIMENTAL PROCEDURES

Materials-[l,10-3H]Oleic acid (2-10 Ci/mmol) was obtained from Du Pont-New England Nuclear. ATP, CoA, fatty acid-free bovine serum albumin, and human LDL were purchased from Sigma. Pre- coated silica gel 60 plates were obtained from Merck. The enzymes used in the direct fluorometric assay of cholesterol masses, cholesterol oxidase, and horseradish peroxidase were purchased from Boehringer Mannheim. Lipoprotein-deficient fetal bovine serum (LPDS) was prepared by Biomedical Technologies, Boston, from KBr serum so- lutions by ultracentrifugation as described (8). ITS (insulin/transfer- rin/selenium) was obtained from Collaborative Research, Inc., Bed- ford, MA.

Cell Cultures-Normal and mutant Type C Niemann-Pick fihro- blasts represented established secondary cell lines derived from su- perficial skin biopsies of normal volunteers and confirmed patients of the Developmental and Metabolic Neurology Branch of the Na- tional Institutes of Health. Cell cultures were maintained in Eagle’s

The abbreviations used are: LDL, low density lipoprotein; LPDS, bovine lipoprotein-deficient serum.

341 1

3412 Type C Niernann-Pick Disease

minimal essential medium supplemented with 10% (v/v) complete fetal bovine serum, 2 mM L-glutamine, 100 units of penicillin, and 100 pg streptomycin/ml in humidified 95% air and 5% CO, at 37 "C. Cells were harvested by washing monolayers three times with phos- phate-buffered saline (PBS) and subsequent treatment with 0.05% trypsin (Sigma) for 5 min at 37 "C. Specific experimental culture manipulations and conditions are described in the appropriate leg- ends.

Fimrescent Cytochemical and Immunocytochemical Staining of Cholesterol and Lysosomes-Cells were seeded and cultured directly on microscopic slide chambers (Lab Tek). In experiments designed to measure only unesterified cholesterol, cell mololayers were fixed with 10% phosphate-buffered formalin and subsequently stained with 0.05 mg/ml of filipin (generously supplied by The Upjohn Co.) for 60 min as described previously (8). Fluorescence of stained preparations was photographed with excitation from a 100-watt mercury arc lamp passed through UG-1 filter and emission viewed through a 510-nm filter using a 60-8 exposure. Concurrent filipin-cholesterol staining and rhodamine-labeled anti-lysosomal antibody fluorescence detec- tion were carried out as follows. Cells in the plastic slide chambers were washed with PBS, fixed in 3% paraformaldehyde for 30 min at room temperature, and washed three times with PBS. All subsequent steps were carried out in a 10% solution of normal fetal calf serum in PBS with 0.05 mg/ml of filipin. The use of filipin in all incubation solutions served to permeabilize the cells to the antibody preparations and to fluorescently label unesterified cholesterol. The primary an- tibody was rat antibody specifically directed against human lysosomal membrane protein and was a generous gift of Dr. J. W. Chen, Department of Pharmacology and Experimental Therapeutics, Johns Hopkins University School of Medicine, Baltimore, MD (13). Cells were incubated with the primary antibody at a 1:4 dilution for 30 min. The cells were washed free of unbound primary antibody and incubated with affinity-purified goat anti-rat IgG conjugated to rbo- damine (Jackson Labs, Avondale, PA) at a dilution of 1:40 or 30 min. Cells were washed, mounted in para-phenylenediamineglycerol, and viewed with a Leitz fluorescence microscope using an excitation filter (band pass 350-410) for filipin and (band pass 530-560) for rhoda- mine. Control for the immunocytochemical study was the replace- ment of the specific primary monoclonal antibody to human lysoso- mal membrane protein with a nonspecific monoclonal antibody to mouse lysosomol membrane protein and subsequent treatment of the cells with rhodamine-conjugated antibody. No rhodamine fluores- cence was noted with the human cells. Controls for discrete visuali- zation of fluorescent signal were: 1) cells exposed to filipin alone and viewed to the rhodamine (band pass 530-560) showed no signal; 2) cells exposed to specific primary antibodies and second rhodamine- conjugated IgG using saponin as the permeabilizing agent and viewed at the filipin (band pass 350-410) showed no signal.

Determination of Unesterified Cholesterol in Plasma Membranes- Advantage was taken of the observation of Lange (14, 15) that cholesterol oxidase efficiently and selectively oxidizes only plasma membrane cholesterol when intact cell suspensions are first treated with glutaraldehyde. Freshly harvested cell pellets (5 mg of protein) were suspended in 1 ml of PBS and aliquoted into separate 0.20-ml samples. Cells were pelleted and resuspended in 0.20 ml of PBS & 1% glutaraldehyde. Following gentle mixing and incubation in an ice bath for 10-15 min, cells were pelleted by low speed centrifugation and washed three times with 1 ml of 310 mM sucrose and 0.5 mM phosphate buffer, pH 7.5, at 10 'C. The individual cell pellets were subsequently suspended in 0.20 ml of this buffer in the presence of 2.5 mg/ml of cholesterol oxidase (6000 units/gm; Breuibacterium sp, Beckman Instruments). Cell suspensions were incubated for 45 min at 10 "C with gentle agitation. Cells were pelleted, washed, and suspended in 0.20 ml of PBS. Cell suspensions not exposed to gluta- raldehyde were used to measure protein by the method of Lowry (16).

The remaining cell suspensions were extracted with 4 ml of chloro- form/methanol (2:l v:v) and the lower lipid-containing phase evapo- rated under a nitrogen stream. Lipid residues were taken up in 0.20 ml of isopropanol and the unesterified cholesterol measured with an enzymatic fluorescence assay (17). Cholesterol levels in non-oxidase- treated cells equaled total cellular free sterol. Cholesterol levels re- maining in oxidase-treated cells represented intracellular free sterol. Free cholesterol specifically associated with the plasma membrane was calculated from the total minus the intracellular levels.

In Vitro Esterification of CeUular Cholesterol-Freshly harvested and washed cell pellets (5 mg of protein) were suspended in 1 ml of 250 mM sucrose and 10 mM Tris-HC1, pH 7.4. The cell suspensions were divided into separate aliquots of 0.20 ml. Some of the suspen- sions were centrifuged and the cell pellets frozen in liquid nitrogen for 1 h. The frozen cell pellets were subsequently taken up in 0.20 ml of 10 mM Tris-HC1, pH 7.4, and frozen and thawed an additional three times in liquid nitrogen and at 37 'C at 5-min intervals. These lysed cells were further disrupted by vigorous homogenization for 1 min at 0 "C in a small glass-fritted homogenizing tube with a motor- ized tight-fitting glass pestle at lo00 rpm. Other portions of the fresh cell suspensions in the isotonic sucrose buffer were placed in a small NZ cavitation chamber (Kontes) at 40 p.s.i. for 5 min at 4 'C. These partially lysed cell suspensions were further homogenized gently in a smooth-surface glass homogenizing tube fitted with a loose Teflon pestle a t 100 r.p.m. for 30 s a t 0 "C. It has previously been shown that such controlled disruption allows cell-free extracts to essentially retain intact subcellular organelles (18). Aliquots (0.010 ml and 50 pg of protein) of the respective total cell-free extracts were incubated in 0.19 ml of 250 mM sucrose, 2 mM dithiothreitol, 5 mM KF, and 10 mM Tris-HC1, pH 7.4, containing 6 mM ATP, 0.6 mM CoA, 15 mM MgCI, and 0.40 mM [3H]oleate (370 dmp/pmol in 14% fatty acid-free bovine serum albumin). Incubations were carried out for 2 h at 37 "C and the lipids subsequently extracted with chloroform/methanol (2:l v:v). The level of [3H]oleate incorporated into cellular cholesterol to form chole~teryl-[~H]oleate was measured by thin layer chromatog- raphy as described previously (8).

RESULTS

Fluorescence Microscopic Studies of the Intracellular Storage of Unesterified Cholesterol-LDL uptake was monitored over a period of 24 h in normal and mutant Niemann-Pick C fibroblasts conditioned by an extended prior period of lipo- protein deprivation. At indicated intervals, cell cultures were washed, fixed, and stained with filipin to follow the intracel- lular deposition of unesterified LDL-derived cholesterol. (Fig. 1). Prior to the uptake of LDL only low levels of filipin staining were found in the cultures indicating a minimal cellular content of unesterified cholesterol. Following 2 h of LDL uptake, a distinctive perinuclear filipin staining devel- oped in both cell lines which was somewhat more intense in the Niemann-Pick C cells. After 6 h of LDL uptake, perinu- clear fluorescence in the mutant cells had increased to levels which were now significantly higher than those in normal cells. Between 6 and 24 h of LDL uptake, a further significant increase of perinuclear filipin-cholesterol staining was noted in mutant but not in normal cells. The current fluorescent studies expand our earlier observations of excessive unester- ified cholesterol storage in mutant cells (9, 10) and clearly now document the temporal and sequential fashion of LDL cholesterol accumulation in NP-C cells.

Flc. 1. Fluorescent filipin staining of the sequential intracellular accumulation of LDL-derived cholesterol in normal and Niemann-Pick C fibroblasts. Confluent flasks of normal and mutant cell cultures were placed on McCoy's medium with penicillin (100 units/ml) and streptomycin (100 mg/ml) + 1% glutamine, 0.1% ITS, 0.2% bovine serum albumin and no serum for 8 days. Cells were harvested and seeded in tissue culture wells (pretreated with 2.5 pg of fibronectin/cm') at a density of 2.0 X 10' cells/9.5-cm2 chamber in 4 ml of McCoy's medium and 0.2% bovine serum albumin. These particular conditions of cholesterol deprivation were found to maximize most consistently a low base-line level of cholesterol in the cultured cells. The Niemann-Pick C phenotype of abnormal cholesterol homeostatic responses is fully expressed under these particular culture conditions (data not presented). Cells were cultured for 2 days and subsequently incubated in fresh medium f LDL (50 pg/ml). Cell monolayers were washed at the indicated intervals with PBS and subsequently fixed and stained with filipin for cholesterol as described in the experimental procedures. Magnification X 60.

0 Y e E C C

Q, z

m E b Z

Type C Niemann-Pick Disease 3413

r 0

E r hl

E r hl

E

mc. 1.

3414 .l!ype C Niemann-Pick Disease

Rhodamine Immuno-Staining of Lysosomes

Filipin Staining for Unesterified Cholesterol

I FIG. 2. C w kdhti~~ Of m a a d ~holeoterol md mm in -4 md NIenuan-

Pick C ceh cultured with LDL. Normal and mutant cells were cultured and prepared for LDL uptake as described in the legend to Fig. 1. Cells were incubated with LDL (50 dml) for 24 h and subsequently washed, fixed, and prepnred for cytochemical staining of unesteri6ed cholesterol with filipin and immunocytochemical stainiq of lyeoeomea with rhodamine-labled antibodies as described under 'Experimental pmceduree." Magni- fication X 94.

The perinuclear storage depots for the unesterifkd choles- terol were identified as lysosomes (Fig. 2). Filipin fluorescence in the perinuclear region showed a very high degree of identity with a second separate fluorescent signal (rhodamine antibody complex) targeted specifically to the lysosomes.

Previous observations of mutant cells had indicated that excessive perinuclear accumulation of LDL cholesterol was contrasted with a deficient cholesterol replenishment of plasma membranes when compared to normal cells (10). These preliminary observations could now be confirmed with cell cultures that were extensively deprived of medium cho- lesterol prior to their uptake of LDL (Fig. 3). This extensive prior cholesterol depletion enhances the differences in inten- sity of filipin staining in LDL-treated and nontreated fibro- blasts. Presumably, this pretreatment minimizes endogenous cellular cholesterol levels prior to lipoprotein loading. LDL uptake by normal cells was shown to be associated not only with notable perinuclear filipin staining but also with a fainter but discemable development of filipin staining at the outer plasma membrane region of the cells (Fig. 3). In comparably treated Niemann-Pick C fibroblasts, a significantly lower level of filipin fluorescence staining was noted in the plasma mem- brane even though very intense filipin-cholesterol staining formed within the perinuclear region of the mutant cells.

Although these photomicrographs are made at a single plane and show a somewhat diffuse intracellular fluorescence at the outer boundaries of the cholesterol-loaded cells, appli- cation of through focus analysis at many planes in the cell allowed one to clearly identify a peripheral plasma membrane fluorescent line. Other ongoing studies with filipin at the electron microscopic level indicate that filipin-cholesterol complexes are present in the plasma membrane of normal LDL-loaded cells? Independent biochemical verification of specific plasma membrane cholesterol enrichment is pre- sented in the next section.

Relative Mass Measurement of Unesterified Cholesterol in

* J. Sokol, E. J. Blanchette-Mackie, H. S. Kruth. N. K. m e r , L. M. Amende, J. D. Butler, E. Robinson, S. Patel. R 0. Brady, M. E. Comly, M. T. Vanier. and P. G. Pentchev. unpublished data.

Plasma Membmes-In order to directly quantitate relative distributions of unesterified cholesterol in the mpective in- tracellular and plasma membrane domains of cultured fibro- blasts, advantage was taken of the selective oxidation by cholesterol oxidase of plasma membrane cholesterol in intact. glutaraldehyde-treated cells (14,151. In cells grown with 10% lipoprotein-deficient serum for 4 days, comparable levels and distributions of unesterified cholesterol were seen in normal and mutant cells with somewhat higher intracellular levels found in the mutant cells (Table I). Following 24 h of LDL uptake, total cellular unesterified cholesterol rose in mutant cells to levels substantially higher than those found in normal cells. However, in association with this internalization of excessive LDL cholesterol by mutant cells, there was less enrichment of the plasma membranes with cholesterol when compared to normal cells.

Both the relative maas measurements as well as fluorescent filipin staining show plasma membrane cholesterol levels to be substantially lower (1646% of total) than reported by others for cultured fibroblasts (>90% of total) (14,151. These former studies were carried out with confluent cells cultured with LPDS for 24 h. The present experiments were specs- caUy desiied to study intracellular distribution of cholesterol in extensively sterol-depleted, sparsely populated, and ac- tively growing cultures during the active phase of LDL uptake. It is likely that specific culture conditions play a major role in determining the disposition of cellular cholesterol between intracellular and plasma membrane pools.

Comparative Esterifications of InternaliEed Cholesterol in Normal and Mutant CeU Preparations-This documented ex- cessive lysosomal storage and tardy intracellular mobilization of cholesterol in Niemann-Pick C fibroblasts suggested that LDL uptake by the mutant cells resulted in aequeatration of exogenously derived cholesterol within a metabolically silent or trapped pool. In order to explore this possibility, advantage was taken of the reported in oitro modulations of cholesterol ester formation through direct alterations of cholesterol within membranes that contain acyl-CoA.cholestero1 acyl- transferase (19,201, the enzyme responsible for intracellular

Type C Niemnn-Pick Disease 3415

NL

N P-C

FIG. 3. Fluoreecent filipin etaining of the differential intracellular distribution of LDL-derived cholesterol in normal and Niemann-Pick Type C fibroblasts. Stock normal and mutant cells were harvested and seeded in 25-cm' flasks at a density of 1 X 106 cells in 5 ml of McCoy's medium containing 1% glutamine, 100 units/ml penicillin, and 100 pg/d streptomycin and 5% LPDS. Medium was replaced every 4 days for 12 days. Cultures were harvested and seeded at a 1:2 dilution in 25-cm' flasks in 5 ml of above complete culture medium. After 24 h of culture, medium was exchanged with fresh medium in which LPDS was exchanged with 0.2% bovine serum albumin. After 5 days of culture, cells were trypsinized and seeded in tissue culture chambers (pretreated with fibronectin, 1 pg/cm') at a density of 30,000 cell49.5 cm' of chamber area in 4 ml of above McCoy's medium containing 0.2% bovine serum albumin. Following 2 days of culture, the cells were treated with fresh medium f LDL (50 &ml) for 24 h. Cell monolayers were washed three times with 4 ml of PBS. Fixation and the specific 6lipin staining techniques for cholesterol are described under "Experimental Procedures." Magnification X 114.

cholesterol estefllcation (21). In mutant Niemann-Pick C cells, the availability of cholesterol for interaction with acyl- CoAcholesterol acyltransferase was considered potentially latent because of possible topological hinderances which could be envisioned to block the translocation of sterol to the catalytic site of acyl-CoAcholesterol acyltransferase. Follow- ing in vivo uptake of LDL, subsequent in vitro esterification of internalized cholesterol could be modulated in cell-free extracts of mutant, but not normal, cells by regulating the extent of secondary organelle disruption (Table II). Following LDL uptake, in vitro synthesis of cholesterol [%]oleate from endogenous cholesterol stores could be varied in mutant cell- free extracts from levels below ((20%) to above (150%) those of comparably treated control cell extracts by controlliig the extent of subcellular organelle disruption. It should also be noted that the relative in vitro deficiency of cholesterol ester- ification observed in mutant cell-free extracts with intact subcellular organelles corresponded to the relative deficiency of cholesterol esterification obge~ed in situ with intact Nie- mann-Pick C cells.

In principle, activation of in uitro cholesterol esterification secondary to a disruption of intracellular membranous struc-

tures may just as readily reflect latency on the part of acyl- CoAcholesterol acyltransferase as it could the lack of avail- able cholesterol. Acyl-CoAcholesterol acyltransferase has been shown to reside normally on the cytoplasmic side of the rough endoplasmic reticulum (22). With regard to the latency of cholesterol esterification in Niemann-Pick C fibroblasts, the evidence strongly favors the existence of a sequestered and metabolically unavailable pool of exogenously derived cholesterol rather than a topologically misplaced acyl- CoAcholesterol acyltransferase enzyme: (a) the phenotypic abnormalities presented by the Niemann-Pick C mutation reflect not only deficient acyl-CoAcholesterol acyltransferase catalysis but also deficient down-regulation of two other cho- lesterol-regulated proteins, the LDL receptor and hydroxy- methylglutaryl-CoA reductase (ll), (b) the histochemical findings clearly show abnormal sterol accumulation, (c) nor- mal orientation of acyl-CoAcholesterol acyltransferase on the cytosolic side of the endoplasmic reticulum in mutant cells is supported by the finding that acyl-CoAcholesterol acyltrans- ferase of mutant cells was as susceptible, in gently disrupted cell homogenates, to proteolytic inactivation by added pro- teases as the enzyme of normal cell extracts (data not shown);

3416 Type C Niemann-Pick Disease

( d ) in viuo and in vitro cholesterol esterification was normal or even somewhat elevated in mutant cells not cultured with LDL (Table 11).

DISCUSSION

Type C Niemann-Pick disease appears to represent a newly defined and unique cholesterol storage disorder. The patho- genic abnormalities include major disruptions of intracellular cholesterol processing. In mutant fibroblasts, extracellular LDL is carried by receptor-mediated endocytosis to lysosomes where apparently normal proteolytic and lipolytic processing of the exogenous lipoprotein initially takes place (11). How- ever, the subsequent intracellular fate of the lysosomal cho- lesterol and the normal cellular responses to cholesterol up- take are compromised by the mutation. A translocation of exogenously derived cholesterol from lysosomes appears de- ficient. The internalized and sequestered cholesterol of Nie- mann-Pick C fibroblasts fails to initiate the prompt homeo-

TABLE I Cellular distribution of unesterified cholesterol in normal and

Niemann-Pick C fibroblasts Stock normal and mutant Niemann-Pick C fibroblasts were har-

vested and seeded at a density of 9 X 10’ cells in 850-cm2 roller culture bottles with 100 ml of Eagle’s minimal essential medium, 2 mM glutamine, and 10% fetal bovine serum for 2 days. The culture medium was replaced with fresh McCoy’s medium with 10% lipopro- tein-deficient human serum and 2 mM glutamine for 4 days. This medium was replaced with fresh medium + 50 pg/ml of LDL protein/ ml and monolayers incubated an additional 24 h. Roller bottles were rinsed three times with 20 ml of PBS and subsequently harvested with 10 ml of 0.05% trypsin in PBS for 5 min at 37 “C. Cell suspen- sions were subsequently pelleted at 700 X g for 5 min and cells washed three times with 10 ml of PBS. Cell pellets (4-5 mg of protein) were suspended in 1.0 ml of PBS and kept on ice for subsequent analytical procedures described under “Experimental Procedures.” Each data point is the average of two separate cell cultures.

Cell culture LDL Unesterified cholesterol levels

Total in cell Plasma membrane % total

50 pgfrnlf.24 h nmolfmg cell protein

Normal (2) - 55 9 16 Mutant (2) - 73 13 18 Normal (2) + 123 57 46 Mutant (2) + 167 28 17 a No additional LDL.

static responses that serve to control and to balance intracel- lular cholesterol levels in normal cells. There is associated with the Niemann-Pick C mutation a tardy down-regulation of the LDL receptor, a delayed suppression of 3-hydroxy-3- methylglutaryl-coenzyme A reductase, and a defective stimu- lation of acyl-CoAcholesterol acyltransferase expression (11). As would be predicted, these delayed metabolic responses lead to excessive intracellular accumulation of unesterified choles- terol which is primarily stored in lysosomes (Fig. 2).

This defective lysosomal translocation of cholesterol is not only associated with delayed homeostatic responses but also with an impaired enrichment of cholesterol in the plasma membranes of mutant cells (Fig. 3 and Table I). Abnormal intracellular cholesterol sequestration can also be inferred from the finding that additional extensive organelle disrup- tion in cell-free extracts greatly enhances the in vitro availa- bility of cellular cholesterol for esterification in cell-free ex- tracts of mutant fibroblasts cultured with LDL (Table 11).

The molecular basis for the abnormal lysosomal sequestra- tion of LDL-derived cholesterol in Niemann-Pick C disease is not known. LDL-cholesterol released in lysosomes is thought to reach the endoplasmic reticulum and the Golgi apparatus (23). Saturation of a limited sterol pool within the endoplasmic reticulum presumably initiates the numerous cellular homeostatic responses that enable normal cells to regulate intracellular cholesterol levels. The components of this cholesterol transport process from lysosomes to the en- doplasmic reticulum are not known. It has been speculated that active vesicular or carrier-mediated transport may be involved (23).

It is likely that Niemann-Pick C disease will prove to be a useful pathological model for elucidating additional steps of intracellular cholesterol processing. Earlier documentation of induced or genetic pertubations of the LDL pathway at the lysosomal step has been limited to the observations that blocked hydrolysis of LDL cholesterol esters leads to lysoso- mal accumulation of unhydrolyzed esters and retarded ho- meostatic responses (24-27). The Niemann-Pick C mutation clearly affects a step subsequent to hydrolytic lysosomal proc- essing (11). Accumulation of unesterified cholesterol within the lysosomes of mutant cells begins to exceed the levels ‘found in normal cells as early as 2 h after initiation of LDL uptake, and by 24 h an extensive lysosomal cholesterol pool

TABLE I1 Accessibility of cellular cholesterol to in vitro and in vivo esterification in normal and Niemann-Pick C fibroblnsts

Stock cell cultures were harvested and seeded at 9 X 10‘ cells in 850-cm2 roller bottles and at 3 X lo6 cells in 25- cm2 flasks in 100 and 7 ml, respectively, of Eagle’s minimal essential medium + 10% fetal bovine serum for 2 days. The cultures were depleted of cellular cholesterol by culturing in McCoy’s medium + 10% LPDS for 4 days. Medium was replaced with fresh medium f 50 pg LDL protein/ml for 12 h. To the smaller 25-cm2 culture flasks, 0.012 ml of 6 mM [3H]oleate (200 dpm/pmol) in 14% acid-free bovine serum albumin was added for the last 2 h of the incubation. These particular cultures were washed, harvested, and subsequently analyzed for in vivo cholesterol [3H]oleate formation by lipid extraction and thin layer chromatography (8). The larger cell cultures were also incubated f LDL (50 pg/ml) for 12 h and subsequently washed and harvested as described in Table I. These cell suspensions were analyzed for unesterified cholesterol levels and in vitro cholesterol [3H]oleate formation as described under “Experimental Procedures.” The determinations represent the average of two separate cell cultures.

Cholesterol [3H]oleate synthesis

Cell culture LDL co-culture

Cellular levels of unesterified cholesterol In vivo Intact Disrupted

In vitro

organelles organelles

50 ~ f m l f 1 2 h nmolfmg protein pmol PHloleate incorporatedfmgf2 h

Normal (2) - 20 7 0 10 Mutant (2) - 30 6 5 30 Normal (2) + 40 2000 540 400 Mutant (2) + 90 500 80 530

No additional LDL.

Type C Niemann-Pick Disease 3417

has formed (Fig. 1). Previous studies have shown that the 4. Schneider, p. B., and Kennedy, E. p. (1967) J. Lipid Res. 8,202- total cellular accumulation of LDL cholesterol in these mu- 209

tant cells does not exceed that of normal cells during the first 5. Sloan, H. R., Uhlendorf, B. W., Kanfer, J. N., Brady, R. O., and

6 h of lipoprotein uptake (8). Consequently, excessive storage Fredrickson, D. S. (1969) Biochem. Biophys. Res. Commun. 34 ,

of cholesterol within lysosomes of the affected cells at this 6. Vanier, M. T., Rousson, R., Zeitouni, R., Pentchev, P. G., and early phase of lipoprotein uptake would suggest a delay in the Louisot, P. (1986) in Enzymes in Lipid Metabolism, Part II translocation of cholesterol from lysosomes to further intra- (Freysz, L., Dreyfus, H., Massarelli, R., and Galt, S., eds) pp. cellular sites of distribution rather than over active endocytic 791-802, Plenum Press, New York

7. Pentchev, P. G., Comly, M. E., Kruth, H. S., Vanier, M. T., uptake. The relationship between lysosomal storage and deficient

Wenger, D. A., Patel, S., and Brady, R. 0. (1985) Proc. Natl. Acad. Sei. U. S. A. 8 2 , 8247-8251

intracellular mobilization of cholesterol in Niemann-Pick c 8. Kruth, H. S., Comly, M. E., Butler, J. D., Vanier, M. T., Fink, J. fibroblasts suggests several possible disruptive mechanisms. K., Wenger, D. A., Patel, S., and Pentchev, P. G. (1986) J. Biol. Excessive lysosomal cholesterol accumulation may represent Chem. 2 6 1 , 16769-16774 a primary lesion at the ~ysosome itself. The diffusion of 9. Pentchev, P. G., Kruth, H. S., Comb, M. E., Butler, J. D., Vanier,

hydrolyzed metabolites from lysosomes is often under the M. T., Wenger, D. A., and Patel, S. (1986) J. Biol. Chem. 261 ,

control of Carrier-Imdiated Processes (28). Lysosomal ~ c u - io. Butler, J. D., Comly, M. E., Kruth, H. S., Vanier, M. T., Filling- mulation of cystine in cystinosis (29) and sialic acid in Salla Katz, M., Fink, J., Barton, N., Weintroub, H., Quirk, J. M., disease (30) are examples of blocked translocations of meta- Tokoro, T., Marshall, D. C., Brady, R. O., and Pentchev, P. G. bolic products from loaded lysosomes. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 556-560

On the other hand, it is also possible that lysosomal choles- 11. Pentchev, P. G., Comly, M. E., Kruth, H. S., Tokoro, T., Butler,

terol accumulation simply reflects the capacity and availabil- J., Sokol, J., Filling-Katz, M., Quirk, J. M., Marshall, D. C.,

ity of lysosomes to store cholesterol when they are called upon Patel, S., Vanier, M. T., and Brady, R. 0. (1987) FASEB J. 1,

to do so because of some more distal primary block. Potential 12. Brown, M. S., and Goldstein, J. L. (1986) Science 232,34-47 primary post-lysosomal abnormalities could include deficient 13. Chen, J. w., Pan, w., D’Souza, M. p., and August, J. T. (1985) sterol carrier proteins or lesions in membrane interactions which normally serve to transport cholesterol to specific tar- 14. Lange, Y., and Ramos, B. V. (1983) J. Bid. Chem. 258, 15130-

get sites. A Partial and temporaw manifestation of excessive 15. Lange, Y., and Matthies, H. J. G. (1984) J. Biol. Chem. 259 , cholesterol storage and deficient sterol transport in hetero- 14624-14630 zygous mutant Niemann-Pick C fibroblasts during only the 16. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. early active phase of LDL uptake (8) tends to favor the (1951) J. Biol. Chem. 193,265-275

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