Type C Niemann-Pick disease: cellular uncoupling of cholesterol homeostasis is linked to the severity of disruption in the intracellular transport of exogenously derived cholesterol

319

Biochimica et Biophysica Acta, 1096 (1991) 319-327 © 1991 Elsevier Science Publishers B.V. 0925-4439/91/$03.50 ADONIS 092544399100084P

BBADIS 61041

Type C Niemann-Pick disease: cellular uncoupling of cholesterol homeostasis is linked to the severity of disruption in the intracellular transport of exogenously derived cholesterol Charles E. Argoff 1, Marcella E. Comly 1, Joan Blanchette-Mackie 2, Howard S. Kruth 3, Harold T. Pye 1, Ehud Goldin 1, Chris Kaneski 1, Marie T. Vanier 4, Roscoe O. Brady 1, and Peter G. Pentchev 1 i Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, 2 Endocrinology Section, Laboratory of Cellular and Developmental Biology, National Institute of Diabetes Digestive and Kidney Disease, 3 Section of Experimental A therosclerosis, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD (U.S.A.) and 4 Department of Biochemistry and 1NSERM U 189, Facultd de Mddecine Lyon-Sua~ Oullins (France) (Received 22 November 1990)

Key words: Niemann-Pick disease type C; Intracellular cholesterol transport; Skin fibroblast culture; Lipid storage disorder; (Human)

A uniquely attenuated disruption of cholesterol homeostasis has been characterized in certain Niemann-Pick, type C (NP-C) fibroblasts. Uptake of LDL-cholesterol by cultured fibroblasts derived from two clinically affected brothers with this variant biochemical phenotype led to less intracellular accumulation of unesterified cholesterol than found in other typical cell lines. This limited cholesterol lipidosis in the variant NP-C cells reflected cholesterol processing errors that differed from the cellular lesions in classical NP-C cells in the following ways: (1) a more limited intracellular distribution of the excessive unesterified cholesterol; (2) shorter and more transient delays in the induction of cholesterol-mediated homeostatic responses; and (3) more efficient intracellular transport of exogenously derived cholesterol to the plasma membrane and the endoplasmic reticulum. Activation of acyl-CoA cholesterol acyltransferase (ACAT) was greater than 100-fold in both control and NP-C fibroblasts when cell cultures were preconditioned with 25-hydroxycholesterol, but the subsequent esterification of exogenous non-lipoprotein [3H]cholesterol remained deficient in all NP-C cells. In the variant NP-C cells conditioned with the oxysterol, this esterification of exogenous [3H]cholesterol was less affected than in classical NP-C cultures. The NP-C mutation affects a broad spectrum of metabolic responses related to the processing of exogenously derived cholesterol. Among this pleiotropic array of deficient responses, retarded intracellular cholesterol transport appears most closely linked to the primary mutation. This conclusion is supported by two current observations: (1) the degree to which sterol transport is affected in mutant cells in turn reflects the extent to which cholesterol-homeostatic responses are compromised; and (2) sterol transport remains deficient despite concurrent normal activation of other cellular responses, such as cholesterol esterification. Introduction Niemann-Pick disease, Type C (NP-C) is an autosomal recessive neurovisceral lipid storage disorder with

Abbreviations: NPC, Niemann-Pick, type C; LDL, low-density lipoprotein; LPDS, lipoprotein-deficient serum; ACAT, acyl CoA: cholesterol acyltransferase; PBS, phosphate-buffered saline; BSA, bovine serum albumin. Correspondence: P.G. Pentchev, Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, Bdg 10, Room 3D-12, National Institutes of Health, Bethesda, MD 20892, U.S.A.

progressive deterioration of the central nervous system usually presenting in childhood or early adolescence [1]. Because of clinical, histochemical and biochemical similarities to other metabolic disorders that show excessive and abnormal sphingomyelin accumulation in tissues, NP-C disease was originally delegated to a family of sphingomyelin lipidoses that includes type A and B Niemann-Pick disease [2]. While subsequent biochemical characterization of Niemann-Pick A and B disease led to the discovery of a primary sphingomyelinase deficiency in these disorders [3], no convincing and consistent studies support the classification of NP-C disease as a primary disorder of sphingomyelin metabolism [4].

320 Studies over the past six years have suggested the proper classification of that disorder as a cholesterol lipidosis. These investigations were initiated by the discovery of a mutant mouse in which intracellular cholesterol storage was associated with a deficiency in the esterification of exogenously derived cholesterol [5,6] and the recognition of similarities between this mutant and human NP-C disease. These studies soon led to the key finding that NP-C fibroblasts were uniquely and severely deficient in their ability to esterify LDL-derived cholesterol, with a partial expression in obligate NP-C carriers [7-10]. Further characterization of cholesterol metabolism in mutant NP-C cell lines demonstrated that delayed induction of essentially all metabolic controls governing cellular cholesterol homeostasis was associated with a retarded intracellular translocation of exogenously derived cholesterol [11,12], in turn associated with an abnormal storage of unesterilied cholesterol in lysosomes and in the Golgi system as well as a delay in subsequent translocation to the plasma membrane [13,14]. This anomalous processing of cholesterol in NP-C fibroblasts was in part confirmed by the studies of Liscum and co-workers [15,16]. The primary molecular lesion of NP-C disease is not known. Phenotypic variation in the clinical presentation of NP-C disease is broad, with a spectrum ranging from accelerated and early deterioration to delayed and protracted decline [9,17]. Similarly, a phenotypic variation in the alterations of intracellular cholesterol processing was suggested in early reports [9,18], and has now been characterized in an accompanying paper (Vanier et al. (1991) Biochim. Biophys. Acta 1096, 328-337). Our current studies show that the variable presentation of abnormal cholesterol-processing phenotypes takes place in a concordant fashion that suggests a tight causal association among the deficient cellular responses with deficient sterol transport most proximately linked to the primary NP-C mutation. Implications of these findings are discussed with regard to the cellular pathogenesis and the biochemical lesion of NP-C disease. Material and Methods

Cells Normal and mutant Niemann-Pick C fibroblasts were derived from superficial biopsies of normal volunteers and confirmed patients of the Developmental and Metabolic Neurology Branch of the National Institutes of Health under guidelines approved by a clinical research committee. Cells were also obtained from the National Institute of General Medical Science, Human Genetic Mutant Cell Repository (Camden, N J). The two 'variant' NP-C cell lines were obtained in two male siblings aged 26 and 30, in whom a combination of deficits referable to cerebellar, cortical and extrapyramidal dysfunctions accompanied by the presence

of supranuclear vertical gaze paresis, foam cells in the bone marrow and mild hepatosplenomegaly allowed for an unambiguous clinical diagnosis of NP-C disease. The clinical course developed by these two brothers was not dramatically different from that seen in other chronic NP-C patients.

Lipoproteins Freshly prepared native human low-density lipoprotein (LDL) and 125I-LDL (400 d p m / n g protein) were purchased from Bionetics Research Institute (Rockville, MD). Reconstituted human [3H]cholesteryl-linoleateLDL (300 d p m / p m o l ) was prepared by incubation of native LDL (1 mg) and 1 mCi of [3H]cholesteryl-linoleate together in 10% dimethylsulfoxide as previously described [19]. Radioactive compounds and other lipids [9,10, 3H]oleic acid (2-20 Ci/mmol), [3H]acetate (75-100 Ci/mmol), [1,2,3H]cholesterol (40-60 C i / m mol) and [1,2,6,7, 3H]cholesteryl-linoleate (60-100 C i / mmol) were purchased from New England Nuclear, Wilmington, DE. 25-Hydroxycholesterol and cholestenone (cholest-4-en-3-one) were purchased from Research Plus (Bayonne, N J). 25-Hydroxycholesteryl-linoleate was synthesized as previously described [20]. Tissue culture and cytochemical supplies Fetal bovine serum was obtained from Hyclone Lab. (Logan, Utah). Lipoprotein-deficient bovine serum was prepared by Biomedical Technologies (Boston, MA). Other tissue culture supplies were obtained from Biofluids (Rockville, MD). Filipin was purchased from Polysciences (Warrington, PA). Microscopic slide chambers (Lab-Tek plastic microscope culture wells) were obtained from Nunc via Thomas Scientific. Maintenance of cell cultures Fibroblast cultures between the third and fifteenth passage were cultured in Eagle's minimal essential medium supplemented with 10% fetal bovine serum, 1% non-essential amino acids, 2 mM glutamine, 100 units penicillin and 100 /tg streptomycin/ml in humidified 95% air and 5% CO 2 at 37°C. Cells were harvested by trypsinization (0.05%, Sigma) and reseeded according to the appropriate specific protocols. Study of intracellular cholesterol homeostatic responses Normal and mutant fibroblasts were seeded at a density of 40 000 cells/35 mm well (20-40 /~g protein) and were cultured for 5 days in 5% LPDS and McCoy's medium. Individual cell responses were subsequently monitored by incubating monolayers with and without LDL for the indicated periods of time. De novo [3H]cholesterol synthesis was initiated by the addition of 10/~Ci of [3H]acetate between 4 - 6 h of incubation of

321 cell cultures with and without LDL. LDL-receptor binding was carried out by incubating cultures for 24 h with and without native L D L and subsequently incubating washed cells with 125I-LDL (5 /~g/ml) for 3 h at 3 7 ° C as previously described [13]. Cholesteryl ester synthesis was carried out be incubating cultures with 10 /~M [3H]oleate (200 d p m / p m o l ) with and without LDL for the specified periods.

Cytochemical analysis of cellular cholesterol accumulation Stock cell cultures were placed in 5% lipoprotein-deficient serum (LPDS) and McCoy's medium for 4 days and subsequently harvested and seeded at a density of 2 . 0 . 1 0 4 cells per 9.5 cm 2 in microscopic chamber wells. After 24 h of attachment, monolayers were incubated an additional 4 days in 4 ml of McCoy's medium and 0.2% bovine serum albumin. LDL (50 # g / m l ) was added to some cultures for 24 h and monolayers were subsequently washed 3 x with PBS, fixed and stained with filipin (0.05 m g / m l ) as described previously [14]. Stained cells were washed, mounted with p-phenylenediamineglycerol and viewed with a Leitz fluorescent microscope using excitation filters of BP 350-410 for filipin. Cellular lipid analysis Fibroblast monolayers (20-80/~g protein) in 35 mm wells were washed 3 x with PBS and subsequently extracted at room temperature for 1 / 2 h with 0.5 ml of isopropanol in closed wells by slow rocking. Radioactive lipids extracted into isopropanol were analyzed by thin-layer chromatography on 0.25 mm thick, precoated silica gel 60 plates (EM Science, Gibbstown, N J) in a solvent of n-hexane/ethyl ether/glacial acetic acid ( 9 0 : 1 0 : 1 , by vol.). Lipids were visualized in iodine vapor and were subsequently scraped and quantitated by liquid scintillation counting. The lipid-extracted cell monolayers were air dried and subsequently dissolved in 0.5 ml of 0.5 M N a O H for 30 min at room temperature with slow rocking in closed wells. Protein was quantified by the method of Lowry [21]. Unesterified and esterified cholesterol were quantified fluorometrically in aliquots of the isopropanol lipid extract corresponding to 10-20 #g of protein [22]. Measurements of the intracellular translocation of exogenously derived cholesterol to plasma membrane Stock cells were initially depleted of cholesterol by culturing with 5% LPDS and McCoy's medium for 4 days and were subsequently seeded in 35 mm wells at a density of 1-105 cells. These cultures were then incubated with 2 ml of 5% LPDS and McCoy's medium for 3 days. Fresh McCoy's medium containing recon3 stituted [ H]cholesteryl-linoleate LDL (10 /~g/ml) was added to the cultures for 16 h. Monolayers were washed free of extracellular LDL and were incubated with

McCoy's medium at 37°C for 15 min. Cultures were placed on ice and washed 2 x with cold Hank's buffered saline, 2 x with cold PBS and 2 m g / m l BSA and finally 2 x with cold PBS. Monolayers were then treated with freshly prepared 1% glutaraldehyde in PBS on ice for 10 min. Following fixation, monolayers were washed 3 x with iced 310 mM sucrose in 1.0 mM phosphate (pH 7.4) and incubated in this buffer containing 2.0 m g / m l cholesterol oxidase (6000 units/g, Brevibacterium sp., Beckman Instruments, Carlsbad, CA) for 30 min at 10°C. Cells were subsequently washed with cold PBS and extracted with isopropanol. Levels of [3H]cholesterol, oxidized [3H]cholesterol (cholest-4-en-3-one) and [3H]cholesteryl ester were analyzed by thin-layer chromatography on silica gel plates in a solvent of chlorof o r m / m e t h a n o l (100: 0.75, v / v ) where the lipids migrated with R v values of 0.65, 0.40 and 0.91, respectively. This procedure represents minor adaptations of the previously reported observations of Lange [23,24] which described in detail how cholesterol oxidase can be employed to efficiently and selectively oxidize plasma membrane cholesterol. The studies of Freeman have shown that this method of cell-surface cholesterol analysis can be applied as effectively to monolayers attached to plasticware [25].

Transport to rough endoplasmic reticulum Cell cultures at a density of 3 • 1 0 4 cells/35 mm wells were prepared as described above. Cells were initially incubated for 16 h with or without 2 / ~ g / m l of 25-hydroxycholesteol in 5% LPDS and McCoy's prior to the addition of 10 mM [3H]oleate (200 d p m / p m o l ) for 2 h intervals at 16, 22 h and 38 h to measure activation of ACAT towards the esterification of endogenous cholesterol [26]. To other cultures also conditioned with 25-hydroxycholesterol, 5 /~g/ml of [3H]cholesterol (30 d p m / p m o l ) were added for 2, 8 and 24 h to measure the ability of exogenous cholesterol to reach the rough endoplasmic reticulum as determined through its esterification at this subcellular compartment by an activated resident ACAT enzyme [27]. Monolayers were washed with PBS and extracted with isopropanol as described earlier. Radioactive [3H]cholesteryl ester and cholesteryl-[3H]oleate ( R v = 0.84), 25-hydroxycholesteryl-[3H]oleate (R v = 0.09) and [3H)cholesterol (R v = 0.07) were separated by thin-layer chromatography in a solvent of n-hexane/ethyl ether/glacial acetic acid ( 9 0 : 1 0 : 1 , by vol.). Results

Kinetics of the induction of cellular cholesterol esterification The cumulative and intermittent rates of cholesterol esterification induced in normal and in mutant NP-C cells by LDL uptake were measured over a period

322 extending to 24 h (Table I). Both typical and variant NP-C cells were deficient in the induction of cholesterol esterification during the early phase (first 6 h) of LDLcholesterol uptake. However, a significant level of cholesteryl ester (35% of normal after 4 h) was formed in variant NP-C cultures compared to the strikingly deficient (less than 5% of normal) classical NP-C cell lines. After 24 h of incubation with LDL, esterification in the variant NP-C cultures had normalized, while only a modest increase was found in typical NP-C cells. In the variant NP-C cells, the impairment in cholesteryl ester formation was best evidenced by the study of intermittent rates between 4 and 6 h of L D L uptake (13% of normal values).

Cytochemical and quantitative evaluation of LDLcholesterol accumulation in cultured fibroblasts Filipin is a fluorescent cytochemical probe specific for unesterified cholesterol [29] and it has previously been shown that the accumulation of free cholesterol in NP-C fibroblasts is associated with an intense filipincholesterol staining of perinuclear vesicles that were identified as lysosomes [13,14]. The characteristic pattern observed in typical NP-C cells can be seen in Fig. lB. In normal cells incubated with LDL, filipincholesterol fluorescence is dramatically less intense (Fig. 1A). In the two variant NP-C cell lines incubated with LDL (Fig. 1C and D), an intermediate level of filipincholesterol staining was observed. In all cholesterol-depleted cells, there is only a weak fluorescence (not shown, [13]). The excess filipin-cholesterol fluorescence in both types of NP-C cells was shown cytochemically to occur in lysosomes (not shown, 13, 14). To better characterize the biochemical abnormalities in the variant NP-C cells in relation to classical NP-C,

quantitative studies were performed. Total cholesterol accumulation during the initial 24 h of L D L uptake did not appear to differ significantly in normal and in NP-C (typical or variant) fibroblasts. However, when compared to normal cells, typical NP-C cultures did accumulate a disproportionally high level of unesterified cholesterol at the expense of esterified cholesterol, as previously d o c u m e n t e d [10,11]. This a b n o r m a l processing of exogenous cholesterol was not observed in the two variant NP-C cultures whose quantitative and qualitative storage of LDL-derived cholesterol was essentially indistinguishable from that of normal cells (Table II). After 48 h of L D L uptake (Table III), unesterified cholesterol concentrations dropped back to nearly basal levels both in normal and in NP-C variant cells, at variance with the situation in classical NP-C cells. A cytochemical documentation of a deficiency in the normal intracellular distribution of LDL-derived unesterified cholesterol was thus obtained in the variant NP-C fibroblasts in spite of a cellular unesterified cholesterol mass that appeared to be in the normal range during the whole period of L D L uptake studied.

Induction of cholesterol-mediated homeostatic responses in normal, classic and variant NP-C fibroblasts Experimental conditions for challenge with LDL were chosen which previously have shown to highlight the deficient induction of cholesterol homeostatic responses in NP-C cells [12]. Normal human fibroblasts responded to LDLcholesterol uptake as outlined by the well-defined LDL-receptor pathway of Goldstein and Brown [28], while LDL-cholesterol uptake by typical NP-C fibroblasts was associated with a characteristic delayed induc-

TABLE I

Cumulative and intermittent rates of cholesteryl ester synthesis during the early and late stages of LDL-uptake Cells were prepared as described in Materials and Methods, and incubated with and without L D L ( 5 0 / ~ g / m l ) for the indicated periods of time. [3H]Oieate (10 #M, 300 d p m / p m o l was present for the entire incubation period for the study of cumulative rates, and during the terminal 2 h of incubation for the study of intermittent rates. The results (mean + S.E.; range in brackets) are expressed as LDL-stimulated cholesteryl [3H]oleate formation. LDL addition

Oleate addition

Cholesteryl [3H]oleate formation ( p m o l / m g protein) controls

typical N P C

variant N P C

(n = 3)

(n = 3)

(n = 2)

470 + 170 (300-630) 56000+6000 (51000-63 000)

18 + 3 (0-53) 4 1 0 0 + 1300 (2 800-5 400)

160 + 90 (100-220) 7 2 0 0 0 + 19000) (59 000-86 000)

3100 + 1 100 (2 0 0 0 - 4 200) 6000 + 200 (5 800-6 200)

45 + 17 (27-60) 1700 + 100 (1600-1800)

430 + 100 (330- 520) 6 300 + 600 (5 900-6 700)

Cumulative rates 0-4 h

0-4 h

0-24 h

0-24 h

0-6 h

4-6 h

0-24 h

22-24 h

Intermittent rates

323 TABLE II

Cellular cholesterol accumulation in fibroblasts cultured with L D L Cell cultures were prepared as described in Materials and Methods. Monolayers were incubated with LPDS (basal level) and with LDL (50 /*g/ml) for 6 h or 24 h. Washed cultures were extracted with isopropanol and lipid and protein measurements were carried out as described in Materials and Methods. Values are expressed in n m o l / m g cell protein.

Basal levels free ester

Normal

Typical NPC

Variant NPC

(n = 3)

(n = 3)

(n = 2)

49 + 6 0

56 + 8 0

48 + 9 0

70 + 14 0

84 + 16 0

193_+30 10 + 8

134_+6 44_+ 17

LDL addition, 6 h free 80 + 8 ester 0 LDL addition, 24 h free 115+26 ester 42 _+15

tion of these responses [12,15] resulting in an excessive cellular accumulation of unesterified cholesterol (Table III). However, under the same experimental conditions,

the two variant NP-C cell lines responded to LDLcholesterol uptake in a manner that was essentially indistinguishable from the responses observed in normal cultures (Table III).

Relative efficiencies in the intracellular transport of endocytosed cholesterol to the plasma membrane and rough endoplasmic reticulum Following endocytic uptake and lysosomal processing of [3H]cholesteryl-linoleate LDL, subsequent intracellular translocation of [3H]cholesterol from lysosomes to the plasma membrane could be evaluated by the levels of cellular [3H]cholesterol susceptible to oxidation in cultures exposed to cholesterol oxidase. The data was calculated as the percent of total cellular [3H]cholesterol oxidized in order to equalize for the 30% greater uptake of reconstituted LDL by the mutant cells. The translocation of endocytosed, LDL-derived, [3H]cholesterol from lysosomes to the plasma membrane was 36% lower in typical NP-C cultures than in normal cells (Fig. 2). In contrast to this partial retardation in classic NP-C fibroblasts, no comparable deficiency through this

A

Fig. 1. Localization of unesterified cholesterol in cultured fibroblasts. Cells were treated as described in Materials and Methods. All micrographs were taken and printed at the same exposure at a magnification of × 175. All cell cultures were grown with LDL ( 5 0 / t g / m l ) for 24 h. Micrograph A represents normal cells and micrograph B cells from a typical NP-C patient. C and D are NPC variant cells from the older (30-year-old) and the younger (26-year-old) brothers, respectively.

324 T A B L E 11I

Homeostatic responses induced in cultured fibroblasts by uptake of LD L-cholesterol Cell cultures were prepared as described in Materials and Methods. De-novo [3H]cholesterol synthesis in the absence of added LDL, studied by i n c o r p o r a t i o n of [3H]acetate into unesterified [3H]cholesterol, was 21, 25 and 20-104 d p m / m g cell protein for normal, typical and variant N P C cultures, respectively. LDL-receptor specific binding in the absence of LDL down-regulation was 10, 8 and 8 ng of 1251-LDL/mg cell protein in normal, typical NPC and variant NPC cell lines, respectively. Cholesteryl ester synthesis in the absence of L D L was less than 0.1 n m o l / m g cell protein for all cell cultures. LDL-cholesterol accumulation was taken to represent the increase in cellular cholesterol mass after treatment with LDL. Levels of unesterilied cholesterol in the absence of LDL were in the range of 40-50 n m o l / m g protein for all cell genotypes. Measurements of all cell responses were carried out in triplicate for 3 normal and 5 NP-C cell lines as indicated. LDL uptake period

Cellular response measured

Cell culture normal typical (n=3) NPC (n = 3)

6h

De-novo cholesterol synthesis

(% of non LDL-treated cells)

Plasma membrane LDL-receptor levels

(% of non LDL-treated cells)

48 h

Induction of cholesteryl ester synthesis

(nmol cholesteryl e s t e r / m g p r o t e i n / 48 h) 58_+ 7 17+ 1 40_+14

48 h

Accumulation of unesterified cholesterol

(nmol cholesterol/mg protein/48 h)

24 h

13+ 3

58 + 15

1614

_~£ ~t

t2-

[~- 115+12%

~ -100±15% 64±10% (P£001)

to-

8~ ~

8-

~_~ ~2-

4

6 2

Normal (4)

40+ 2

182 + 20

18

~ cO

NP-~ (4)

NP-C Variants (2)

Fig. 2. Oxidation of plasma m e m b r a n e [3H]cholesterol generated in cultured fibroblasts following endocytic uptake of [3H]cholesteryl-ester LDL. Normal and mutant cell cultures were incubated with r e c o n s t i t u t e d [ 3 H ] c h o l e s t e r y l l i n o l e a t e - L D L (15 / ~ g / m l , 300 d p m / p m o l ) for 16 h as outlined in Materials and Methods. Monolayers were subsequently washed free of excess LDL, fixed, treated with cholesterol oxidase and analyzed for free, oxidized and esterified [3H]cholesterol as described. The average recovery of free, oxidized and esterified [3H]cholesterol in the normal fibroblast cultures after such treatment was 6.35_+1.19,1.04_+0.18 and 5.604-0.90 n m o l / m g protein, respectively. In typical NP-C cultures the recovery of free, oxidized and esterified [3H]cholesterol was 8.75 _+2.79, 0.87 + 0.19 and 6.0+2.0 n m o l / m g , respectively. The average values for the same compounds in NP-C variant fibroblasts were 7.35+1.09,1.46+0.13 and 7.30+1.0 n m o l / m g protein, respectively. The relative translocation of [3H]cholesterol to the plasma m e m b r a n e was determined from the fractional oxidation of total cellular free [3H]cholesterol generated in the cells and was calculated according to the formula: [3H]cholestenone/[3H]cholestenone + [3H]cholesterol x 100. These determinations were carried out in triplicate with the indicated number of cell lines in three separate experiments.

21+ 3

62_+ 2

20-

~ P_

variant NPC (n = 2)

83+ 5

34___ 4

o

gg

70 _+ 15

ACYL-COA: CHOLESTEROL ACYLTRANSFERASE

Esterification of Exogenously Transported Cholesterol {[~H] CholesteryI-Ester Formationl

Activation of Endogenous Cholesterol Esterification (Cholesteryl-[3H] Oleate Formation) Additions to Culture Medium 25-OH Cholesterol: [~H] Cholesterol: [{HI Oleate:

[

~

I

(hours)

( )

(+) 0-18

(+) 0-24

(+) 0-40

II (+) 0-18

(+) 0 24

(+) 0-40

( )

(-}

(-)

I (+) 16-18

(+) 16 24

(+) 16-40

(+) 16 18

(+) 16-18

(+) 22-24

{+) 38-40

(-)

(-)

I

I

(_)

Ib

.,v ..... '" c'2'rh I

400[

I

(-)

1600 [ [] Normal(4;

•

]

(hours)

HI

8000

o

~0o,~

',

]7000 ~

16000 ,T,ooo, 1 s°°° I ~oo44°°° ~

Iij ~

~ i ~

ii I I

°t3ooo

M 12000 1 looo v

Fig. 3. Transport of exogenously derived [3H]cholesterol to the rough endoplasmic reticulum for esterification by acyl-CoA: cholesteryl acyhransferase activated with 25-hydroxycholesterol. Normal and mutant cell cultures were prepared and incubated as described in Materials and Methods. Cells were treated with 25-hydroxycholesterol to activate A C A T located in the RER. Exogenous non-lipoprotein [3H]cholesterol was subsequently added to the conditioned cultures to measure the intracellular transport of the exogenous cholesterol to the RER as defined by the level of interaction with A C A T to form [3H]cholesteryl-esters.

325 intracellular route was detected in the variant NP-C lines (Fig. 2). In a separate experimental approach to the study of the intracellular routing of exogenously derived cholesterol, advantage was taken from the fact that ACAT is specifically localized in the RER [27]. If ACAT were activated prior to the uptake of exogenous cholesterol, the rate of esterification of the internalized cholesterol would reflect the cellular efficiency of its transport to the RER. Such activation of ACAT can be achieved by treatment of cell cultures with oxysterols such as 25-hydroxycholesterol [26]. In the present studies, cellular activation of ACAT was measured by the levels of endogenous cholesterol esterified with added [3H]oleate. Addition of 25-hydroxycholesterol resulted in comparable activation of ACAT in normal and in typical or variant NP-C cells, with levels of cholesteryl [3H]oleate 100-fold greater than in non oxysterol-treated cultures (Fig. 3). Since 25-hydroxycholesterol down-regulates the LDL-receptor [30], cells preconditioned with this compound could not be subsequently challenged with [3H]cholesteryl-linoleate LDL. To obviate this problem, the cultures were alternatively incubated with non-lipoprotein [3H]cholesterol, whose cellular uptake has been shown not to require functional LDL receptors [31]. The level of non-lipoprotein [3H]cholesterol that was found associated with the cell monolayers after 24 h of incubation was in the range of 400 n m o l / m g of cell protein, and to a certain extent represented nonspecifically adhering cholesterol, since filipin studies revealed high fluorescent staining of the surface of these cells (data not shown). In these ACAT-activated cells, esterification of [3H]cholesterol rose gradually during the initial 8 h of incubation and increased rapidly during the remaining 16 h of treatment. Esterification of [3H]cholesterol in these cells most likely reflects the efficiency with which exogenous [3H]cholesterol can be transported to a depleted ACAT substrate pool of sterol at the RER. The relative efficiency in the intracellular transfer of the non-lipoprotein [3H]cholesterol to the RER was evaluated by the total levels of [3H]cholesteryl esters formed in the various cell cultures during 24 h of incubation with [3H]cholesterol. The actual amounts of [3H]cholesterol esterified by the various cultures indicated that the relative efficiency in the intracellular transfer of the exogenous sterol to the RER was in the following order: normal (100%), variant NP-C (63%) and typical NP-C (40%) (Fig. 3). Discussion

The Niemann-Pick C mutation disturbs intracellular cholesterol processing by a generalized uncoupling of cholesterol-mediated homeostatic responses as well as by a delayed intracellular mobilization of exogenous cholesterol [12,15]. These cellular lesions clearly do not

reflect any apparent dysfunction in the initial endocytic uptake or hydrolytic processing of LDL [12,15]. It has generally been assumed that these errors in cellular cholesterol metabolism are closely linked. Disrupted cholesterol homeostasis itself appears to represent a delayed induction of cellular responses rather than absolute deficiencies of the individual regulatory steps [12,15]. Considerable attention has now been shifted to the defect in intracellular cholesterol mobilization as a potential fundamental factor in the cellular and molecular pathogenesis of NP-C disease [13,16]. An early indication of the possible pivotal involvement of a structurally related defect in NP-C disease was the finding that the intracellular processing of LDL-cholesterol in obligate heterozygous NP-C cells was intermediate between the relative capacities of normal and homozygous affected cells [9,10]. Subsequent and current studies have clearly documented a sterol-transport lesion that affects the intracellular distribution of exogenously derived cholesterol. These investigations have shown that deficient intracellular mobilization in NP-C fibroblasts is characterized by premature and excessive accumulation of cholesterol in the Golgi complex [14] and in lysosomes [13-15] and by an accompanying delay in translocation of exogenous cholesterol to other cellular compartments, such as the plasma membrane (Refs. 13 and 16 and Fig. 2) and endoplasmic reticulum (Fig. 3). The results of the current comparative studies in typical and variant NP-C cells, clearly different in their ability to respond to internalized cholesterol, have suggested that a direct cause and effect relationship exists between the deficiency in cholesterol transport and the delayed induction of homeostatic responses. Compared with typical NP-C cells, variant cells exhibited less cellular accumulation of unesterfied cholesterol upon exposure to LDL (Table II and Fig. 1) and, in turn, showed a shorter and more transient delay in the induction of homeostatic responses (Tables I and III). This more facile induction of cellular responses mirrored a level of intracellular cholesterol transport that was clearly less affected than the mobilization of cholesterol in typical NP-C cultures (Figs. 2 and 3). Thus, the pathogenic series of events highlighting the disruption of cellular processing of exogenous cholesterol in NP-C cells likely can be traced through the following sequence: primary lesion > disrupted intracellular trafficking of exogenous cholesterol > delayed induction of homeostatic responses > excessive intracellular accumulation of unesterified cholesterol. The accumulation of excess exogenous cholesterol primarily within lysosomes [13,14] has suggested that sterol-trapping within this organelle reflects a primary block in further intracellular mobilization of cholesterol. 'Lysosomal sequestration' mutations which exist in cystinosis [32] and Salla disease [33] have been shown to represent specific transport blocks in lysosomal release

326 of cystine and sialic acid, respectively. Unfortunately the post-lysosomal pathways of intracellular LDLcholesterol transport remain largely undefined. Although L D L uptake has been shown to enrich cholesterol in a number of post-lysosomal compartments (endoplasmic reticulum, Golgi, plasma membrane) [34,35,13,14] the pathways and mechanisms through which this cholesterol pool is channeled remains largely unknown. This paucity of data has restricted a more detailed delineation of the lesion in intracellular cholesterol trafficking in NP-C cells. Considering a lysosomal cholesterol accumulation in NP-C cells which has been shown to reach ten-times the normal level [12,15], it was surprising to learn of the extensive capacity of the LDL-derived [3H]cholesterol to escape from a more persistent lysosomal-trapping during its intracellular routing through this organelle (Fig. 2). The rate of intracellular translocation of LDLderived cholesterol from lysosomes to the plasma membrane was found to be reduced by only 40% in typical NP-C cells and by the present methods could, in fact, not be shown to be affected in the variant NP-C cultures (Fig. 2). Other studies have also reported a relatively high level of residual transport of exogenous cholesterol in classical NP-C cells [13,16]. These observations might suggest a leaky mutation that permits significant levels of cholesterol to escape the cellular transport block. On the other hand, the appearance of a consistently measurable partial lesion in cholesterol processing in heterozygous NP-C cells (Refs. 9 and 10 and accompanying paper, Vanier et al.) suggests a primary genetic mutation that is tightly linked to the abnormal cellular phenotypes of this disorder. The extensive permissiveness reflected in the intracellular transport of cholesterol through lysosomes may actually represent a specific block in only one of several alternative pathways. Characterization of the deficient mobilization of exogenous cholesterol in NP-C cells has in fact brought forth data which suggests that affected sterol transport may not be exclusively highlighted by a lysosomal transport lesion. For example, recent studies have revealed that LDL-cholesterol uptake in NP-C cells is also associated with premature sterol enrichment in the Golgi [14]. Other studies have shown that weak-base amphiphilic amines, such as U18666A [36], imipramine, propranolol, W7 or chlorpromazine [37] disrupt cellular cholesterol transport and processing in normal cells in a manner that mimics the NP-C mutation, without substantially affecting known pH-dependent functions, such as receptor-mediated uptake or lysosomal hydrolytic processing of LDL. Under specific experimental conditions these drugs may partition into other acidic nonlysosomal compartments where their presence could affect routing of exogenous cholesterol to the plasma membrane and RER. The present study has shown that

in NP-C cells, intracellular translocation of endocytosed non-lipoprotein cholesterol to the R E R was affected to a greater extent than the translocation of LDL derived cholesterol from lysosomes to plasma membrane (Fig. 2). This finding suggests that the burden of deficient sterol transport can be magnified in mutant cells when cholesterol passes through cellular compartments or pathways that may extend beyond lysosomes. Regardless of the specific intracellular location(s) of the transport block, the NP-C mutation appears to compromise most specifically the pathway (s) through which the early and rapid phase of LDL-cholesterol uptake is channeled, as illustrated by study of cholesterol esterification kinetics in variant NP-C cells (Tables I and III). Further understanding of the underlying molecular basis responsible for the variable expression of deficient cellular cholesterol processing in NP-C fibroblasts will depend to a large extent on the identification of the primary gene mutation. Unravelling the sequence of cellular events that can now be traced back to the cholesterol transport block would appear to represent a useful approach toward achieving this goal.

Acknowledgements We are grateful to the National Tay-Sachs and Allied Diseases Foundation of Chicago for their generous support for research on Type C Niemann-Pick disese.

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