Amino Acid Substitution in NPC1 That Abolishes Cholesterol Binding Reproduces Phenotype of Complete NPC1 Deﬁciency in Mice

Amino acid substitution in NPC1 that abolishes cholesterol binding reproduces phenotype of complete NPC1 deﬁciency in mice

Xuefen Xiea, Michael S. Browna,1, John M. Sheltonb, James A. Richardsonb, Joseph L. Goldsteina,1, and Guosheng Lianga,1

Departments of aMolecular Genetics and bPathology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9046

Contributed by Joseph L. Goldstein, August 4, 2011 (sent for review July 18, 2011)

Substitution mutations in adjacent amino acids of the N-terminal The truncated N-terminal domain of NPC1, referred to as domain of NPC1, a lysosomal membrane protein, abolish its NPC1(NTD), can be expressed and purified as a soluble protein cholesterol binding activity and impair its ability to export (4). Crystallographic studies show that this domain forms a stable cholesterol from lysosomes of cultured cells lacking npc1 [Kwon structure that is largely α-helical (2). Cholesterol binds to this HJ, et al. (2009) Cell 137:1213–1224]. Here, we show that the same domain in an orientation opposite to its orientation in NPC2 (i.e., the 3β-hydroxyl group is buried in the protein, where it two mutations (proline-202 and phenylalanine-203, both changed fi to alanine) reproduce the phenotype of complete NPC1 deficiency makes speci c contacts with certain amino acids, whereas the when knocked into the mouse npc1 gene by homologous recom- isooctyl side chain projects to the surface). When NPC1(NTD) is pf/pf incubated with cholesterol in solution containing ethanol and bination. Homozygous npc1 mice exhibited neurodegenera- dilute detergent, the cholesterol binds to the protein very slowly tion beginning at day 49 and died at a median age of 84 d, as at 4 °C (8). Slow binding is attributed to the presence of several previously reported for mice that lack npc1. Liver and other organs α npc1pf/pf -helices that partially occlude the sterol binding pocket (2). of the mice accumulated excess cholesterol in lysosomes. Binding is accelerated by orders of magnitude if the cholesterol In liver, mRNAs encoding several lysosomal proteins were ele- is first bound to NPC2 and then transferred to NPC1(NTD) (8). vated, including NPC1 and NPC2 and several digestive enzymes We speculate that NPC2 interacts with NPC1(NTD) so as to (acid lipase, β-glucuronidase, and cathepsins B and D). Weekly move aside the obstructing helices, allowing cholesterol to slide treatment with hydroxypropyl-β-cyclodextrin (HPCD) beginning from NPC2 to NPC1 without having to enter the water phase. at 7 wk reduced hepatic cholesterol accumulation and diminished The sliding model is supported by crystallography showing the the lysosomal mRNAs. We conclude that the cholesterol binding opposite orientation of cholesterol in the two binding sites (2, 3). site in the N-terminal domain of NPC1 is essential for cholesterol The cholesterol handoff model is supported by alanine-scanning export from lysosomes in living animals as it is in cultured cells. mutagenesis of NPC2 and NPC1(NTD) (2, 3). These mutations The HPCD-mediated reduction of excess lysosomal enzymes may revealed two classes of defects. Replacement of amino acids in contribute to the ability of this drug to delay the progression of either of the sterol binding pockets led to a loss of cholesterol NPC disease in mice. binding. The other class of mutations localized to patches on the surface of both proteins that are not required for cholesterol cholesterol binding domain | cyclodextrin | knockin mutation | lysosomal binding but are required for cholesterol transfer from NPC2 to storage disease | Niemann-Pick disease NPC1(NTD). In modeling studies, these patches could be aligned so as to bring the two cholesterol binding sites into close proximity, in such a way that the plane of the cholesterol would be held n Neimann-Pick C disease, a mutation in either of two lyso- constant as the sterol slid from NPC2 to NPC1(NTD) (3). Isomal proteins, NPC1 or NPC2, causes cholesterol and other The physiologic relevance of the identified amino acids re- lipids to accumulate in lysosomes throughout the body, resulting quired for binding and transfer was confirmed by introducing the in death from multiorgan failure (1). To explain this dual re- mutations into plasmids encoding NPC2 or full-length NPC1. quirement, we proposed a handoff model in which NPC2 and Proteins encoded by these mutant plasmids failed to rescue de- NPC1 act sequentially to remove cholesterol from lysosomes (2, fective sterol transport in cultured cells deficient in NPC2 or 3). Malfunction of either protein causes a primary accumulation NPC1, respectively (2, 3). of cholesterol with secondary accumulation of other lipids, and In the present studies, we have sought to confirm the physiologic this leads to cell death, especially in liver, lung, and cerebellum. relevance of the cholesterol binding site on NPC1(NTD) in organs The sequential handoff model for NPC action is supported by of living animals. For this purpose, we chose a mutation that re- biochemical and ultrastructural studies. Cholesterol enters lyso- places two adjacent amino acids in the cholesterol binding pocket somes as a result of the receptor-mediated endocytosis of LDL. (proline-202 and phenylalanine-203) with alanines. In previous Lysosomal acid lipase hydrolyzes the cholesteryl esters of LDL, studies, NPC1(NTD) containing these mutations failed to bind and the resulting insoluble cholesterol is postulated to be scav- cholesterol when tested in vitro, and full-length NPC1 containing enged by NPC2 before it has a chance to form crystals. NPC2 is these mutations failed to complement the defect in NPC1-deficient a soluble protein of 132 amino acids that is localized to the lyso- CHO cells (2). Here, we show that knockin mice homozygous for pf/pf somal lumen. Sterol binding studies (4) and X-ray crystallography the P202A/F203A mutation (designated as npc1 mice) exhibit (5) have demonstrated that NPC2 binds cholesterol in a specific a clinical and pathologic syndrome indistinguishable from that orientation, with the isooctyl side chain buried and the 3β-hydroxyl group exposed. The model postulates that NPC2 carries cholesterol to the lysosomal lining membrane, but it is prevented from Author contributions: X.X., M.S.B., J.L.G., and G.L. designed research; X.X., J.M.S., and G.L. accessing the membrane because the membrane is coated by performed research; X.X., M.S.B., J.M.S., J.A.R., J.L.G., and G.L. analyzed data; and M.S.B., a dense glycocalyx (2). At this point NPC2 transfers its cholesterol J.L.G., and G.L. wrote the paper. to NPC1, a 1,278-amino acid protein with 13 transmembrane he- The authors declare no conflict of interest. lices that is embedded in the lysosomal membrane (6, 7). The 1To whom correspondence may be addressed. E-mail: Joe.Goldstein@UTSouthwestern. cholesterol acceptor site on NPC1 is contained in the N-terminal edu, [email protected], or [email protected]. domain, a sequence of 240 amino acids that is postulated to project This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. into the lumen of the lysosome (2, 4). 1073/pnas.1112751108/-/DCSupplemental.

15330e15335 | PNAS | September 13, 2011 | vol. 108 | no. 37 www.pnas.org/cgi/doi/10.1073/pnas.1112751108 Downloaded by guest on September 29, 2021 nih/nih +/pf pf/pf reported for npc1 mice, the classic model for NPC1 deficiency WT, npc1 , and npc1 mice was 87:148:79. The WT and pf/pf pf/pf (9). Treatment of the npc1 mice with hydroxypropyl-β-cyclo- npc1 littermates were indistinguishable at birth, and both dextrin (HPCD) restores tissue cholesterol homeostasis, just as it z nih/nih showed normal growth and weight gain until 45 d of age (Fig. does in the npc1 mice (10–12). Our data also show that livers 4A). Thereafter, the WT mice continued to gain weight, whereas pf/pf pf/pf of the knockin mice have elevated levels of several lysosomal the npc1 mice began to lose weight. By 85 d, npc1 mice enzymes, including cathepsin B and D, that are restored to normal (16.1 ± 0.6 g) weighed 41% less than their WT littermates fi pf/pf by treatment with HPCD. The latter nding may explain the ex- (27.4 ± 0.9 g). At z50 d of age, npc1 mice began to exhibit fi tended lifespan in HPCD-treated NPC1-de cient mice. tremor and ataxia that progressed until death at an average age of 84 ± 3d(n = 30) (Fig. 4B). The age of onset of weight loss Results pf/pf and locomotor dysfunction in npc1 mice was comparable to Fig. 1A shows the gene-targeting strategy that was used to gen- nih/nih pf/pf that in npc1 mice, the classic mouse model of NPC disease npc1 npc1 pf/pf erate a line of knockin ( ) mice in which the adjacent (9). The average lifespan of the npc1 mice (84 d) also agreed nih/nih proline and phenylalanine residues 202 and 203 were replaced with that of npc1 mice (85 d) reported by Li et al. (14) in nih/nih with alanines. These two residues are part of the cholesterol studies of npc1 mice in the same C57BL/6 and 129Sv/Ev binding pocket in the NPC1(NTD) (2). Fig. 1B shows represen- +/pf mixed genetic background. Heterozygous npc1 mice were tative Southern blot analysis of EcoRI-digested genomic DNA indistinguishable from WT littermates. extracted from the tails of mice with the indicated genotype. For pf/pf At 9 wk of age, npc1 mice showed pathological features WT mice, a single 5.5-kb band was observed. In mice homozy- nih/nih pf/pf npc1 gous for the knockin allele (designated npc1 ), a single 7.5-kb identical to those of mice, including loss of Purkinje cells +/pf D band was detected. Both bands were present in the npc1 in the cerebellum (Fig. 5 ) and numerous large foam cells in liver +/pf E F heterozygous mice. For all experiments described here, npc1 (Fig. 5 ), spleen (Fig. 5 ), lung, and kidney. At 9 wk of age, npc1pf/pf heterozygous male and female mice were crossed to obtain WT mice accumulated excess cholesterol in many organs (Fig. pf/pf and npc1 littermates. 6A). The increases ranged from 2.5-fold in kidney to 7.4-fold in pf/pf Compared with those of WT littermates, livers of npc1 liver. Compared with other organs, the cholesterol content in brain was relatively high in WT mice, and there was no increase in npc1 mice exhibited increases in NPC1 and NPC2 mRNAs (1.7- and pf/pf nih/nih 4.7-fold, respectively) (Fig. 2A). Hepatic NPC1 and NPC2 pro- mice. Similar results have been reported for npc1 mice tein levels were also markedly increased (Fig. 2B). Fig. 2C (15). compares the glycosylation patterns of WT NPC1 and mutant A noteworthy feature of NPC disease is the buildup of lipids NPC1(P202A/F203A). Membrane fractions prepared from livers such as gangliosides GM2 and GM3 (16). As shown in Fig. 6B,in pf/pf pf/pf of WT and npc1 mice were treated with endoglycosidase H liver and brain of the npc1 mice, levels of gangliosides GM2 MEDICAL SCIENCES nih/nih (Endo H) or peptide:N-glycosidase F (PNGase F) and then and GM3 were elevated to a similar extent as in npc1 mice. subjected to immunoblot analysis with anti-NPC1. To compen- npc1pf/pf pf/pf Fig. 7 shows an experiment in which WT and mice sate for the increased protein level in npc1 mice, we used only were treated with three weekly injections of saline or HPCD half as much membrane protein from the mutants. In both WT beginning at 7 wk of age. At 9.5 wk of age, the content of total pf/pf pf/pf and npc1 mice, the N-linked carbohydrate chains of NPC1 and free cholesterol in livers of saline-injected npc1 mice was were partially resistant to Endo H, indicating that both WT and 7.4-fold and 7.5-fold higher than that in the saline-injected WT fi mutant NPC1 proteins had traf cked from the endoplasmic re- mice, respectively (Fig. 7A). HPCD treatment reduced the total ticulum (ER) to the Golgi apparatus. PNGase F, which removes npc1pf/pf N and free cholesterol content in the livers of mice by 64% all -linked carbohydrates, decreased the molecular weights of and 65%, respectively (Fig. 7A) and greatly reduced the number WT and mutant NPC1 proteins to the same degree. of lipid-laden foam cells (Fig. 7B). Thus, HPCD administration fi pf/pf To con rm that the P202A/F203A mutation did not alter the can overcome the cholesterol transport defect in npc1 mice, subcellular localization of NPC1, we examined its location by as it does in mice deficient in either npc1 or npc2 (10–12). double-labeled immunofluorescent confocal laser scanning mi- In addition to elevations in the mRNAs for NPC1 and NPC2 croscopy in transfected cells (Fig. 3). Human SV-589 cells were pf/pf (Fig. 2A), the livers of npc1 mice maintained elevated levels transfected with plasmids encoding Flag-tagged WT NPC1 or the P202A/F203A mutant. The Flag-tagged NPC1 was immunola- of mRNAs encoding a variety of lysosomal enzymes, including fl acid lipase, cathepsin B, cathepsin D, and β-glucuronidase (Fig. beled with anti-FLAG plus a red uorescent secondary antibody C (Figs. 3 A and D). Lysosomal-associated membrane protein 1 7 ). A notable exception was acid sphingomyelinase, whose ex- (LAMP1), a known lysosome marker (13) was immunolabeled pression level was not changed. Up-regulation of two of these with anti-LAMP1 plus a green fluorescent secondary antibody mRNAs (cathepsin D and cathepsin B) was observed previously (Figs. 3 B and E). WT NPC1 (Fig. 3C) and the mutant P202A/ in human fibroblasts homozygous for the I1061T NPC1 mutation F203A NPC1 (Fig. 3F) colocalized with LAMP1. (17) and in NPC1-deficient mice (18). Importantly, as shown in pf/pf The npc1 mice were born at the expected Mendelian ratio Fig. 7C, the increases in the cathepsin mRNAs, as well as those +/pf from intercrosses of the npc1 heterozygous male and female for NPC2, acid lipase, and β-glucuronidase, were abolished when mice. At the time of weaning (3 to 4 wk of age), of a total of 314 cholesterol accumulation was reversed by HPCD treatment of pf/pf offspring derived from 57 such intercrosses, the observed ratio of the npc1 mice.

Fig. 1. Generation of npc1 knockin mice harboring two point mutations in NPC1 that abolish cholesterol binding activity. (A) Schematic of the mouse WT npc1 allele and the targeting construct used to generate an npc1pf allele by introducing two point mutations (P202;F203 to A202;A203) into exon 5 of npc1. The targeting vector also contains a loxP- ﬂanked pgkneopA cassette and two copies of the HSV-TK gene as selection markers. The probe used for the Southern blot is denoted by the horizontal ﬁlled rectangle labeled “probe.” (B)Representative Southern blot analysis of EcoRI-digested genomic DNA from tails of mice with the indicated genotypes.

Xie et al. PNAS | September 13, 2011 | vol. 108 | no. 37 | 15331 Downloaded by guest on September 29, 2021 Fig. 2. NPC1 and NPC2 levels in livers of WT and npc1pf/pf mice. Nine-week-old male WT and npc1pf/pf littermates were fed an ad libitum chow diet before study. (A) mRNA analysis. Relative amounts of NPC1 and NPC2 mRNAs in livers were determined by quantitative real-time PCR with apoB as the invariant control. Values represent the amount of mRNA relative to that in WT littermates, which is arbitrarily deﬁned as 1. Each bar represents the mean ± SEM of data from 4 mice. Asterisks denote the level of statistical signiﬁcance (Student t test) between the WT and npc1pf/pf mice. **P < 0.01. The number of PCR cycles

(cycle threshold, Ct) required to reach the threshold line of 0.15 is shown inside the bar graph. (B) Immunoblot analysis. For each genotype, whole-cell lysates were prepared from four mouse livers. Aliquots of the pooled lysates (40 μg protein) were subjected to 8% SDS/PAGE and immunoblot analysis. β-Tubulin was used as a loading control. (C) Glycosidase digestion. For each genotype, membrane fractions from four mouse livers were pooled. Aliquots of the pooled fractions (40 μg and 20 μg for WT and npc1pf/pf, respectively) were incubated in the absence or presence of the indicated glycosidase and subjected to SDS/ PAGE and immunoblot analysis.

Discussion minal domain of NPC1 reproduces the lethal phenotype of The studies in this article demonstrate that a knockin mutation complete NPC1 deficiency in mice. This observation provides (P202A/F203A) that abolishes cholesterol binding to the N-ter- strong evidence that the cholesterol binding site on NPC1(NTD) that was demonstrated previously to be essential for NPC1 function in cultured cells (2, 4) is also essential for the function of the protein in the whole animal. pf/pf The npc1 mice harboring the knockin P202A/F203A mutation accumulated cholesterol in various organs in a pattern that nih/nih resembles that seen in NPC1-deficient npc1 mice, the standard model in the field (9). This accumulation led to organ dysfunction and death at the same age as previously observed in nih/nih nih/nih the npc1 mice. Like the npc1 mice (10–12, 15), the pf/pf npc1 mice showed dramatic improvement when treated with the cholesterol solubilizer HPCD. Weekly injections caused a marked fall in hepatic cholesterol content (Fig. 7 A and B). In important quantitative studies, Dietschy and coworkers observed that weekly injections of HPCD, beginning at 7 d of age, nih/nih markedly prolonged the lifespan of npc1 mice (15). This improvement was associated with a marked reduction in the content of unesterified cholesterol in liver. In another study they showed that a single injection of HPCD at 49 d of age lowered tissue cholesterol to a much lesser extent (19). Our results indicate that a reduction in liver cholesterol can be attained when the initial injection is given at 7 wk of age and repeated twice at weekly pf/pf intervals. We have not yet studied the npc1 mice to determine whether lifespan can be extended under these circumstances. We considered the possibility that cholesterol accumulation in pf/pf the npc1 mice was caused by impaired folding of the mutant NPC1, thereby precluding its exit from the ER and its transport to the lysosome. Two observations argued against this possibility. First, we used endoglycosidase H treatment to show that the N- linked carbohydrates of the mutant NPC1 were processed in the Golgi to the same extent as wild-type NPC1, indicating that the protein left the ER (Fig. 2C). Second, when an epitope-tagged version of the mutant NPC1 was transfected into cultured cells, the protein colocalized with the lysosome marker LAMP1 (Fig. 3). A unique observation in the current studies was the elevation in the hepatic content of mRNAs encoding a subset of lysosomal Fig. 3. Colocalization of transfected NPC1(P202A/F203A) with endogenous npc1nih/nih fi proteins. A previous study showed that brains of mice LAMP1. SV-589 broblasts were transfected with TK-driven plasmids had elevations in two lysosomal enzymes, cathepsins B and D expressing Flag-tagged versions of either WT NPC1 (A–C) or mutant NPC1 npc1pf/pf (P202A/F203A) (D–F) as described in Materials and Methods. Twenty-four (18). In livers of the mice, we found elevations in the fi mRNAs encoding these two enzymes as well as the mRNAs hours after transfection, the cells were xed and immunostained for Flag- β tagged NPC1 (red) and endogenous LAMP1 (green). Nuclei were stained encoding other lysosomal enzymes, such as -glucuronidase, ca- blue with DAPI. Fluorescence images were viewed and processed by confocal thepsin L1, lysosomal acid lipase, neuraminidase, and acid microscopy as described in Materials and Methods. Each image represents β-glycosidase. However, not all lysosomal mRNAs were elevated. a maximum intensity projection of a confocal z-stack of 32 images. (Scale Most importantly, there was no elevation in the mRNA encoding bars, 10 μm; magnification: 190×.) acid sphingomyelinase. Cultured cells lacking NPC1 are reported

15332 | www.pnas.org/cgi/doi/10.1073/pnas.1112751108 Xie et al. Downloaded by guest on September 29, 2021 Fig. 4. Growth and survival of WT and npc1pf/pf mice. (A) Littermate WT (n = 30, 18 male and 12 female) and npc1pf/pf (n = 30, 18 male and 12 female) mice were weighed weekly beginning at 5 wk of age. (B) The same cohort of WT (n = 30) and npc1pf/pf (n = 30) mice were followed up to 112 d of age, and the percent survival of each group was plotted as a function of time. For comparison, the reported survival curve for npc1nih/nih mice (14) was replotted in red.

to have reduced acid sphingomyelinase activity despite normal associated with a maldistribution of the enzyme from lysosomes levels of the enzyme protein (20, 21). Reduced sphingomyelinase to the cytoplasm. They postulated that these proteolytic enzymes activity is likely responsible for the elevated sphingomyelin levels initiated a series of reactions leading to neuronal death. Con- in NPC1-deficient lysosomes (1, 22). sidered together with their data, our observation that cathepsin In addition to the elevations in certain lysosomal enzyme mRNA levels are reduced by HPCD treatment raises the pos- mRNAs, we also found elevations in the mRNAs encoding both sibility that the improvement in lifespan in HPCD-treated mice is NPC1 and NPC2 (Figs. 2A and 7C). The elevations in NPC1 and related to the lowering of cathepsin levels that occurs when ly- NPC2 protein were at least as profound as the mRNA elevations sosomal cholesterol is reduced. This hypothesis can be tested in (Fig. 2B). These data raise the possibility that the accumulation future experiments in the mouse model. MEDICAL SCIENCES of cholesterol triggers a compensatory response designed to up- regulate the proteins that might enhance cholesterol removal Materials and Methods from lysosomes. Consistent with this hypothesis was the obser- Generation of npc1 Knockin Mice. The targeting vector used to generate npc1 vation that all of the elevated lysosomal mRNAs were restored knockin mice was constructed in pJB1, a gene replacement vector with toward normal when cholesterol was depleted from the lyso- a loxP-flanked pgkneopA cassette and two copies of the HSV-TK gene as somes by HPCD treatment (Fig. 7C). selection markers (23). The two regions of homology were generated by PCR Amritraj et al. (18) provided evidence that the up-regulation using genomic DNA derived from SM-1 ES cells (derived from 129SvEv fi blastocysts). PCR products were cloned into pJB1. For the short-arm region of cathepsins B and D in cerebellum of NPC1-de cient mice was containing exon 5 of npc1, the codons for a proline (CCA) and a phenylalanine (UUU) at adjacent residues 202 and 203 of NPC1 were mutated to alanine codons (GCA and GCU, respectively) by site-directed mutagenesis (Stratagene QuikChange Site-Directed Mutagenesis Kit). The integrity of all constructs was confirmed by restriction analysis and DNA sequencing. SM-1 ES cells were cultured, transfected with the linearized targeting vector, and selected with 250 μg/mL G418 as described previously (24). Three independent ES clones that had undergone homologous recombination as determined by PCR and Southern blotting were injected into C57BL/6J blastocysts to generate chimeric mice. When crossed to C57BL/6J females, male chimeras from all three clones gave offspring that carried the npc1 knockin allele through the germ line.

Animal Studies. All experiments were carried out with WT and npc1 knockin (npc1pf/pf) littermates obtained from intercrosses of npc1+/pf heterozygous male and female mice, all of which are hybrids of C57BL/6J and 129Sv/Ev strains. The npc1nih/nih mice used in Fig. 6 (obtained from Dr. John Dietschy, University of Texas Southwestern Medical Center, Dallas, TX) were on a pure BALB/c background (9, 11). All mice were housed in colony cages with a 12-h light/12-h dark cycle and fed ad libitum with Harlan Teklad Global Diet 2018 (Harlan Laboratories) and killed in the nonfasting state during the early phase of the light cycle. For the growth and survival study, the WT and npc1pf/pf littermates were weighed weekly, and their general appearance was monitored daily. Starting at 7 wk of age when the npc1pf/pf mice started to show tremor and ataxia, food pellets were placed directly in the cage. When a mouse exhibited visible difficulty in ambulating to reach water or take food, it was killed, and this was noted as the day of death. All animal experiments were performed with the approval of the Institutional Animal Care and Research Advisory Committee at University of Texas Southwestern Medical Center at Dallas. Fig. 5. Histology of various tissues from WT and npc1pf/pf mice. Tissues from 9-wk-old female WT (A–C) and npc1pf/pf (D–F) littermates were fixed, sec- Cyclodextrin Treatment. HPCD (CTD Holdings, catalog no. THPB-EC) in a 20% tioned, and stained with H&E as described in Materials and Methods. Arrows (wt/vol) saline solution was administered to mice at a dose of 4,000 mg/kg (A) denote Purkinje cells that are present in cerebellum of WT mice but body weight by s.c. injection as described previously (11). Male WT and absent from npc1pf/pf mice. (Scale bars, 50 μm; magnification: 40×.) npc1pf/pf littermates were injected with HPCD or saline at 7, 8, and 9 wk of

Xie et al. PNAS | September 13, 2011 | vol. 108 | no. 37 | 15333 Downloaded by guest on September 29, 2021 Fig. 6. Accumulation of cholesterol and gangliosides in organs of npc1pf/pf mice. (A) Total cholesterol content in various organs of 9-wk-old male WT and npc1pf/pf littermates was determined as described in Materials and Methods. Each bar represents mean ± SEM of data from four mice. Asterisks denote level of statistical signiﬁcance (Student t test) between the WT and npc1pf/pf mice. **P < 0.01. (B) Gangliosides from tissues of 9-wk-old male npc1pf/pf, npc1nih, and WT mice were subjected to TLC and visualized by orcinol staining as described in Materials and Methods. For each genotype, equal weights of liver or brain tissue from four mice were pooled. Each lane corresponds to the ganglioside fraction isolated from 3 mg of brain or 1.5 mg of liver (wet weight). Position of migration of standards for GM1, -2, and -3 are indicated.

age. Three days after the ﬁnal injection the mice were killed to obtain Glycosidase Treatment and Immunoblot Analysis. Membrane proteins were plasma and tissues for various analyses. prepared from frozen tissues as described previously (25). For glycosidase treatment, the 100,000 × g membrane pellet from 50 mg liver or brain was Quantitative Real-Time PCR Analysis. Total RNA was prepared from mouse liver resuspended in 100 μL buffer containing 10 mM Tris$chloride at pH 6.8, 100 using RNA STAT-60 (Tel-Test) and subjected to quantitative real-time PCR as de- mM NaCl, and 0.5% (wt/vol) SDS and shaken for 30 min at room temperature. scribed previously (24). All reactions were carried out in triplicate, and the relative Equal amounts of solubilized membrane protein from the liver or brain tissues

amounts of mRNAs were calculated using the comparative Ct method with of four mice were then pooled. Aliquots of the pooled membrane protein were apolipoprotein B (apoB) as the invariant control. Primers for apoB were described incubated in the absence or presence of 2,500 U Endo H or 2,500 U PNGase F previously (24). Primers not previously described can be found in Table S1. (New England Biolabs) and subjected to 8% SDS/PAGE and immunoblot

Fig. 7. Cyclodextrin treatment of WT and npc1pf/pf mice. Male WT and npc1pf/pf littermates were treated with three weekly injections of saline or HPCD beginning at 7 wk of age as described in Materials and Methods. At 9.5 wk of age (3 d after the final injection), the mice were killed to obtain plasma and organs for analysis. (A) Content of total and free cholesterol in livers of WT and npc1pf/pf mice injected with saline or HPCD. Each bar represents the mean ± SEM of data from five mice. (B) Histol- ogy of representative livers from WT and npc1pf/pf mice injected with saline or HPCD. Livers were fixed, sectioned, and stained with H&E. (Scale bars, 40 μM; magnification: 40×.) (C) Relative amounts of mRNAs in livers of the indicated groups were determined by real-time PCR, with apoB as the invariant control. Values represent the amount of mRNA relative to that in WT littermates injected with saline, which is arbitrarily defined as 1 and denoted by the dotted line. Each bar represents the mean ± SEM of data from five mice. Statistical analysis (A and B) was performed with two-tailed Student ttest.*P < 0.05; **P < 0.01; ***P < 0.001.

15334 | www.pnas.org/cgi/doi/10.1073/pnas.1112751108 Xie et al. Downloaded by guest on September 29, 2021 analysis as described previously (3). The primary antibodies for immunoblot bodies. After three washes with PBS, the cells were incubated with 300 nM analyses were 0.5 μg/mL rabbit polyclonal antibody directed against a synthetic DAPI (Invitrogen, catalog no. D3571) in PBS for 10 min to stain the nuclei. peptide corresponding to C-terminal region of human NPC1 (catalog no. Fluorescence was viewed with a Leica TCS SP5 laser scanning confocal mi- μ 36983, Abcam), 1 g/mL rabbit polyclonal anti-NPC2 (3), and 1:1,000 dilution of croscope using an Argon Laser with excitation wavelength of 488 nm for β rabbit monoclonal anti- -tubulin (catalog no. 2128, Cell Signaling). Bound Alexa Fluor 488 (green) and a HeNe1 laser with excitation wavelength of 561 antibodies were visualized by chemiluminescence (SuperSignal West Pico nm for Alexa Fluor 568 (red). DAPI (blue) ﬂuorescence was viewed using Chemiluminescent Substrate; Thermo Scientiﬁc) using a 1:3,000 dilution of a HeNe1 laser with excitation wavelength of 405 nm. Colocalization analysis anti-rabbit IgG (GE Healthcare) conjugated to horseradish peroxidase. was done using the Imaris software (version 7.2.3; www.bitplane.com).

Histology. For histological analysis, anesthetized mice were transcardially Lipid Measurements. The content of total and free cholesterol in tissues was perfused with normal saline and then with 10% (vol/vol) formalin. After measured using Wako kits (catalog no. 439-17501 and 435-35801, re- perfusion, the tissues were fixed in 10% formalin overnight at 4 °C. The fixed tissues were embedded in paraffin, sectioned at 5 μm, and stained spectively). Extraction and analysis of gangliosides were carried out as pre- fl with H&E. viously described (26). Brie y, total lipids from brain and liver tissues were extracted in chloroform:methanol:PBS (1:1:0.9, vol/vol/vol) by the method of Immunofluorescence. Human SV-589 fibroblasts were grown at 37 °C in 5% Bligh and Dyer (27). Equal amounts (based on wet tissue weight) of total

CO2 in DMEM containing 100 U/mL penicillin, 100 μg/mL streptomycin sul- lipid extracts from four mice per group were pooled, and aliquots of the fate, and 10% FCS. On day 0, the cells were set up at 65 × 103 cells/35-mm pooled samples were then separated into neutral and anionic lipid fractions glass-bottom dish (MatTek, catalog no. P35G-1.5-14-C). On day 1, the cells using DEAE Sephadex A-25 columns (GE Healthcare). The anionic lipid were transfected with 1 μg pTK-NPC1-His8-Flag3 or pTK-NPC1(P202A/ fractions (containing gangliosides) were desalted with PD-10 Sephadex G-25 F203A)-His8-Flag3 using FuGENE 6 transfection reagent (Roche Applied Sci- columns (GE Healthcare). Aliquots of the ganglioside fraction corresponding ence) as described previously (2). Twenty-four hours after transfection, the to 3 mg brain or 1.5 mg liver tissue were analyzed by TLC using high-per- ﬁ cells were xed for 10 min at room temperature in 4% (wt/vol) para- formance TLC plates (catalog no. 818140, Macherey-Nagel). The plates were formaldehyde, washed with PBS (three times; 1 min per wash), and treated developed in a choloroform:methanol:0.2% CaCl2 (60:35:8, vol/vol/vol) so- with 0.1% (vol/vol) Triton X-100 in PBS for 90 s. After washing 3 times with lution. Gangliosides were visualized by staining with 0.5% (wt/vol) orcinol PBS, the cells were incubated for 30 min in a blocking solution containing dissolved in 3 M sulfuric acid. Aliquots (4 μg each) of GM1, GM2, and GM3 10% (vol/vol) normal goat serum (Invitrogen, catalog no. 50-062Z). There- standards (Avanti) were loaded on the same TLC plate. after, the cells were incubated in the same blocking solution for 16 h at 4 °C with 1 μg/mL mouse monoclonal anti-LAMP1 (Abcam, catalog no. ab25630) and 0.5 μg/mL rabbit polyclonal anti-Flag (Sigma-Aldrich, catalog no. F7425) ACKNOWLEDGMENTS. We thank Monica Mendoza and Isis Soto for invalu- able help with animal studies, Liz Lummus and Robert Hammer (University to detect endogenous LAMP1 and the transfected Flag-tagged NPC1 pro-

of Texas Southwestern Transgenic Core) for ES cell injections, and Kate Luby- MEDICAL SCIENCES teins, respectively. After washing three times with PBS, the cells were in- Phelps (University of Texas Southwestern Live Cell Imaging Core) and Lina μ cubated for 1 h at room temperature with 5 g/mL Alexa Fluor 488 (green) Abi Mosleh for help with immunoﬂuorescence. This work was supported by donkey anti-mouse IgG (Invitrogen, catalog no. A-21202) and 5 μg/mL Alexa National Institutes of Health Grant HL20948 and grants from the Ara Fluor 568 (red) goat anti-rabbit IgG (Invitrogen, catalog no. A-11036) anti- Parseghian Medical Research Foundation and Moss Heart Fund.

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