Human Molecular Genetics

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Human Molecular Genetics, 2003, Vol. 12, No. 3 257-272
© 2003 Oxford University Press

Defective endocytic trafficking of NPC1 and NPC2 underlying infantile Niemann–Pick type C disease

Titta S. Blom¹, Matts D. Linder¹, Karen Snow², Helena Pihko³, Michael W. Hess^4,5, Eija Jokitalo⁵, Ville Veckman⁶, Ann-Christine Syvänen⁷ and Elina Ikonen^1,*

¹Department of Molecular Medicine, National Public Health Institute, Helsinki, Finland, ²Molecular Genetics Laboratory, Mayo Clinic, Rochester, MN, USA, ³Department of Child Neurology, Hospital for Children and Adolescents, University of Helsinki, Helsinki, Finland, ⁴Institute of Anatomy, Histology and Embryology, University of Innsbruck, Austria, ⁵Institute of Biotechnology, University of Helsinki, Helsinki, Finland, ⁶Department of Microbiology, National Public Health Institute, Helsinki, Finland and ⁷Department of Medical Sciences, Uppsala University, Uppsala, Sweden

Received September 16, 2002; Accepted November 22, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION SUBJECTS AND METHODS REFERENCES

 
Niemann–Pick type C (NPC) disease is a fatal recessively inherited lysosomal cholesterol-sphingolipidosis. Mutations in the NPC1 gene cause 95% of the cases, the rest being caused by NPC2 mutations. Here the molecular basis of a severe infantile form of the disease was dissected. The level of NPC1 protein in the patient fibroblasts was similar to that in control cells. However, the protein was partially mislocalized from late endocytic organelles diffusely to the cell periphery. In contrast, NPC2 was upregulated and accumulated in cholesterol storing late endocytic organelles. Two point mutations and a four-nucleotide deletion were identified in the NPC1 gene, leading to the amino acid substitutions C113R, P237S and deletion of 37 C-terminal amino acids (delC). Overexpression of individual NPC1 mutations revealed that delC produced an unstable protein, wild-type and NPC1-P237S colocalized with Rab7-positive late endosomes whereas NPC1-C113R localized to the ER, Rab7-negative endosomes and the cell surface. Expression of wild-type or NPC1-P237S cleared the lysosomal cholesterol accumulation in NPC1-deficient cells whereas C113R or delC did not. In the Finnish and Swedish population samples, alleles carrying C113R or delC were not identified, whereas 5% of the alleles carried P237S. Our studies identify P237S as a prevalent NPC1 polymorphism and delC and C113R as deleterious NPC1 mutations. Moreover, they show that delC leads to rapid degradation of NPC1 and C113R to endocytic missorting of the protein. These changes are accompanied by lysosomal accumulation of NPC2, suggesting that NPC1 governs the endocytic transport of NPC2.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION SUBJECTS AND METHODS REFERENCES

 
Niemann–Pick type C (NPC) disease is an autosomal recessively inherited neurodegenerative lysosomal storage disease. Its cellular hallmark is the accumulation of low-density lipoprotein-derived unesterified cholesterol in the late endocytic organelles (1). The clinical manifestations of NPC are heterogeneous, with the age of onset ranging from early infancy to adulthood. Most patients develop progressive neurological dysfunction but the disease course is variable. Children with infantile neurological onset of the disease invariably show pronounced abnormalities in cellular cholesterol trafficking (1–3).

In NPC disease, two genetic complementation groups, NPC1 and NPC2, have been identified (4,5). NPC1 is the gene defective in 90–95% of the cases (6). HE1/NPC2 was recently identified as the protein mutated in the minor complementation group (7). The NPC1 cDNA sequence predicts a 1278 amino acid protein with 13 transmembrane spans (8) and several domains conserved during evolution. These include an N-terminal region with high species conservation designated as the NPC domain and a sterol-sensing domain that is shared by the key regulators of cholesterol metabolism (6). The NPC1 protein has been localized to late endocytic organelles that are positive for the small GTPase Rab7 and lysosome-associated membrane protein (Lamp)-2 but negative for cation-independent mannose-6-phosphate receptor (MPR) (9,10). The protein was also found to be localized to peripheral cellular sites and the plasma membrane, potentially regulating transport to and from the cell periphery (11,12). Present evidence suggests that NPC1 controls not only the trafficking of cholesterol but also other lipids, in particular sphingolipids (13–15). In addition, a possible function of NPC1 as a transmembrane pump has been introduced (16). However, the precise itineraries of the protein and its function remain elusive.

Mutational analysis of NPC1 has revealed over 100 different alterations widely distributed throughout the gene (6,17). The majority are missense mutations, several of which result in essentially normal amounts of NPC1 protein (18–20). One of the most prevalent mutations, I1061T substitution, is a frequent mutant allele in patients of Western European descent and correlates with a classic juvenile phenotype of the disease (21). To our knowledge, this is the only NPC patient mutation for which subcellular localization and functional studies of the encoded protein have been carried out at the cellular level. This mutant protein reached the storage lysosomes but was incapable of removing the cholesterol accumulation (22).

NPC2 is a small 151-amino acid soluble, cholesterol-binding protein that was initially identified as the second NPC gene based on its ability to bind the mannose-6-phosphate receptor (MPR) (7). Biochemical evidence suggests that the protein is lysosomal but can also be secreted from cells (7,23). The majority of the cases in the NPC2 complementation group show a severe clinical course and exhibit deleterious NPC2 mutations, leading to early termination of the protein (24). The cellular trafficking of NPC2 in normal or mutant cells has not been characterized.

In this study, we determined the mutations causing an infantile form of NPC disease in a Finnish patient. We generated cDNA constructs harboring each of the NPC1 mutations identified and, upon overexpression in cells, dissected the subcellular localization, biochemical behavior and functional consequences of the individual mutant proteins. In addition, the frequencies of the identified mutations in Finnish and Swedish population samples were determined. Characteristics of the in vitro expressed NPC1 mutant proteins were compared with those of the endogenous NPC1 protein in the patient fibroblasts. Further studies in the patient cells led to the recognition of MPR and NPC2 as likely downstream targets of the causative mutation.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION SUBJECTS AND METHODS REFERENCES

 
NPC1 mutation analysis
The nucleotide changes and corresponding amino acid changes identified in the NPC1 gene of the patient are shown in Figure 1A and their segregation in the family in Figure 1B. Intriguingly, the C113R change was absent in the samples from both the mother and the paternal grandparents, suggesting that the substitution represents a de novo mutation either in the proband or in the father. DNA fingerprinting using multiallelic markers indicated that false paternity was highly unlikely (97% probability of correct paternity; data not shown). The C-terminal deletion (delC) derives from the mother and the P237S substitution presumably from the father. Figure 1C demonstrates the localization of the mutations in the predicted topology model of the NPC1 protein (8). Both of the point mutations affect the N-terminal luminal loop, where few mutations have been reported so far (20). DelC truncates the NPC1 sequence in the predicted 12th membrane span and as a result of the frame shift, leads to the incorporation of 36 novel amino acids as indicated in Figure 1C.

View larger version (36K): [in this window] [in a new window]   Figure 1. (A) Nucleotide and corresponding amino acid changes detected in the Finnish family, (B) segregation of the alterations in the pedigree and (C) schematic illustration of the amino acid changes in the NPC1 protein. Numbers refer to pedigree members in Table 1 (B). Amino acids in the insert correspond to the novel sequence introduced by the frameshift at nucleotides 3611–3614 and starting after T1202 of the NPC1 protein (C).

 
Characterization of the NPC1 mutant protein in the patient fibroblasts
Immunofluorescence microscopy of control fibroblasts revealed good correlation of anti-NPC1-positive dots and the well-known distinct distribution of the late endosomal marker protein Lamp-1 (lysosome-associated membrane protein), in accordance with previous reports (9,25). By contrast, the patient cells showed a considerable amount of diffuse anti-NPC1-staining extending to the very cell periphery, including weak plasma membrane staining—in addition to the perinuclear anti-NPC1/anti-Lamp1-staining (Fig. 2A). The late endosomes of the patient cells accumulated free cholesterol and glycolipids as visualized by the characteristic staining patterns with the fluorescent antibiotic filipin and anti-3'LM1 antibodies, respectively (Fig. 2B) (14). To study these endosomes at the ultrastructural level, cryofixation and freeze-substitution were used. This technique preserves lipid-enriched structures more reliably than conventional ambient-temperature chemical fixation. The features observed were typical for NPC cells: highly proliferated spherical and less frequently pleiomorphic endosomal/lysosomal compartments concentrated around the nucleus, usually almost completely filled with multi-lamellar/vesicular structures and a granular matrix (Fig. 2C and D). Double-immunolabeling of thawed ultrathin cryosections from chemically fixed patient cells showed that both NPC1 and 3'LM1 co-localized specifically to these structures (Fig. 2E). Other binding sites of anti-NPC1 and anti-3'LM1, however, could not be assigned unambiguously to particular organelles due to the low specimen contrast. Together, our immunofluorescence and immuno-electron microscopic data show that the patient NPC1 protein is partially mislocated but some of it still resides in the late endocytic organelles.

View larger version (104K): [in this window] [in a new window]   Figure 2. Morphological characterization of NPC patient fibroblasts. (A) Control and NPC fibroblasts were immunostained with anti-NPC1 and anti-Lamp-1 antibodies. (B) The cells were also stained with filipin and anti-glycolipid 3'-LM1 antibodies. (A) represents confocal images, (B) wide-field images. (C and inset, D) Ultrastructure of the cryofixed, freeze-substituted NPC cells with abundant multi-lamellar/-vesicular structures in the perinuclear region; scale bar=500 nm (C). (E) Immuno-electron microscopy shows specific co-localization of NPC1 (5 nm gold: arrows) and 3'LM1 (10 nm gold: arrow-heads) within multi-lamellar/-vesicular structures (asterisks); scale bar=200 nm.

 
The amount of NPC1 protein in the patient fibroblasts was analyzed by Western blotting using anti-NPC1 antibodies. The patient NPC1 protein was expressed at a level comparable to that of the wild-type NPC1 in control fibroblasts (Fig. 3A). Also the size of the mutant NPC1 was similar to that of the wild-type (wt) protein (180 kDa). Distribution of NPC1 between the plasma membrane and intracellular compartments was then studied by surface biotinylation. This revealed that, in the patient cells, there was about 2-fold more NPC1 protein on the cell surface (Fig. 3B and C). This is in accordance with the more pronounced anti-NPC1 plasma membrane staining observed by immunofluorescence microscopy in the patient cells.

View larger version (32K): [in this window] [in a new window]   Figure 3. Biochemical analysis of NPC1 protein in the patient fibroblasts. (A) Equal amounts of protein (20 µg) from control and NPC fibroblast lysates were analyzed by SDS–PAGE and Western blotting using NPC1-antibodies. (B) Control and NPC1 cells were surface-biotinylated, one-tenth of the sample representing the ‘total’ was TCA precipitated and from the rest, biotinylated proteins were precipitated with Streptavidin-agarose (‘surface’). The samples were analyzed by western blotting. (C) Densitometric scanning of the intensity of the NPC1 protein band on the surface as a percentage of total NPC1 (mean±SEM from three experiments), *P<0.05 between control and NPC (student's t-test).

 
Transient overexpression of the mutant NPC1 polypeptides
To analyze the contribution of the identified NPC1 gene mutations to the cellular characteristics of the protein, the respective mutations were generated individually in NPC1 cDNA and the encoded proteins expressed in COS-1 and Chinese hamster ovary (CHO) cells. [³⁵S]Met pulse-labeling of COS-1 cells for 30 min followed by 4 h of chase and anti-NPC1 immunoprecipitation showed that the C113R and P237S mutants were expressed at levels comparable to NPC1-wt. In contrast, the delC mutant was unstable with only very minor amounts of the polypeptide immunoprecipitated (Fig. 4A). Increased degradation of delC was evident already at 1 h of chase (data not shown). Subcellular localization studies of the mutant proteins revealed that NPC1-P237S distributed largely to Lamp-1 positive organelles similarly to the wt protein while NPC1-C113R colocalized with Lamp-1 immunoreactivity only juxtanuclearly, the peripheral NPC1-C113R positive organelles being Lamp-1 negative (Fig. 4B). In addition, the perinuclear staining of the C113R mutant overlapped extensively with the endoplasmic reticulum (ER) chaperone protein disulfide isomerase (Fig. 4B). The distribution of the NPC1 proteins was then compared with that of the late endosomal Rab7 by co-overexpression of NPC1 and Rab7-GFP in CHO cells. Again, the NPC1-wt and -P237S colocalized extensively with the late endosomal marker whereas NPC1-C113R was not efficiently distributed to Rab7-GFP positive structures (Fig. 4C).

View larger version (97K): [in this window] [in a new window]   Figure 4. Overexpression of wt and individual mutant NPC1 proteins. Wt NPC1 and C113R, P237S and delC mutants were transiently expressed in COS-1 (A, B, D and E) and CHO cells (C). Cells were labeled with [35S]Cys/Met for 1 h, chased for 4 h and NPC1 was immunoprecipitated using anti-NPC1 antibodies. The samples were analyzed by SDS-PAGE and autoradiography (A). To study the subcellular localization of the proteins, transfected cells were stained with anti-NPC1, anti-Lamp-1 and anti-PDI antibodies and visualized by confocal microscopy (B). Alternatively, cells were co-transfected with wt or mutant NPC1 together with Rab7-GFP and stained with anti-NPC1 antibodies (C). Glycosylation of newly synthesized mutant NPC1-C113R was studied by Endo H digestion. After pulse-labeling of cells with [35S]Cys/Met for 30 min and chasing for 4 h, NPC1 was immunoprecipitated, digested with EndoH and analyzed by SDS-PAGE and autoradiography (D). The intracellular versus plasma membrane localization of wt and C113R was compared by surface biotinylation and western blotting as in Fig. 3E.

 
Subcellular localization of NPC1-C113R
The perinuclear ER and sometimes nuclear envelope labeling of NPC1-C113R (Fig. 4B and C) suggested that the mutant protein does not leave the ER efficiently. We therefore analyzed the arrival of the newly synthesized NPC1 proteins to the Golgi complex by Endoglycosidase H (Endo H) digestion of the [³⁵S]Met pulse-labeled and immunoprecipitated polypeptides. Both NPC1-wt and -C113R acquired Endo H resistance only gradually within the course of several hours, demonstrating slow exit from the ER. Furthermore, a slight delay in the development of the Endo H-resistant form was noted with NPC1-C113R compared with the wt protein, indicating delayed transport of the mutant to the Golgi complex (Fig. 4D). Eventually a fraction of both proteins was distributed to the plasma membrane as shown by surface biotinylation. Interestingly, NPC1-C113R was localized to the plasma membrane more avidly than the NPC1-wt (Fig. 4E).

As NPC1-C113R colocalized poorly with late endocytic markers we further characterized its distribution upon ER exit. The punctate morphology of the NPC1-C113R positive structures and the biochemically observed plasma membrane localization of the protein suggested delivery to endosomal compartments. The NPC1-C113R containing structures were negative for fluorescent transferrin, a classical marker of the endocytic recycling compartment (Fig. 5). However, a minor proportion of the C113R organelles colocalized with the early endosomal antigen 1 (EEA-1) indicating that some of the protein localizes to early endocytic organelles. To further address the potential endocytic nature of the NPC1-C113R containing organelles, a fluid phase tracer was internalized into the cells. Several of the NPC1-C113R positive vesicular profiles colocalized with fluorescent dextran (Fig. 5) indicating they were indeed of endocytic origin.

View larger version (163K): [in this window] [in a new window]   Figure 5. Subcellular localization of NPC1-C113R in HeLa cells. Cells transfected with NPC1-C113R-GFP were immunostained with anti-EEA1 antibodies, or incubated with TexasRed-Transferrin for 30 min at 37°C, or labeled with TRITC-Dextran for 1 h followed by chasing for 17 h at 37°C. Panels represent confocal images. Note that high overexpression and/or GFP-tagging of NPC1 leads to ER/nuclear envelope retention more easily than low level expression of the nontagged/endogenous protein.

 
Complementation of NPC1-deficient cells with the NPC1 proteins
The functionality of NPC1 proteins can be studied by expressing them in NPC1 deficient cells exhibiting late endosomal cholesterol accumulation. The wt NPC1 protein is capable of clearing the cholesterol deposits within the time frame of a transient transfection. We next compared the ability of the wt and mutant NPC1 proteins to complement the NPC phenotype in NPC1 deficient CHO cells (26). The transfected cells were stained by filipin and anti-NPC1 antibodies, viewed by fluorescence microscopy and the ability of the different NPC1 proteins to unload the cholesterol accumulation was scored (n=200 cells per construct except n=20 cells for delC). Owing to the instability of delC NPC1, it was difficult to find cells expressing the protein. In the few cells found, the protein was localized in a diffuse or reticular ER-nuclear envelope pattern and failed to produce any reduction in the number of cholesterol-filled deposits (Fig. 6A and B). NPC1-C113R was partly found in punctate endosomal structures but partly retained in the nuclear envelope (Fig. 6A). It was unable to reduce the lysosomal cholesterol accumulations (Fig. 6B). Instead, NPC1-P237S complemented the NPC1 phenotype similarly to the wt protein (Fig. 6A and B).

View larger version (71K): [in this window] [in a new window]   Figure 6. Removal of cholesterol deposits by wt and mutant NPC1 proteins. (A) The ability of wt and mutant NPC1 proteins to complement the NPC phenotype was studied by transfecting the NPC1 constructs into NPC1 deficient M12 cells. Cells were fixed at 3 days post-transfection, and stained with filipin and anti-NPC1 antibodies. (B) Cells were viewed (n=200 per construct, except n=20 cells for delC) by wide-field microscopy and the ability of the different proteins to unload the cholesterol accumulation was scored into three categories as detailed in Materials and Methods.

 
Population frequencies of the identified mutations
The complementation analysis indicated that C113R and delC are the disease-causing NPC1 mutations while P237S appears to be a polymorphism. To assess whether the corresponding base changes occur in the normal population, pooled DNA samples representing the Swedish and Finnish populations were analyzed by solid-phase minisequencing (Table 1). The minisequencing method allows quantitative detection of a mutant allele present as 1% of a pooled DNA sample (27). The proportion of the mutant allele in a pooled sample corresponds directly to its frequency in the population from which the pooled sample is derived. For the C113R and delC NPC1 mutations the signal ratios measured in the pooled DNA samples were indistinguishable from the corresponding ratios in a sample from a homozygous individual, showing that the mutations are not prevalent (over 1%) in the normal population. For the NPC1-P237S mutation, however, the minisequencing analysis revealed about 3-fold elevated signal ratios in the pooled samples, compared with those in the samples from homozygous individuals. Using the mean value of the ratios obtained when analyzing the heterozygous pedigree members 2 and 3 as reference value, we calculated that the P237S mutant allele occurs with a frequency of 4.3–4.7% in the normal Finnish and Swedish populations (Table 1). Next, the evolutionary conservation of the C113 and P237 residues and the adjacent polypeptide regions were studied in NPC1 orthologs from other organisms. The residue corresponding to C113 of human NPC1 resides in the NPC domain and was found to be conserved in all the species analyzed, further supporting its significance for an apparent evolutionarily conserved function (Fig. 7). Interestingly, P237 is also conserved in all the mammalian organisms analyzed and resides within a highly conserved stretch of amino acids, albeit outside the NPC domain (Fig. 7).

View this table: [in this window] [in a new window]   Table 1. Determination of the Finnish and Swedish population frequencies of the NPC1 mutations by quantitative analysis of pooled DNA samplesa

 

View larger version (71K): [in this window] [in a new window]   Figure 7. Evolutionary conservation of human NPC1 residues C113 and P237. Evolutionary conservation of the mutated amino acids C113 and P237 was studied in NPC1 orthologs using the CluctalW algorithm (44). Numbers of the first and last amino acids shown of human NPC1 are indicated, NPC domain corresponds to amino acids 55–165. The Genbank accession numbers used in this alignment are: Chinese hamster, Cricetulus griseus, AF182744; mouse, Mus musculus, AF003348; rabbit, Oryctolagus cuniculus, AF202730; human, Homo sapiens, AF002020; cat, Felix catus, AF258783; dog, Canis familiaris, AF315034; bovine, Bos taurus, AF299073; porcine, Sus scrofa, AF169635; fruit fly, Drosophila melanogaster, AJ249606; Arabidopsis thaliana, NP_174975; yeast, Saccharomyces cerevisiae, L42821; Caenorhabditis elegans, U53340.

 
NPC2 protein in the NPC1 patient cells
The indistinguishable clinical, cellular and biochemical phenotypes between NPC1 and NPC2 patients suggest that the two gene products may function in tandem or sequentially (5,6,28). We therefore studied the level and distribution of the endogenous NPC2 protein in control and patient fibroblasts. In control fibroblasts, anti-NPC2 antibodies visualized punctate and perinuclear staining that was effectively inhibited by preincubation of the antiserum with the purified protein (Fig. 8A). In some cells, the punctate staining extending towards the cell periphery predominated whereas in others, the perinuclear aspect was most intensely visualized (Fig. 8B). Double-immunostaining revealed colocalization of the punctate NPC2 staining with Lamp-1 while the perinuclear fluorescence colocalized with gamma-adaptin, a marker for the trans-Golgi network (TGN) (Fig. 8C). In addition, the anti-NPC2 staining was pronounced in cellular extensions and, in these areas, the punctate staining often did not colocalize with Lamp-1 (see Fig. 9D, control cells). Thus, in control fibroblasts the NPC2 immunoreactivity was found to be distributed to the TGN as well as Lamp-1 positive endosomes and Lamp-1 negative peripheral organelles.

View larger version (137K): [in this window] [in a new window]   Figure 8. Localization of NPC2 protein in human fibroblasts. (A) The specificity of anti-NPC2 staining was confirmed by preincubating the antibody with purified NPC2 protein or BSA followed by immunofluorescence staining of control fibroblasts. (B) Control fibroblasts were double immunostained with anti-NPC2 and anti-Lamp-1 or (C) anti-NPC2 and anti-gamma-adaptin. Panels represent confocal images.

 

View larger version (94K): [in this window] [in a new window]   Figure 9. Characterization of NPC2 protein in the patient fibroblasts. (A) Equal amounts of protein (20 µg) from control and NPC fibroblasts were analyzed by SDS–PAGE and western blotting using anti-NPC2-antibodies. (B) Densitometric scanning of the intensity of the NPC2 protein bands (mean±SEM; n=4 from two independent experiments; *P<0.01, Student's t-test). (C) Equal amounts of total RNA (20 µg) from control and NPC fibroblasts were analyzed by northern blotting using the coding region of the NPC2 cDNA as a probe. Ethidium bromide staining of ribosomal RNA bands was used to control equal RNA loading (below). (D) Control and NPC fibroblasts were stained with anti-NPC2 and anti-Lamp-1 antibodies and filipin (wide-field images). The trans-Golgi area (arrowheads) and NPC2-positive Lamp-1 negative peripheral organelles (arrows) are indicated.

 
In Western blot analysis, the anti-NPC2 antibodies recognized a typical doublet between 20 and 25 kDa in both control and NPC patient fibroblasts (Fig. 9A). The bands represent differentially glycosylated isoforms of the protein (data not shown). Interestingly, the level of immunoreactive NPC2 in the patient cells was slightly (1.5-fold) higher than in control cells (Fig. 9A and B). This was paralleled by increased NPC2 transcript levels in the patient cells as shown by Northern blot analysis (Fig. 9C). Immunofluorescence microscopy revealed that in contrast to control cells, in the patient fibroblasts the anti-NPC2 staining in the perinuclear, Lamp-1 negative TGN area was not evident and the majority of immunoreactivity was concentrated in Lamp-1 positive, cholesterol-filled late endosomes/lysosomes. Very few peripheral NPC2-positive structures were observed in the patient cells (Fig. 9D). These results suggest that one of the cellular consequences of the NPC1 defect is the upregulation of NPC2 and accumulation of the NPC2 protein in the cholesterol engorged late endocytic compartments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION SUBJECTS AND METHODS REFERENCES

 
In this study, we identified the mutations causing an infantile form of NPC disease, determined their prevalence in the general population and dissected their consequences at the cellular level. Surprisingly, we found the two candidate proteins NPC1 and NPC2 to be expressed at normal or elevated levels in the patient cells. We therefore reasoned that this NPC case would be particularly suitable for dissecting the consequences of mutations on the cellular itineraries of the two proteins. Mutation screening resulted in the identification of two point mutations (NPC1-C113R and NPC1-P237S) and a C-terminal deletion (NPC1-delC) in the NPC1 gene of the patient. At present, the best evidence for the functional importance of a particular residue of NPC1 is its effect on the ability of the protein to reverse the cholesterol accumulation in NPC1-deficient cells. Based on this criterion, delC and C113R were found to result in nonfunctional NPC1 proteins, whereas NPC1-P237S produced a functional protein complementing the NPC phenotype. These findings agree with the morphological and biochemical data obtained upon overexpression of the individual proteins as well as with the characteristics of the endogenous protein in the patient cells.

NPC1-delC resulted in an unstable NPC1 protein and presumably does not contribute significantly to the NPC1 immunoreactivity of the patient cells. This mutation results in the disappearance of a C-terminal dileucine motif, and deletion of this putative lysosomal targeting signal has previously been shown to cause the retention of the protein in the ER (29).

The majority of NPC1-C113R failed to localize correctly to late endocytic compartments and was mislocalized to the ER, early endosomes and the plasma membrane. This agrees with the diffuse NPC1 staining pattern of the patient cells. Moreover, surface biotinylation experiments indicated that both the endogenous NPC1 protein in the patient cells and the overexpressed NPC1-C113R were more readily found on the cell surface than the wt protein. Owing to a number of Cys residues in the NPC domain, disulfide bonds are likely to be important for its proper conformation. Indeed, C113R may participate in the formation of a disulfide bridge. We have found C113R to result in the loss of a number of putative NPC1 interaction partners in a yeast two-hybrid screen, speaking in favor of a major conformational change induced by the amino acid substitution (our unpublished data). The finding that NPC1-C113R was localized to atypical endocytic organelles points to the importance of the NPC domain in determining the endocytic routing of the protein.

Considering the inheritance of the mutant alleles in the pedigree, an unusual situation prevails. The maternally inherited delC makes the proband a compound heterozygote for deleterious, non-functional NPC1, similarly to the carrier mother. P237S present in the paternal allele would alone produce a functional NPC1 protein; however, a novel mutation, C113R, was generated either in the father or in the proband. Roughly normal amounts of the mutant protein were produced in the patient cells and some of it evidently reached the endocytic circuits and the plasma membrane. However, the amount of functional NPC1 protein reaching its correct destination and interaction partners probably fell under a critical threshold level. This would represent a typical scenario envisaged in recessively inherited loss-of-function mutations.

Previous reports have identified the NPC1-P237S substitution in several other NPC patients. It has been found in combination with one or two other putative pathogenic NPC1 mutations but also represented the only identified NPC1 patient mutation (30). NPC1-P237S was reported to be a deleterious NPC1 mutation with no occurrence in 100 unaffected Japanese samples (18) nor in 95 unaffected French samples (19). Instead, Sun and coworkers suggested it to represent a benign polymorphism based on its identification on approximately 2% (two out of 98) of chromosomes from the US general population (30). Our demonstration that NPC1-P237S complements the NPC phenotype strongly supports this idea. Moreover, identification of this substitution in 4–5% of alleles in Finnish and Swedish population samples (of 360 and 500 chromosomes, respectively) distinguishes P237S as a prevalent NPC1 change in these populations. This finding opens up interesting questions regarding the potential modulatory role of this residue in NPC1 function. Pro is an alpha helix breaker and its substitution with Ser could result in significant conformational changes; moreover, this residue is conserved between mammals. Thus, NPC1-P237S could potentially be associated with more subtle alterations of cholesterol balance.

Our findings emphasize the nature of NPC disease as an endocytic trafficking defect (31). Firstly, the causative mutation NPC1-C113R represents a novel type of natural NPC1 transport defect with preferential localization of the protein in atypical endocytic compartments and on the cell surface. Secondly, NPC1-C113R substitution underscores the importance of the protein as a regulator of membrane trafficking. Malfunctioning of NPC1 due to this mutation apparently leads to the mistrafficking of NPC2. Furthermore, we also found the MPR to be partially redistributed from its predominant TGN localization towards late endocytic compartments in the patient cells (our unpublished data). Thus, it is conceivable that the NPC1-dependent disturbance in membrane trafficking results in defective recycling of the MPR from late endocytic organelles and this, in turn, promotes the stagnation of NPC2 in the lysosomes. The increased NPC2 levels observed in the patient cells may represent a compensatory upregulation of the protein.

Interestingly, MPR was found to be redistributed to Lamp-positive organelles in another NPC fibroblast line (32) but not in CHO cells phenocopying the NPC1 mutation (25). Cells lacking MPR or the enzyme generating the mannose-6-phosphate recognition signal develop late endosomal cholesterol accumulation (33,34), suggesting that functioning of the MPR pathway is needed for normal endocytic cholesterol transport. This could be attributed, at least in part, to the trafficking of the cholesterol-binding NPC2 protein. Whether lysosomal sequestration of NPC2 is associated with the development of the particular disease manifestations described here or represents a more widespread characteristic in the phenotypic spectrum of the disease remains to be determined. Notably, the majority of patients lacking NPC2 function develop a severe infantile form of the disease with early pulmonary involvement that was also characteristic to the case analyzed here (24,35).

In summary, we have here dissected the functional consequences of NPC1 mutations in a rapidly progressive infantile form of NPC disease. A prevalent NPC1 polymorphism and a novel NPC1 trafficking defect were characterized. In addition, a potential role for NPC1 as a regulator of NPC2 transport was uncovered. Future studies will be directed at understanding how the molecular pathology eventually culminates in the clinical manifestations of the NPC disease.

	SUBJECTS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION SUBJECTS AND METHODS REFERENCES

 
Clinical phenotype of the subject
The patient was the first child of the parents. At the age of 1 year, splenomegaly was noted and interstitial peribronchial attenuation found in a chest X-ray that was taken because of recurrent respiratory infections. At the age of 1.5 years he was investigated because of slow motor and intellectual development. He had not learned new skills since the age of 1. Computer tomography showed brain atrophy, the changes in the lung parenchyma were augmented and the liver and spleen were enlarged. Serum cholesterol level was low (2.14 mmol/l). At the age of 2 years he could not sit unsupported, he was ataxic and his lower extremities were spastic. Electromyography showed neuropathic changes. Bone marrow aspirate and liver and lung biopsies revealed large macrophages with a foamy cytoplasm, typical of Niemann–Pick disease. Electron microscopy revealed intralysosomal lamellar dark bodies. The diagnosis of NPC disease was confirmed from cultured skin fibroblasts by the laboratory of Dr Marie Vanier (University of Lyon, France), based on the staining of cellular free cholesterol with filipin and demonstration of impaired cholesterol esterification (34). The patient represents a severe infantile form of the disease (onset of first neurological symptoms at age <2 years), according to the classification by Vanier and Suzuki (2). A case report of the patient has been published in a Finnish medical journal (36).

Materials
Cell culture media, L-glutamine, antibiotics and filipin were from Sigma. Fetal bovine serum (FBS) was from Gibco, Lipofectamine2000 from Invitrogen Life Technologies, FuGENE6 from Roche, and Texas Red Transferrin and TRITC-Dextran from Molecular Probes. EZ-Link Sulfo-NHS-LC-Biotin and Immunopure Immobilized Streptavidin were from Pierce. Redivue Promix [³⁵S], [³H]-labeled dNTPs, [-³²P]dATP, ProteinA Sepharose CL-4B, Amplify and Hybond-N nylon membrane were from Amersham Pharmacia Biotech, X-OMAT film from Kodak and Endoglycosidase H (Endo H) from New England Biolabs. Gold particles, 5 and 10 nm, were from British BioCell International. RNeasy Protect Mini Kit was from Qiagen and Ultrahyb buffer from Ambion.

Antibodies
Rabbit polyclonal antibodies against NPC1 have been described (14). Rabbit polyclonal antibodies against NPC2 were produced by amplifying human NPC2 cDNA (amino acids Glu20-Leu151, signal sequence excluded) by PCR using the primers 5'-TGCGGGATCCGAACCGGTGCAGTTCAAGG-3' and 5'-TCGTGGATCCTTAGAGATGAGAAACGATCTG-3'. The resulting fragment was subcloned into the BamHI-site of the pGAT-4 expression vector (kindly provided by Johan Peränen, Institute of Biotechnology, University of Helsinki, Finland) and expressed as a His₆-GST fusion protein in Escherichia coli JM 109(DE3). The insoluble protein was purified from cell debris on preparative SDS–polyacrylamide gels (SDS–PAGE) and used for immunization of New Zealand White rabbits. Mouse monoclonal antibodies against human Lamp-1 were from Developmental Studies Hybridoma Bank, mouse monoclonal antibodies against EEA-1 from Transduction laboratories and mouse monoclonal antibodies against protein disulfide isomerase (PDI) were from Stressgen. Mouse IgM antibodies against 3'-LM1 have been described previously (37). Anti-rabbit and anti-mouse FITC- and TRITC-conjugated secondary antibodies were from Immunotech, and Alexa568- and Alexa488-conjugated secondary antibodies from Molecular Probes. Anti-goat TRITC-conjugated secondary antibodies were from Santa Cruz and IgG-HRP conjugates from Bio-Rad.

Cell culture
Control fibroblasts (F92-99) obtained as described (38) and fibroblasts (F92-116) from a skin biopsy of a Finnish NPC patient were cultured in Eagle's minimum essential medium (MEM) supplemented with 10% FBS, 2 mM L-glutamine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10 mM Hepes pH 7.4. CHO-K1 and CHO-K1 cells harboring a deletion in NPC1 locus (M12 cells) (26) were generously provided by Daniel S. Ory (Washington University School of Medicine, St Louis, MO, USA). CHO, COS-1 and HeLa cells were cultured in Dulbecco's MEM supplemented with 10% FBS, 2 mML-glutamine, 100 µg/ml penicillin, 100 µg/ml streptomycin, as well as non-essential amino acids for CHO cells.

Detection of mutations in genomic DNA
Genomic DNA was isolated from cultured fibroblasts or peripheral blood leukocytes. The DNA samples were screened for mutations in NPC1 using conformation sensitive gel electrophoresis (CSGE) as previously described (30). Briefly, primers flanking each exon were used to generate PCR products that were then separated by electrophoresis under mild denaturing conditions (39,40). For detection of any homozygous alterations, PCR products from test samples were mixed with PCR products from normal control prior to CSGE analysis. All band shifts identified by CSGE were further investigated by cycle sequencing of new PCR products.

DNA constructs and transient transfection
NPC1/pCR3.1 has been described previously (6). NPC1 mutation constructs NPC1-C113R/pCR3.1 ( point mutation C113R; nt 337 TC of NPC1 cDNA), NPC1-P237S/pCR3.1 (P237S; nt 709 CT) and NPC1-delC/pCR3.1 (deletion TTAC; nt 3611–3614) were created using Chameleon kit (Stratagene) following the manufacturer's protocol. Mutations were confirmed using cycle-sequencing kit (BIGDYE) and automated ABI377 sequencer (Applied Biosystems). GFP-wtRab7 was a generous gift from Angela Wandinger-Ness. CHO and HeLa cells were transfected using Lipofectamine2000 and COS-1 cells using FuGENE6 according to the manufacturer's instructions.

RNA isolation and northern blot analysis
Total cellular RNA was isolated from patient and control fibroblasts using the RNeasy Protect Mini Kit according to manufacturer's protocol. Equal amounts (20 µg) of total cellular RNA were size-fractionated on 1% formaldehyde–agarose gels, and transferred to Hybond-N nylon membrane. To control equal sample loading ethidium bromide staining was used. The probe was produced by amplifying human NPC2 cDNA by PCR using the primers 5'-GTGCAAGCTTGGATGCGTTTCCTGGCAGC-3' and 5'-CCGCTGATATCTTAGAGATGAGAAACGATCTG-3' and labeled with [-³²P]dATP (3000 Ci/mmol) using random primed DNA labeling kit. The membrane was hybridized overnight at 42°C in Ultrahyb buffer, washed three times with 1x SSC/0.1% SDS at 42°C for 30 min and once at 65°C for 30 min, and exposed to Kodak AR X-Omat film at -70°C with intensifying screens.

Immunofluorescence microscopy
Immunofluorescence microscopy was performed essentially as described (41). Briefly, cells fixed with 4% paraformaldehyde were incubated in 10% FBS supplemented with 0.05% filipin for 30 min at 37°C to permeabilize cells and visualize unesterified cholesterol. Alternatively, the cells were permeabilized with 0.01% Triton X-100 (for anti-PDI labeling). The coverslips were incubated with primary and secondary antibodies diluted in 5% FBS and viewed with a Zeiss Axiophot photomicroscope or Leica TCS SP confocal microscope. Complementation of M12 cells by the NPC1 constructs was assessed by filipin staining and scored into three categories as follows. Not complemented=no decrease in the filipin staining of NPC1-positive organelles; partially complemented=50–80% of NPC1-positive organelles lack filipin-positive storage material; fully complemented=>80% of NPC1-positive organelles lack filipin-positive storage material. For specific inhibition of NPC2 immunofluorescence, anti-NPC2 serum was preincubated with nitrocellulose-immobilized His₆-GST-NPC2 fusion protein, or BSA as a negative control (10 µg blotted from SDS–PAGE/100 µl antiserum diluted 1:400), for 6 h at room temperature, prior to immunostaining of cells.

Electron microscopy
For morphological analysis, cryofixation by means of slam-freezing, followed by freeze-substitution and Epon-embedding were performed essentially as described (42), except that the substitution media consisted of acetone supplemented with both 1% (w/v) OsO₄ and 0.5% (w/v) uranyl acetate, a prerequisite for good preservation of NPC multi-lamellar/-vesicular structures. For on-section immunolabeling, cells were fixed with 2% (w/v) formaldehyde, 0.2% (v/v) glutaraldehyde in 0.1 M sodium-phosphate buffer, pH 7.4, embedded in 10% gelatin, infiltrated with 2.3 M sucrose in phosphate buffer and frozen in liquid nitrogen. Ultrathin thawed cryosections were double immunolabeled using rabbit polyclonal antiserum against NPC1 (diluted 1:60) and mouse monoclonal antibodies against 3'LM1 (diluted 1:10), visualized by anti-rabbit IgG conjugated with 5 nm and anti-mouse IgG+IgM conjugated with 10 nm gold particles.

Surface biotinylation
Cells were washed with PBS+ ( phosphate buffered saline supplemented with Ca²⁺ and Mg²⁺) and incubated on ice with 1 mg/ml of Biotin for 30 min. Cells were blocked with 0.1 M glycine, 0.3% BSA in PBS+ twice for 5 min, and solubilized in lysis buffer (2% NP40, 0.2% SDS in PBS supplemented with 25 µg/ml of each chymostatin, leupeptin, antipain and pepstatin A). One-tenth of the volume was removed to represent the ‘total’ sample, which was TCA precipitated. The biotinylated proteins were precipitated with Streptavidin-agarose over night at 4°C, followed by washes at 4°C (once with lysis buffer, three times with 1% NP40 in PBS and twice in 0.1% NP40, 0.5 M NaCl in PBS). The precipitated complexes were incubated for 5 min in 50 mM Tris–HCl, pH 7.5, 25 mM DTT and washed with 50 mM Tris–HCl, pH 7.5. Total and biotinylated proteins were separated by SDS-PAGE and immunoblotted as in (14) using polyclonal anti-NPC1 antibodies.

Metabolic labeling, immunoprecipitation and glycosidase digestion
At 48 h post-transfection, COS-1 cells were incubated in Cys- and Met-free medium for 30 min at 37°C. Cells were then pulse-labeled for 30 min pulse by Redivue Promix [³⁵S] Cys/Met labeling mix (100–140 µCi/ml) in Cys- and Met-free medium containing 0.2% BSA, 10 mM Hepes pH 7.4, and chased for 4 h in medium containing 300 µg/ml of unlabeled Cys and Met and 20 µg/ml cycloheximide. The cells were washed on ice with PBS and lysed in immunoprecipitation buffer (50 mM Tris–HCl pH 7.4, 2 mM EDTA, 150 mM NaCl, 1% NP40, 0.4% NaDOC, 0.4% SDS) in the presence of protease inhibitors (25 µg/ml of each chymostatin, leupeptin, antipain, and pepstatin A). Anti-NPC1 antiserum was added, the samples were rotated overnight at 4°C, and after incubation with protein A-sepharose beads (for 2 h), washed five times with washing buffer (like immunoprecipitation buffer, except 0.8% SDS) and twice with 50 mM Tris–HCl, pH 7.4. The immunoprecipitated proteins were separated by SDS–PAGE, the gels were fixed, incubated in Amplify reagent, dried and exposed on X-OMAT film. For Endo H digestion, the samples were divided into two after the last immunoprecipitation washing step. Digestion was performed for one of the samples according to the manufacturer's instructions for 6 h with 1000 U of the enzyme and the nondigested and digested samples were analyzed by SDS–PAGE and autoradiography as above.

Transferrin and dextran uptake
To analyze the endocytic trafficking of dextran, HeLa cells were incubated at 3 days post-transfection for 30 min on ice in air medium (MEM containing 0.35 g/l NaHCO₃, 10 mM Hepes, pH 7.4, 5% FBS) to prevent adsorption of the dextran conjugate to the cell surface. The cells were then labeled for 1 h at 37°C with 5 mg/ml TRITC-Dextran in DMEM and chased for 17 h in medium containing 5% FBS. To study transferrin uptake, the transfected cells were incubated for 30 min at 37°C in serum-free medium supplemented with 0.2% BSA, 50 µg/ml Texas Red Transferrin.

Analysis of pooled DNA samples
Equal amounts of DNA from 150 female and 100 male blood donors from the Uppsala region were combined into two pooled DNA samples, respectively. The Finnish pooled DNA sample originated from a batch of pooled leukocytes from 180 blood donors from the Helsinki region (43). The NPC1 mutations were analyzed in the three pooled DNA samples by solid-phase minisequencing in a microtiter plate format using [³H]-labeled dNTPs ([³H]-dATP 69 Ci/mmol, [³H]-dCTP 57 Ci/mmol, [³H]-dGTP 33 Ci/mmol, [³H]-dTTP 127 Ci/mmol) as described previously (43). DNA from NPC-pedigree members of known genotype and control individuals were included in the analysis. The proportion of mutant sequence in the pooled DNA samples was determined by comparing the signal ratio obtained with the [³H]-labeled dNTPs corresponding to the mutant and normal alleles in a pooled sample to that in a heterozygous individual, in which the two alleles are present in equal amounts.

	ACKNOWLEDGEMENTS

 
We thank Liisa Arala, Marie Lindersson, Mervi Lindman, Birgitta Rantala and Arja Strandell for skillful technical assistance, Kurt von Figura for the anti-MPR antibody, Dan Ory for the M12 cells and Matti Lukka for help with paternity testing. This work was financially supported by the Ara Parseghian Medical Research Foundation (E.I. and K.S.), the Academy of Finland (grants 43184, 49987), and the Swedish Research Council (A.-C.S.). T.S.B. is a fellow of the Helsinki Graduate School in Biotechnology and Molecular biology and M.D.L. of the Helsinki Biomedical Graduate School.

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Department of Molecular Medicine, National Public Health Institute, Biomedicum Helsinki, Haartmaninkatu 8, 00251 Helsinki, Finland. Tel: +358 947448469; Fax: +358 947448960; Email: elina.ikonen{at}ktl.fi

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				N. H. Pipalia, A. Huang, H. Ralph, M. Rujoi, and F. R. Maxfield Automated microscopy screening for compounds that partially revert cholesterol accumulation in Niemann-Pick C cells J. Lipid Res., February 1, 2006; 47(2): 284 - 301. [Abstract] [Full Text] [PDF]

				G. Chinetti-Gbaguidi, E. Rigamonti, L. Helin, A. L. Mutka, M. Lepore, J. C. Fruchart, V. Clavey, E. Ikonen, S. Lestavel, and B. Staels Peroxisome proliferator-activated receptor {alpha} controls cellular cholesterol trafficking in macrophages J. Lipid Res., December 1, 2005; 46(12): 2717 - 2725. [Abstract] [Full Text] [PDF]

				M. Willenborg, C. K. Schmidt, P. Braun, J. Landgrebe, K. von Figura, P. Saftig, and E.-L. Eskelinen Mannose 6-phosphate receptors, Niemann-Pick C2 protein, and lysosomal cholesterol accumulation J. Lipid Res., December 1, 2005; 46(12): 2559 - 2569. [Abstract] [Full Text] [PDF]

				A. C. Berger, T. H. Vanderford, K. M. Gernert, J. W. Nichols, V. Faundez, and A. H. Corbett Saccharomyces cerevisiae Npc2p Is a Functionally Conserved Homologue of the Human Niemann-Pick Disease Type C 2 Protein, hNPC2 Eukaryot. Cell, November 1, 2005; 4(11): 1851 - 1862. [Abstract] [Full Text] [PDF]

				E. Rigamonti, L. Helin, S. Lestavel, A.L. Mutka, M. Lepore, C. Fontaine, M.A. Bouhlel, S. Bultel, J.C. Fruchart, E. Ikonen, et al. Liver X Receptor Activation Controls Intracellular Cholesterol Trafficking and Esterification in Human Macrophages Circ. Res., September 30, 2005; 97(7): 682 - 689. [Abstract] [Full Text] [PDF]

				A.-L. Mutka, S. Lusa, M. D. Linder, E. Jokitalo, O. Kopra, M. Jauhiainen, and E. Ikonen Secretion of Sterols and the NPC2 Protein from Primary Astrocytes J. Biol. Chem., November 19, 2004; 279(47): 48654 - 48662. [Abstract] [Full Text] [PDF]

				E. B. Neufeld, J. A. Stonik, S. J. Demosky Jr., C. L. Knapper, C. A. Combs, A. Cooney, M. Comly, N. Dwyer, J. Blanchette-Mackie, A. T. Remaley, et al. The ABCA1 Transporter Modulates Late Endocytic Trafficking: INSIGHTS FROM THE CORRECTION OF THE GENETIC DEFECT IN TANGIER DISEASE J. Biol. Chem., April 9, 2004; 279(15): 15571 - 15578. [Abstract] [Full Text] [PDF]

				R. A. Nixon Niemann-Pick Type C Disease and Alzheimer's Disease: The APP-Endosome Connection Fattens Up Am. J. Pathol., March 1, 2004; 164(3): 757 - 761. [Abstract] [Full Text] [PDF]

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