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Functional and Structural Biology on the Lipo-network, 2006: 1-15 ISBN: 81-7895-232-7 Editors: Kosuke Morikawa and Shin-ichi Tate

1 Intracellular cholesterol transport by NPC1/NPC2: Mysteries of Niemann-Pick disease type C

Haruaki Ninomiya Department of Neurobiology, Tottori University Faculty of Medicine Yonago 683-8503, Japan

Abstract Niemann-Pick disease type C (NPC) is an inherited lipid storage disorder caused by mutations in NPC1 or NPC2. Loss of function of either gene causes progressive and fatal neurodegeneration, typically in children. The brain pathology of NPC is characterized by accumulation of cholesterol andother lipids, progressive loss of neurons, especially that of cerebellar Purkinje cells, and robust glial infiltration. At the cellular level, the most prominent

Correspondence/Reprint request: Dr. Haruaki Ninomiya, Department of Neurobiology, Tottori University Faculty of Medicine, Yonago 683-8503, Japan. E-mail: [email protected]

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phenotype of NPC1/NPC2 deficiency is accumulation of free cholesterol in the endosomes and lysosomes. NPC1 is a polytopic glycoprotein that contains a sterol-sensing domain whereas NPC2 is a soluble luminal protein that contains an MD-2-like lipid-recognition domain. Based on the phenotype caused by their loss of function, it is apparent that these two proteins facilitate export of cholesterol from the endosomal system. It remains a mystery, however, how they do so. There remains another challenging mystery: why do neurons die when their functions are impaired? An important message of recent studies using mouse and drosophila models is that the principal problem with NPC is not cholesterol storage, but its shortage.

Introduction Niemann-Pick disease type C (NPC) is an autosomal recessive lipid storage disorder characterized by endosomal accumulation of low-density lipoprotein (LDL)-derived cholesterol [1]. It is a rare disease with a prevalence of 1/120,000 newborns. Typically, it is a childhood disease that manifests in early infancy and results in death within the first decade of life. However, clinical onsets and courses are variable and it is classified to infantile, juvenile and adult forms depending on the time of onset. The patients develop both neurological symptoms including cerebellar ataxia, cataplexy and ophthalmoplegia, and visceral symptoms such as hepatosplenomegaly. Their brain pathology is characterized by accumulation of cholesterol and other lipids, progressive loss of neurons, especially that of Purkinje cells, and robust glial infiltration. Cell fusion studies using primary-cultured patients’ skin fibroblasts revealed the presence of two genetic complementation groups, designated as NPC1 and NPC2. Approximately 95% of NPC patients belong to NPC1 and the remaining 5% belong to NPC2. Both clinical and biochemical phenotypes are indistinguishable between these two groups. On 1997, the disease-causing gene of NPC1 was identified by linkage analysis and complementation studies [2]. Since it was a novel gene without any known function, it was given the name NPC1. Three years later, HE1 (human epididymis 1) was identified as the gene responsible for NPC2 [3]. Neither NPC1 nor NPC2 is an enzyme involved in sphingolipid metabolism. Therefore, NPC is a disease entity distinct from Niemann-Pick disease type A and B, which are caused by deficiency of sphingomyelinase. Based on the phenotype of their loss of function, it is obvious that these two proteins play key roles in intracellular cholesterol transport. Since their identification, intensive research efforts have been made to clarify their physiological functions. Fundamental questions, however, remain to be answered. What are the roles of NPC1 and NPC2 in endosomal lipid flow? How do they co-operate? Why do neurons die when their functions are impaired?

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This review summarizes what we have learned in recent years and present current hypotheses that are being addressed to answer these questions. The first section provides a list of experimental animals and cells used for the studies of the disease.

1. Model animals and cells 1.1 Mouse There are two mouse strains that carry loss-of-function mutations of NPC1: a C57/BL strain spm (Fig. 1) and a Balb/C strain npcNIH [4]. In both strains, the homozygous NPC1-deficient mice develop symptoms similar to those of human patients and die within 3 months of age. Their progressive and

Figure 1. Pathology of NPC1-deficient mice. Mice were 6-week old in a-c. a. C57BL spm mice. The homozygous mice are small because of retarded growth. b. Cholesterol accumulation in cerebral neurons (filipin staining). c. Cerebellar atrophy of homozygous mice (HE). d. Progressive loss of Purkinje cells in homozygous mice (calbindin immunostaining). Bar: 0.1 mm.

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severe phenotype corresponds to the infantile form of the human disease. The retroposon insertion site is known for Balb/C npcNIH whereas the genomic mutation is left unknown for C57/BL spm. Therefore, Balb/C npcNIH has been used in most of the studies. Like humans, their brain pathology is characterized by lipid accumulation, Purkinje cell loss and glial proliferation. Transgenic expression of NPC1 in neuronal and glial cells (driven by a prion gene promoter) abolished the symptoms and normalized the life span of NPC1-deficient mice [5], indicating that the primary cause of death of these mice is neurodegeneration. NPC2-deficient mice were generated by gene targeting and have been shown to have the same phenotype with NPC1-deficient mice. These two lines of mice were intercrossed to generate double deficient mice that lack both NPC1 and NPC2. The phenotype of the double deficient mice was indistinguishable from NPC1 or NPC2 single deficient mice, indicating non-redundant roles of the two genes in the same lipid transport pathway [6].

1.2 Drosophila There are two NPC1 orthologs in the flute fly genome, named dnpc1a and dnpc1b. Dnpc1a is highly expressed in the ring gland, an organ that produces a steroid hormone, ecdysone. This steroid hormone is required for molting of larva. Dnpc1a-deficient animals had intracellular accumulation of cholesterol throughout their body as evidenced by filipin staining, could not undergo molting and die at the first trimester of the larval stage. Ring gland-specific expression of dnpc1a rescued these animals. Also effective were exogenous supply of ecdysone, or its precursors cholesterol or 7-hydroxy-cholesterol [7,8]. These findings argue that the most serious problem caused by dnpc1a-deficiency is impaired steroidgenesis. This is consistent with the effects of allopregnanolone in NPC1-deficient mice (see below), providing evidence for the notion that the principal problem of NPC is not lipid storage, but its shortage.

1.3 Mammalian cells lines We and others have established CHO cell lines that lack NPC1 [9]. Also available are skin fibroblasts from patients with mutations in NPC1 or NPC2. The intracellular accumulation of cholesterol and associated biochemical abnormalities have been best characterized in these cell lines.

1.4 Yeasts Ncr1 is the yeast homolog of NPC1. When expressed in cells from patients with NPC1 disease, this protein could correct the cellular phenotype, indicating the conserved function of this family of proteins [10]. Ncr1 in yeast cells resides on the limiting membrane of the vacuole and this targeting depends on


Vps proteins (vacuolar protein sorting proteins) that form the ESCRT complex (endosomal sorting complex required for transport) on the endosomal membrane [11]. Ncr1-null yeast cells lacked any apparent phenotype [10], but recently, it was found that these cells acquired resistance to the ether lipid drug, edelfosine, suggesting that the lipid transport was also affected in these cells [12]. The yeast homolog of NPC2 was identified and has been shown to be able to correct the cellular phenotype of cells from patients with NPC2 disease [13].

2. Impaired lipid trafficking in NPC cells In the following sections, NPC1-deficient cells and NPC2-deficient cells are collectively described as NPC cells. The most prominent phenotype of NPC cells is accumulation of free cholesterol in their abnormal endosomes. There are two sources of cholesterol to cells: one is exogenous supply in the form of LDL, and the other is endogenous synthesis. Esterified cholesterol contained in LDL is hydrolyzed in the endosomes/lysosomes to generate free cholesterol, which is then transported from the late endosomes to other cellular sites, such as the plasma membrane and the endoplasmic reticulum. This process of cholesterol export is retarded in NPC cells [14]. Impairment of the transport results in the formation of aberrant endosomes that contain multiple multivesicular bodies (Fig. 2). The aberrant endosomes contain marker molecules of both late endosomes and lysosomes, suggesting they are the hybrid products [15]. Besides this cholesterol accumulation, NPC cells are characterized by their delayed responses to LDL. Experimentally, this phenotype is most easily seen when cells were once depleted of cholesterol and then loaded with LDL. When cells were depleted of cholesterol, they adapt to the situation by increasing the protein levels of LDL receptor and HMG-CoAR, (3-hydroxy-3-methylglutaryl CoA reductase), the key players in LDL uptake and cholesterol synthesis, respectively. LDL loading results in prompt down-regulation of these molecules. This down-regulation is delayed in NPC cells and, as expected, disappearance of nuclear SREBP (sterol-response element binding protein) is also delayed [9]. NPC cells and tissues accumulate various lipids other than cholesterol [16,17]. Especially in the brain, levels of gangliosides such as GM2 and GM3 are markedly increased whereas levels of cholesterol are barely increased [18]. There is a long lasting controversy whether this glycolipids accumulation is a primary event or secondary to cholesterol accumulation. Regardless of the biochemical mechanism, NPC is classified to sphingolipidosis because of this glycolipids accumulation. However, this classification is misleading because NPC is not caused by a deficiency of any enzyme involved in sphingolipid metabolism. Pathologically, NPC is classified to lysosomal storage diseases.

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Figure 2. Intracellular accumulation of cholesterol in NPC cells. a. Schematic representation of the flow of LDL-derived cholesterol. Retardation of the transport out of the endosomes results in formation of multivesicular bodies in NPC cells. Endogenous cholesterol synthesis (shown in grey arrows) is up-regulated. b. Enlarged macrophages in the bone marrow of NPC patients, designated as sea blue histiocytes or Niemann-Pick cells. c. Transmission electron microscopy of multivesicular bodies in NPC1-deficient CHO cells.

This classification is also misleading because the main biochemical defect resides in the late endosomes, but not in the lysosome.

3. Structure and function of NPC1 3.1 Primary and secondary structures The cDNA for human NPC1 is about 4.9 kb long and encodes 1,278 amino acids. Expression studies using FLAG-tagged proteins revealed a polytopic protein with 13 transmembrane domains [19] (Fig. 3). The N-terminus is on the luminal side and the C-terminus is on the cytosolic side. There is a signal peptide on the N-terminus and a di-leucine motif on the C-terminus. The


sterol-sensing domain (SSD) spans transmembrane domains 3 to 8. The amino acid sequence on the N-terminal loop (a.a.55-165) is highly conserved between species and is called the NPC1 domain. This domain contains a leucine zipper motif. The tertiary structure is not known, but it is predicted that it forms an oligomer.

Figure 3. Secondary structure of NPC1 (a) and its intracellular localization (b). See text for details.

3.2 Genomic structure and regulation of gene expression The human NPC1 gene is encoded on 18q11. It is composed of 24 exons and is 45kb long. There is no TATA box on the 5’ promoter region. Instead, there is a GC rich sequence which is characteristic for a house keeping gene. There are various response elements in the promoter region but a sterol-response element is absent [20]. We have shown that NPC1 mRNA levels are increased in fibroblasts from NPC patients [21]. This finding suggested up-regulation of NPC1 gene transcription in cells with impaired NPC1 function. The signaling mechanism for this up-regulation, however, is left unknown.

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3.3 Tissue distribution NPC1 is a house keeping gene and is ubiquitously expressed. In the brain, it is expressed by both neuronal and glial cells [22,23].

3.4 Intracellular localization NPC1 primarily resides in the late endosome. This localization was revealed by double staining with various endosomal marker proteins [24]. Real-time visualization of NPC1-GFP fusion protein showed dynamic intracellular movement of this protein [25,26]. It moves on tubulovesicular transport vesicles that shuttle between perinuclear endosomes and the cell periphery. The tubulovesicular vesicles appear to undergo relentless fission and fusion.

3.5 Posttranslational modifications and the degradation mechanism It is heavily glycosylated and migrates between 170 and 200 kDa on SDS polyacrylamide gel electrophoresis. The size of the protein differs among the cell lines. There is no evidence for other modifications including ubiquitination, phosphorylation or modification with lipids. The degradation mechanism of this protein is left unknown. An important observation is decreased levels of mutant NPC1 proteins in patients’ cells on Western blotting [27]. These cells expressed rather increased levels of its mRNAs as revealed Northern blotting [21], indicating that the decrease was due to a post-transcriptional event. Cells from patients with juvenile/adult forms retained relatively higher levels of the protein, suggesting a potential correlation between the levels of the protein and the severity of the disease [27]. Therefore, it is tempting to hypothesize accelerated degradation of mutant NPC1 proteins, which may be the major biochemical basis for the disease. This hypothesis also implicates a possibility of a therapeutic approach aiming at stabilization of the mutant proteins.

3.6 Protein function The molecular function of this protein remains a mystery. It has been shown that NPC1 possesses a lipid permease activity when expressed in bacterial cells [28,29] and that this protein can bind cholesterol [30], it remains to be demonstrated that this protein transports cholesterol molecules between different compartments in mammalian cells. It also remains to be clarified whether NPC1 function is regulated by cellular cholesterol, although the functional significance of its SSD has been well documented [31,32]. In addition to NPC1, SSDs are found in other proteins involved in the control of cellular cholesterol homeostasis: SREBP cleavage-activating protein (SCAP) and HMG-CoAR, both of which reside primarily in the endoplasmic reticulum (ER) (Fig. 3). SCAP associates with the ER-retention proteins, Insig-1 and Insig-2, in a sterol-dependent manner: when cells are depleted of cholesterol,


SCAP, together with SREBP, is released from the Insig proteins and transported to the Golgi apparatus, where a mature, active form of SREBP is generated by proteolysis and stimulates transcription of a diverse set of genes involved in cholesterol uptake and synthesis [33,34]. Similarly, HMG-CoAR associates with the Insig proteins in a sterol-dependent manner, but in this case, the association facilitates its ubiquitination and subsequent degradation by the proteasome, thus shutting off endogenous cholesterol biosynthesis [35,36,37]. By analogy, one may hypothesize a cholesterol-dependent regulation of NPC1 function, either by its association with other proteins or post-translational modifications. 4. Structure and function of NPC2 Dr. Lobel and colleagues, in the process of screening lysosomal proteins modified with mannose-6-phosphate (M6P), identified human epididymis-1 (HE-1) and revealed the absence of this protein in skin fibroblasts from patients with NPC2 disease [3]. Analysis of their genome revealed various mutations in this gene, indicating that HE-1 was the disease-causing gene of NPC2. HE-1-deficient mice generated by gene targeting showed a phenotype quite similar to that of NPC1-deficient mice, confirming that HE-1 was the NPC2 gene. HE-1, as it name implies, has been known as a cholesterol-binding protein enriched in the epididymis. It is now referred to as NPC2. 4.1 Protein structure Human NPC2 is encoded on 14q24. The cDNA for human NPC2 is about 0.6 kb long and encodes 151 amino acids (Fig. 4). It has a signal peptide on the N-terminus, indicating that it is a luminal protein. Three arginine residues are glycosylated. Glycosylation of N39, that contains M6P, has been shown to be critical for its function [38]. There are three pairs of cysteine residues predicted to form three intramolecular disulfide bonds. The entire amino acid sequence corresponds to an MD-2-like lipid recognition domain (ML domain) [39]. X-ray chrystarography of a protein purified from bovine milk revealed the presence of six beta sheet structures. The protein was predicted to form a hydrophobic pocket that could accept cholesterol molecule [40], consistent with the cholesterol binding capacity of this protein demonstrated by biochemical studies [41]. 4.2 Tissue distribution and intracellular localization Like NPC1, NPC2 is ubiquitously expressed. It is a secreted protein contained in milk and sperm, and most likely in other body fluids. When expressed in cultured cells, it is secreted to the culture medium and is also localized in the endosomal lumen. The protein can reach the endosome/lysosome

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Figure 4. Primary structure of NPC2. Secondary structure is depicted in Fig. 3b.

from the trans-Golgi network via the M6P-dependent pathway to the endosome/ lysosome. The secreted protein can also reach these compartments presumably by binding to cell-surface M6P receptors [41]. 4.3 Protein function NPC2 can be purified from the conditioned medium of CHO cells stably expressing this protein. In vitro binding assays using the purified protein confirmed the cholesterol binding capacity of NPC2 with a Kd value in a nanomolar range. Mutant proteins that lacked the binding capacity failed to restore cholesterol flow in cells with NPC2 mutations, indicating that the cholesterol binding capacity was critical for its function [41]. It remains to be clarified how this cholesterol-binding protein takes part in the cholesterol efflux from the endosomal system. One may postulate its possible function based on what we know about the functions of ML domain proteins. The ML domain, an amino acid sequence of about 150 amino acids that contains two pairs of cysteine residues, is shared by four kinds of mammalian proteins, MD-1, MD-2, GM2 activator and NPC2 [39]. GM2 activator is a lysosomal protein required for beta-hexosaminidase A to hydrolyze ganglioside GM2 and its deficiency causes Tay-Sachs disease AB variant. It can bind GM2 and is supposed to extract membrane-embedded GM2 so as to make it available to the enzyme. MD-2 is a secreted protein that is required for bacterial lipopolysaccharide (LPS) to activate Toll-like receptor 4. It can bind both LPS and Toll-like receptor 4 and thus works as a bridge between the two molecules. MD-1 is supposed to have a similar function to link LPS and RP105 protein expressed on B cells. By analogy, one may imagine that NPC2 binds and extracts membrane-embedded cholesterol and makes it available to NPC1. Experimental evidence for this transfer model is yet to be demonstrated.

5. Biochemical basis for neurodegeneration: Is NPC a disease of cholesterol shortage? Although it is apparent that accumulation of cholesterol and other lipids is the most prominent feature of NPC cells and tissues and that this phenotype is


a direct consequence of the loss of function of NPC1/NPC2, it remains unknown whether lipid storage can explain other pathological changes in NPC brain including neuronal loss and glial infiltration. The main symptoms of NPC patients and mice are neurological complications and they are the cause of death. In an attempt to examine the cause-effect relationship of glycolipids accumulation and neuronal death, double knockout mice that lacked NPC1 and GalNAcT, the gene encoding the beta-1-4GalNAc transferase, were generated. Deletion of this synthetic enzyme markedly attenuated glycolipids accumulation in the brain but failed to ameliorate neuronal loss or symptoms [42]. Similarly, genetic deletion of LDL receptor in NPC mice reduced cholesterol storage in visceral organs but did not affect the disease progression [43]. A surprising and encouraging finding was that administration of a neurosteroid allopregnanolone to NPC mice ameliorated neuronal loss and prolonged their life span [44]. This reduction in neuronal cell loss was accompanied by amelioration of glial cell proliferation [45]. It has been known that neurons express a series of enzymes that are required for the synthesis of various steroid derivatives. The positive effect of the neurosteroid suggested that the principal problem with NPC neurons is not lipid storage but its shortage, a notion also suggested by the studies using drosophila. However, the functions of neurosteroids in the brain remain mysterious and it is far from clear how they help neuronal cells to survive. Future studies aiming at the metabolic pathways for neurosteroids and their physiological functions may, hopefully, yield a better treatment of NPC. Another recent progress is generation of chimeric mice that have both wild-type and NPC1-deficient Purkinje cells. Studies using these mice showed survival of the wild-type cells surrounded by inflammatory cells [46]. This finding excluded the possibility that any cytokines or toxic substances secreted by inflammatory cells are a primary cause of neuronal death, but it remains possible that such a factor(s) accelerates degeneration of NPC1-deficient neurons.

6. Is NPC childhood Alzheimer’s disease? Although NPC is a very rare disease, it has attracted attention from researchers in a broad range of fields. In the fields of basic researches, this is because the protein products of the two disease-causing genes play key roles in intracellular lipids trafficking. In the fields of clinical researches, this is because there appear to be several pathophysiological aspects shared by NPC and more common diseases, that may be caused by an imbalance of cholesterol metabolism, including Alzheimer’s disease (AD). NPC is clearly different from AD in two points. First, it is typically a childhood disease. Second, the main symptom of infantile patients is cerebellar ataxia, due to loss of Purkinje cells. However, it is also true that clinical

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manifestations of NPC can be highly variable. Some patients in school ages or older show learning difficulties or intellectual disturbances, symptoms similar to those of AD [1]. Besides, it has been known that brains of NPC patients contain neurofibrillary tangles (NFTs), the morphology of which was indistinguishable from those seen in AD brains [47]. Since it has become recognized that abnormal metabolism of cholesterol is a risk factor of AD, several lines of evidence accumulated that suggested a link between NPC and AD. First, intracellular localization of both amyloid beta-protein precursor and presenilin was altered in NPC cells, leading to increased production of amyloid beta-protein, which accumulated in the late endosomes [48, 49]. Second, MAPKs are constitutively activated in NPC cells and brains, resulting in aberrant phosphorylation of tau [50]. Third, amyloid beta-protein disposition was observed in some of the brains of NPC patients who lived over 30 years, and those who had such pathology were homozygous for apolipoprotein E epsilon 4 allele [51]. Based on these findings, one may postulate a following scenario: the impairment of cholesterol trafficking can not only be a primary cause of NFT formation, but also be a risk factor for senile plaque formation, which manifests in the setting of a specific apolipoprotein E genotype or by aging. 7. Perspective It will soon be a decade since the identification of NPC1, the gene product responsible for NPC. The physiological functions of NPC1/NPC2 at the molecular level remain elusive and further studies are warranted. As for the pathogenesis of this disease, recent progress gave rise to an important concept that the principal problem of NPC is not lipid storage, but its shortage. Future studies will be aimed at consolidating this concept, and may generate a successful treatment of this disease.

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