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FEBS Letters 580 (2006) 5518–5524

Minireview imbalance in the neurological disorder, Niemann-Pick C disease

Jean E. Vance* Canadian Institutes for Health Research Group on the Molecular and Biology of , Department of Medicine, 332 HMRC, University of Alberta, Edmonton, Alta., Canada T6G 2S2

Received 11 May 2006; revised 29 May 2006; accepted 1 June 2006

Available online 15 June 2006

Edited by Gerrit van Meer

of [5,6] and other lipids, including bis-monoacyl- Abstract Niemann-Pick C (NPC) disease is a progressive neu- rological disorder in which cholesterol, gangliosides and bis-mon- glycerol phosphate [7] and the gangliosides GM2 and GM3 oacylglycerol phosphate accumulate in late endosomes/ [8–10]. lysosomes. This disease is caused by mutations in either the The brain is the most cholesterol-rich organ in the body: NPC1 or NPC2 gene. NPC1 and NPC2 are involved in egress approximately 25% of the body’s cholesterol is located in the of lipids, particularly cholesterol, from late endosomes/lysosomes brain whereas the brain comprises only 5% of body mass. but the precise functions of these proteins are not clear. An In most tissues, cholesterol is acquired either from endogenous important question regarding the function of NPC proteins is: synthesis or from receptor-mediated endocytosis of plasma why do mutations in these ubiquitously expressed proteins have lipoproteins, particularly low density lipoproteins (LDLs). such dire consequences in the brain? This review summarizes However, cholesterol metabolism in the central nervous system the roles of NPC proteins in lipid homeostasis particularly in (CNS) is distinct from that in peripheral tissues because plas- the central nervous system. Ó 2006 Federation of European Biochemical Societies. Published ma lipoproteins are unable to cross the blood–brain barrier. by Elsevier B.V. All rights reserved. Therefore, all cholesterol in the brain is synthesized within the CNS [11]. The majority (70–80%) of cholesterol in the Keywords: Cholesterol; ; Gangliosides; brain of adult animals resides in myelin. The highest rate of Late endosomes; Lysosomes cholesterol synthesis in the brain occurs during the period of active myelination after which cholesterol synthesis declines to a low level that is thought to reflect primarily that occurring in and astrocytes [12]. The half-life of cholesterol in the brain is 4–6 months. In the face of ongoing synthesis of 1. Niemann-Pick C disease is a progressive neurological disorder cholesterol in the brain, cholesterol homeostasis is maintained by the export of excess cholesterol in the form of 24-hydroxy- Niemann-Pick type C (NPC) disease is an autosomal reces- cholesterol from the brain into the plasma. This derivative of sive lipidosis that is characterized by progressive neurodegen- cholesterol is produced by the action of cholesterol 24-hydrox- eration [1]. This disorder is caused by a mutation in either ylase, an that is expressed only in a specific subset of the NPC1 gene (in 95% of the cases) or the NPC2 gene (in neurons [13]. Interestingly, in NPC disease cholesterol accumu- 5% of the cases). NPC disease is biochemically distinct from lates in all tissues except the brain. Indeed, in NPC-deficient Niemann-Pick disease types A and B which are caused by ge- brains the amount of cholesterol even decreases with increasing netic defects in lysosomal sphingomyelinase activity [2]. The age [14]. The reason that cholesterol does not accumulate in clinical manifestations of NPC disease (vertical gaze palsy, NPC-deficient brains is probably because extensive demyelina- ataxia, dystonia and seizures) usually become evident in early tion occurs in this disease [15]. Since myelin is the major source childhood and death typically occurs in the teenage years. of cholesterol in the brain, the progressive loss of myelin would Many NPC infants also experience neonatal jaundice and be expected to mask any age-related increase in the cholesterol enlargement of the liver and/or spleen and a few of these indi- content of neurons and astrocytes [16]. viduals die from acute liver failure. Currently, no effective treatment is available for NPC disease. Although NPC disease is relatively rare (1 in 150000 live births in the general pop- 2. Two NPC genes in mammalian cells ulation), the incidence in Yarmouth County, Nova Scotia is 1% with an estimated carrier frequency of 10–25% [3]. The The human NPC1 gene was identified and cloned in 1997 most characteristic histological features of the brains of indi- [17]. The NPC1 protein is an integral membrane protein that viduals with NPC disease are neuronal loss, particularly of is primarily localized to late endosomes/lysosomes but also cy- Purkinje cells in the cerebellum, and the widespread appear- cles through the Golgi apparatus [18,19]. This protein contains ance of swollen neurites [4]. The biochemical phenotype of 1278 amino acids and 13 putative transmembrane domains NPC-deficient cells and tissues is the lysosomal accumulation that are separated by lumenal glycosylated loops (Fig. 1). NPC1 also contains a cysteine-rich loop [20], a di-leucine lyso- somal targeting motif and a leucine zipper motif, the latter suggesting that NPC1 interacts with other proteins [21]. One *Fax: +1 780 492 3383. E-mail address: [email protected] candidate as a NPC1-interacting protein was MLN64, a

0014-5793/$32.00 Ó 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.06.008 J.E. Vance / FEBS Letters 580 (2006) 5518–5524 5519

Fig. 1. Topological model of NPC1 protein. NPC1 is a 1278-amino acid transmembrane protein that is present in late endosomal/lysosomal (LE/lys) membranes. The lumenal N-terminus of the protein contains a leucine zipper motif whereas the C-terminus is cytosolic and contains a lysosomal targeting sequence. The protein is predicted to contain 13 transmembrane domains, five of which (red bars enclosed in red box) comprise a sterol- sensing motif. protein that resides in late endosomes/lysosomes and contains the steroidal analog GW707 [43]. These compounds mimic a START domain that has been identified in some lipid trans- NPC1 deficiency in many respects including the sequestration port proteins [22]. However, mice with a targeted mutation in of cholesterol in late endosomes/lysosomes. However, not all MLN64 are healthy and display only minor problems in sterol of the biochemical effects of NPC1 deficiency are recapitulated trafficking [23], suggesting that MLN64 is not an obligatory by these agents [44]. For example, in addition to causing cho- component of the NPC1 pathway. An important feature of lesterol sequestration in late endosomes/lysosomes, U18666A the NPC1 protein is the five transmembrane domains that also inhibits cholesterol biosynthesis at the level of squalene comprise a sterol-sensing domain (Fig. 1) [17,24]. This motif epoxidase [45]. has been identified in other proteins that are involved in cholesterol homeostasis, such as the sterol regulatory ele- ment-binding protein cleavage activating protein (SCAP) and 3. NPC proteins are required for the egress of cholesterol from 3-hydroxy-3-methylglutaryl-Co-A reductase. late endosomes/lysosomes The NPC2 gene (also called HE1) was identified in a study designed to characterize the lysosomal proteome [25]. The The accumulation of large amounts of unesterified choles- 151 amino acid NPC2 protein is a ubiquitously expressed, sol- terol in late endosomes/lysosomes of NPC1-deficient fibro- uble protein that is present in the lumen of lysosomes. This blasts that have been loaded with LDL-cholesterol implies cholesterol-binding protein had previously been identified as that NPC1 is involved in the movement of LDL-derived, une- a major secretory component of epididymal fluid [26]. The sterified cholesterol out of the endosomal pathway [5,6]. This crystal structure of NPC2 has been solved and reveals a hydro- idea is supported by the finding that the NPC1 protein con- phobic binding pocket that has the capacity to bind cholesterol tains a sterol-sensing motif [17]. The intracellular itinerary of [27,28]. The disease progression in patients with genetic defects LDL-derived cholesterol transport, and the proposed site of in NPC2 appears to parallel that of individuals who are defi- action of NPC1 in this pathway, are illustrated in Fig. 2. cient in NPC1 [29,30]. Although the properties of NPC1 and Exogenously-supplied LDLs are endocytosed by the LDL NPC2 have been extensively investigated, the precise function receptor that is concentrated within clathrin-coated pits on of neither of these proteins has yet been defined. the cell surface. Upon reaching the late endosomes/lysosomes, Investigation of the function of NPC1 and NPC2 has been cholesteryl esters within the core of the LDL particles are greatly aided by the development of cell and animal models hydrolyzed to unesterified cholesterol. Subsequently, in an of NPC deficiency. These model systems include mutants of NPC1-dependent pathway, the released cholesterol leaves the yeast [31] and Drosophila [32], skin fibroblasts from NPC pa- late endosomes/lysosomes and is distributed to other mem- tients [5,6], and mutants of Chinese hamster ovary cells defi- branes including the plasma membrane and the endoplasmic cient in NPC1 [33,34]. Two murine models of NPC disease, reticulum. that have null mutations in the Npc1 gene, are widely used Available data are consistent with the idea that NPC1 and [35,36]. These mice are asymptomatic at birth but progressively NPC2 participate together in mediating the egress of choles- develop neurological problems typical of those in human NPC terol from the late-endosomes/lysosomes although the precise disease. These mice usually die by the age of 10–15 weeks. A mechanism by which these proteins act remains unknown. feline model with a spontaneous mutation in NPC1 has also Based on known motifs in the protein, and from functional been used for some studies [37]. More recently, a hypomorphic studies on mutant forms of NPC1, several roles have been pro- mouse expressing only 0–4% of residual NPC2 protein was posed for NPC1 [46–49] including: a cholesterol ‘‘flippase’’, a generated. The progression of neurological symptoms and fatty acid permease, a ganglioside transporter, a cholesterol the lipid accumulation in Npc1À/À mice and Npc2-hypomor- sensor that monitors the level of cholesterol in the lysosomal phic mice, as well as in double mutants deficient in both membrane and promotes the release of cholesterol from the NPC proteins, are essentially identical suggesting that NPC1 membrane to an acceptor carrier protein, and a member of a and NPC2 participate in the same lipid transport pathway. multi-protein complex that mediates cholesterol movement Other useful tools for studying the phenotype of NPC-defi- out of lysosomal membranes. Some recent studies have sug- ciency in cultured cells are the amphiphilic amines U18666A gested that NPC2 might function directly as a cholesterol and imipramine [38–40], as well as progesterone [41,42] and transport protein [50]. NPC2 is a soluble intra-lysosomal 5520 J.E. Vance / FEBS Letters 580 (2006) 5518–5524

4. NPC1 deficiency leads to defects in vesicle trafficking

Loss of NPC1 function appears to have profound effects on multiple vesicle transport processes. For example, NPC1 defi- ciency results in the formation of multilamellar vesicles that are enriched in cholesterol, glycosphingolipids and bis-mono- acylglycerol phosphate [7]. In addition to the formation of lipid-laden endosomes, NPC1-deficient fibroblasts exhibit reduced mobility of endosomal tubulovesicular structures [54]. The trafficking of mannose-6-phosphate receptors is also impaired in NPC1-deficient cells [7]. In a recent study, the ef- fect of cholesterol sequestration in late endosomes/lysosomes on Rab9-dependent export of mannose-6-phosphate receptors Fig. 2. NPC1 is required for the egress of low density lipoprotein was investigated [55]. NPC1 deficiency was found to increase (LDL)-derived cholesterol from late endosomes/lysosomes. (1) LDL is the level of Rab9 by reducing Rab9 degradation. The authors (2) endocytosed by LDL receptors that are clustered in coated pits on concluded that cholesterol enrichment of late endosomal/lyso- the cell surface. (3) In late endosomes/lysosomes (LE/lys) cholesteryl somal membranes disrupts the trafficking of mannose-6-phos- esters in LDLs are hydrolyzed to unesterified cholesterol which is phate receptors and causes the accumulation of Rab9 on these distributed throughout the cell (4), particularly to the plasma mem- brane via an unknown mechanism that requires NPC1. (5) Cholesterol membranes. Since Rabs play important roles in many aspects from the late endosomes/lysosomes is also transported to the endo- of membrane trafficking, these observations indicate that the plasmic reticulum (ER) where an increased cholesterol content results proper targeting of a variety of proteins is likely to be impaired in decreased synthesis of cholesterol and LDL receptors, and increased in NPC deficient cells. Another indication that the normal cholesterol esterification. In addition, cholesterol that is synthesized in the ER is transported to the plasma membrane (6). In NPC1-deficient function of Rabs is altered by NPC1 deficiency is the observa- cells, unesterified cholesterol becomes sequestered in late endosomes/ tion that overexpression of Rab7 or Rab9 in NPC1-deficient lysosomes so that the cholesterol content of the plasma membrane is cells reduces the cholesterol accumulation in late endosomes/ reduced. Moreover, despite an overall accumulation of cholesterol in lysosomes [56,57]. Furthermore, when the Rab9 content of NPC1-deficient cells, the does not sense the NPC1-deficient human fibroblasts and mouse cortical neurons increased cholesterol content in the cell. Consequently, the synthesis of cholesterol and LDL receptors continues unabated and cholesterol was increased by protein transduction, lipid storage was re- esterification is inappropriately decreased. duced [58]. Thus, increased expression of Rab7/9 appears to partially bypass a deficiency of NPC1. protein that binds cholesterol. Storch and co-workers have shown in an in vitro transport assay that NPC2 transports 5. Why is NPC disease primarily a neurological disorder? cholesterol to phospholipid vesicles via a direct interaction with acceptor membranes. Intriguingly, this transfer is pro- An unresolved question is whether the accumulation of cho- moted by inclusion of the lysosomal phospholipid, bis-mono- lesterol in NPC1-deficient cells occurs secondarily to the accu- acylglycerol phosphate, in the acceptor membranes [50]. mulation of gangliosides, or vice versa whether gangliosides Possibly NPC1 acts in concert with NPC2 so that NPC1 is a accumulate in response to the accumulation of cholesterol cholesterol ‘‘sensor’’ whereas NPC2 acts as a cholesterol trans- [9,10,59–61]. Also unknown is whether the neurodegeneration porter. However, it is not clear how the presence of the NPC2 is caused by an accumulation of cholesterol or an accumula- protein within the lumen of late endosomes/lysosomes would tion of gangliosides. Another key question is: are the neurolog- be able to fulfill this role. ical problems characteristic of NPC disease due to an excess of A consequence of the sequestration of cholesterol in lyso- cholesterol (or gangliosides) in late endosomes/lysosomes, or somes of NPC1-deficient cells is that the cholesterol content due to a deficiency of cholesterol (or gangliosides) at other cel- of the plasma membrane is reduced [51]. Thus, despite an over- lular locations such as the plasma membrane, endoplasmic all accumulation of cholesterol in NPC1-deficient cells, the reticulum and/or mitochondria? The most obvious pathologi- endoplasmic reticulum does not sense the increased cholesterol cal feature of NPC disease is the progressive loss of neurons, content in the cell. Consequently, the synthesis of cholesterol particularly Purkinje cells in the cerebellum. Other neurologi- and LDL receptors continues unabated and cholesterol esteri- cal manifestations of the disease are lipid storage, the forma- fication is inappropriately decreased (reviewed in [52]). It was tion of meganeurites and ectopic dendrites, and the presence originally thought that the cholesterol that accumulates in of neurofibrillary tangles (reviewed in [62]). However, the NPC1-deficient cells was derived only from LDLs internalized NPC1 protein is expressed in all tissues and all cell types exam- by receptor-mediated endocytosis. However, endogenously ined to date. It is, therefore, not clear why the loss of function synthesized cholesterol also appears to become sequestered in of this ubiquitously expressed protein affects mainly the CNS the endosomal pathway of NPC1-deficient fibroblasts [53]. and why neurons are particularly vulnerable. Furthermore, in NPC1-deficient neurons, newly-synthesized Since the blood–brain barrier is impermeable to LDLs in the cholesterol appears to be the major source of the cholesterol circulation, all of the cholesterol that accumulates in NPC1- that accumulates in the late endosomes/lysosomes of cell deficient brains is made endogenously within the CNS. Diets- bodies [44]. The accumulation of newly-made cholesterol in chy and colleagues compared the flux of cholesterol in the the endosomal system would not be unexpected since the plas- brains of Npc1À/À and Npc1+/+ mice and found that the overall ma membrane, the site of most cellular cholesterol, is con- rate of cholesterol synthesis in the brain is decreased, and the stantly undergoing endocytosis. rate of cholesterol excretion from the brain is increased, by J.E. Vance / FEBS Letters 580 (2006) 5518–5524 5521

NPC1 deficiency. However, the increased excretion of choles- terol content of axonal plasma membranes [78]. These observa- terol does not depend on an increased removal of cholesterol tions are consistent with the finding that the anterograde by 24-hydroxylation [63]. transport of cholesterol from cell bodies to distal axons of The CNS operates a lipoprotein transport system distinct Npc1À/À sympathetic neurons is impaired [44]. We proposed from that in the plasma. In the CNS, cholesterol is exchanged that the severe neurological problems in NPC disease might among cells via lipoproteins that are secreted by glial cells, pri- occur because NPC1 has another function in neurons in addi- marily astrocytes. The most abundant apolipoprotein (apo) in tion to its established role in transporting cholesterol out of the CNS is apo E. This protein is synthesized primarily by late endosomes/lysosomes in cell bodies. Moreover, defects in astrocytes, although small amounts of apo E also appear to neuronal function might occur either because of lipid accumu- be made by neurons under certain conditions [64,65]. -de- lation in the cell bodies or because of a deficiency of cholesterol rived lipoproteins are the size and density of plasma high den- in distal axons [77]. One defect that was detected in Npc1À/À sity lipoproteins [66]. Lipoprotein particles that contain apo J mouse embryonic striatal neurons was that the responsiveness and apo A1 are also present in the CNS in smaller amounts of these neurons to brain-derived neurotrophic factor, and a [67]. Apo E-containing glial lipoproteins have been shown to signaling pathway initiated by the TrkB receptor, are impaired promote synaptogenesis [68] and stimulate axon growth [66] [79]. of CNS neurons. After a nerve injury, the synthesis of apo E In neurons, late endosomes/lysosomes of the degradative by glial cells increases by 150-fold [69–71]. It has been pro- pathway are thought to be restricted to the cell bodies [80]. posed that apo E-containing lipoproteins secreted by astro- The cholesterol that accumulates in cell bodies of NPC1-defi- cytes are taken up by adjacent neurons via receptors of the cient neurons co-localizes with the late endosomal/lysosomal LDL receptor superfamily [71,72]. A parallel pathway is marker LAMP1 (Fig. 3) [44,77]. In addition to the NPC1 pro- thought to operate in the peripheral nervous system in which tein being present in cell bodies, however, we found using indi- cholesterol is recovered from degenerating myelin and is deliv- rect immunofluorescence [44], immunoblotting [44,81], and ered to neurons for axon growth and repair. Consistent with adenoviral expression of a chimera of NPC1 and green fluores- this model, the re-utilization of cholesterol from degenerating cent protein [82], that NPC1 is also abundant in distal axons. myelin is impaired in the peripheral nervous system of Furthermore, NPC2 is also present in both cell bodies and dis- Npc1À/À mice [73]. tal axons of mouse sympathetic neurons [81]. These findings Only a few studies have been reported on the function of suggested that NPC1 and NPC2 might have -specific NPC1 at the molecular level in cells of the nervous system functions in axons. (i.e. neurons and glia) (reviewed in [74]). As in other NPC1- In support of this idea, we have recently demonstrated that deficient cells, such as fibroblasts, NPC1 deficiency causes an NPC1 is present in recycling endosomes in pre-synaptic nerve accumulation of cholesterol in cerebellar astrocytes [75]. Glial terminals [81]. Synaptic vesicles were isolated from the brains cells (astrocytes, oligodendrocytes and microglia) comprise of Npc1À/À and Npc1+/+ mice. Although the cholesterol con- approximately 90% of the cells in the brain. We suggested, tent of the synaptic vesicles was unaffected by NPC1 defi- therefore, that an impaired production of lipoproteins by ciency, the relative amounts of two synaptic vesicle proteins, NPC1-deficient astrocytes might contribute to the neurological synaptophysin and synaptobrevin, were altered. The formation problems of NPC disease. We tested the hypothesis that of the synaptophysin/synaptobrevin complex is a key event in sequestration of cholesterol in Npc1À/À glial cells [75] inhibits synaptic vesicle recycling and depends on the cholesterol con- the generation of apo E-containing lipoproteins and/or causes tent of the plasma membrane [83]. Interestingly, formation of the release of defective, non-functional lipoprotein particles. this complex is reduced in the brains of Npc1À/À mice com- However, the amount of apo E secreted, the size of the lipo- pared to Npc1+/+ mice [84]. Thus, it is possible that a reduced protein particles, and the cholesterol content of the culture content of cholesterol in the plasma membrane of axons [77] medium of Npc1À/À astroglia were indistinguishable from interferes with the synaptic vesicle recycling process. Electron those of Npc1+/+ glia [75]. Moreover, in a functional assay, microscopy of synaptosomes from the cerebellum of Npc1À/À apo E-containing lipoproteins from Npc1+/+ and Npc1À/À glia mice showed the presence of aberrantly large synaptic vesicles were equally effective in stimulating axonal extension [75].In [81]. Thus, NPC1 deficiency causes biochemical and morpho- other studies, Scott and co-workers have demonstrated that logical abnormalities in the pre-synaptic terminal, suggesting NPC1 function within mouse neurons per se is critical for pre- that NPC1 might play a role in synaptic transmission. Perhaps venting neurodegeneration [76]. Thus, although these experi- a function of NPC1 in axons is to control the cholesterol con- ments do not exclude the possibility of detrimental tent of the nerve terminal and thereby regulate synaptic vesicle consequences of long-term deficits in the production or func- recycling and neurotransmitter release. Although some of the tion of glial lipoproteins, or in other functions of glial cells, neurological problems of NPC disease (e.g. ataxia) are likely the data indicate that the neurological problems characteristic to be caused by neuronal death (particularly of Purkinje cells), of NPC disease are more likely attributable to defects within even modest defects in synaptic transmission might contribute the neurons per se rather than to defects in the glial lipopro- to the other neurological impairments known to occur in this teins. disease. A recent study has reported that NPC1 deficiency does The function of NPC1 in neurons has also recently been not change the characteristics of ion channels or cause gross examined. Cholesterol accumulates in cell bodies of Npc1À/À alterations in electrophysiological properties of neurons [85]. mouse sympathetic neurons [77]. However, the cholesterol Some intriguing histopathological similarities between content of the distal axons of these neurons is diminished by NPC disease and Alzheimer’s disease have recently emerged. NPC deficiency [77]. Similarly, induction of the NPC pheno- Endosomal abnormalities are characteristic of both NPC dis- type by treatment of cultured hippocampal neurons with the ease and Alzheimer’s disease, and cholesterol imbalance has cholesterol transport inhibitor, U18666A, decreases the choles- also been implicated in the aberrant processing of amyloid 5522 J.E. Vance / FEBS Letters 580 (2006) 5518–5524

Fig. 3. Cholesterol accumulates in late endosomes/lysosomes of cell bodies of Npc1À/À mouse sympathetic neurons. Sympathetic neurons from Npc1+/+ (upper panels) and Npc1À/À (lower panels) were stained with antibodies raised against NPC1 (green) or the lysosome-associated membrane protein-1, LAMP-1 (red). The neurons were also incubated with the cholesterol binding compound, filipin (turquoise), then examined by confocal microscopy. In Npc1+/+ neurons, cholesterol is mainly restricted to the cell surface whereas in Npc1À/À neurons cholesterol has been redistributed to, and is sequestered in, intracellular sites that largely co-localize with LAMP-1 (merge panel). Size bar: 20 nm. (Some panels are reprinted from Karten et al. [77] with permission.)

precursor protein that is a feature of Alzheimer’s disease old mice lacking functional NPC1 the cholesterol content is [86,87]. Increased immunoreactivity to amyloid precursor pro- approximately 10-fold higher than in wild-type mice [97]. tein has been observed in the brains of even very young (10- My laboratory has recently investigated the changes in lipid day-old) Npc1À/À mice. Moreover, the levels of Ab40 and metabolism that occur in NPC1-deficient mouse hepatocytes. Ab42 are increased by NPC1-deficiency [88] and by treatment The cholesterol content of hepatocytes from 5-week-old of neurons with the cholesterol transport inhibitor U18666A Npc1À/À mice is 5-fold higher than that of Npc1+/+ hepato- [89]. As in Alzheimer’s disease, neurofibrillary tangles are pres- cytes. However, in marked contrast to the situation in fibro- ent in the brains of adult, and even juvenile, NPC patients blasts, the rate of cholesterol esterification in hepatocytes is [90–92], and these tangles show the same pattern of tau hyper- increased several-fold by NPC1 deficiency but the rate of cho- phosphorylation as occurs in brains of Alzheimer’s disease pa- lesterol synthesis is not increased (A. Kulinski and J.E. Vance, tients. Although Npc1À/À mice do not develop the unpublished data). NPC1 deficiency also approximately dou- neurofibrillary tangles, tau is hyperphosphorylated [93–95]. bles the number of apo B100-containing very low density lipo- protein particles secreted by cultured hepatocytes. Consistent with the enhanced rate of cholesterol esterification, the ratio 6. Liver disease in NPC patients of cholesteryl esters to triacylglycerols in the very low density lipoprotein particles secreted by Npc1À/À hepatocytes is double In addition to the severe neurodegeneration that occurs in that in the lipoproteins secreted by Npc1+/+ hepatocytes (A. individuals with NPC disease, hepatomegaly and cholestasis Kulinski and J.E. Vance, unpublished data). Thus, profound also occur perinatally in approximately 50% of individuals changes in lipid metabolism are induced in hepatocytes by with NPC disease, and some of these infants die from acute NPC1 deficiency, and some of these alterations are quite dis- liver failure by the age of 6 months [96]. Moreover, in tinct from those that occur in NPC1-deficient fibroblasts. It Npc1À/À mice, plasma levels of alanine aminotransferase and is likely that changes in lipid homeostasis induced by NPC1 aspartate aminotransferase (markers of liver damage) increase deficiency in hepatocytes contribute to the hepatic abnormali- markedly after the age of 5 weeks, reaching a level 15-fold ties in NPC disease. higher than that in wild-type mice [97]. Thus, NPC1 appears to play an important role in the liver. Indeed, the level of expression of the NPC1 protein is higher in the liver than in 7. Potential avenues for therapeutic intervention in NPC disease any other tissue, including the brain [98]. Furthermore, the flux of lipoprotein-derived cholesterol So far, no effective therapies have been developed for the through the liver is greater than through any other tissue. treatment of NPC disease. However, the prospects of finding For example, approximately 80% of LDL-cholesterol and a treatment for this disease have increased because of the nearly all of the chylomicron remnants are removed from the large body of research that has been performed during the plasma by the liver [14,99]. The rate of uptake of plasma past 10 years in understanding the function of the NPC pro- LDL cholesterol via receptor-mediated endocytosis in the liv- teins and the metabolic changes that are induced by a defi- ers of Npc1À/À mice is the same as in Npc1+/+ mice [14]. Thus, ciency of NPC1 and NPC2. One potential strategy for cholesterol from LDLs becomes sequestered in late endo- treatment of this disease is a gene therapy approach in which somes/lysosomes in NPC1-deficient livers: in livers of 8-week- the wild-type NPC gene would be delivered to NPC patients J.E. Vance / FEBS Letters 580 (2006) 5518–5524 5523 to restore the missing function. 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R.D., Sakuraba, H., Parton, R.G. and Gruenberg, J. (1999) Nat. A hallmark of NPC1 deficiency at the cellular level is an Cell Biol. 1, 113–118. aberrant accumulation of cholesterol and gangliosides in late [8] Watanabe, Y., Akaboshi, S., Ishida, G., Takeshima, T., Yano, endosomes/lysosomes. Therefore, initial attempts to find a T., Taniguchi, M., Ohno, K. and Nakashima, K. (1998) Brain Dev. 20, 95–97. treatment for NPC disease have focused on methods that [9] Zervas, M., Dobrenis, K. and Walkley, S.U. (2001) J. Neuro- would reduce the amount of cholesterol and/or gangliosides pathol. Exp. Neurol. 60, 49–64. in tissues, particularly in the brain. Introduction of a null [10] te Vruchte, D., Lloyd-Evans, E., Veldman, R.J., Neville, D.C., mutation in the LDL receptor into Npc1À/À mice partially re- Dwek, R.A., Platt, F.M., van Blitterswijk, W.J. and Sillence, D.J. (2004) J. Biol. 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