Niemann-Pick C Disease and Mobilization of Lysosomal Cholesterol by Cyclodextrin. University, Halifax, NS, Canada

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1 Niemann-Pick C Disease and Mobilization of Lysosomal Cholesterol by Cyclodextrin Jean E. Vance a and Barbara Karten b a The Group on Molecular and Cell Biology of Lipids and Department of Medicine, University of Alberta, Edmonton, AB, Canada and b Department of Biochemistry and Molecular Biology, Dalhousie University, Halifax, NS, Canada Correspondence to: Dr. Jean E. Vance 328 HMRC, University of Alberta Edmonton AB T6G 2S2 Canada jean.vance@ualberta.ca Tel: (780) Fax: (780) Abbreviations: ACAT, acyl-coa:cholesterol acyltransferase; CYCLO, 2-hydroxypropyl-βcyclodextrin; ER, endoplasmic reticulum; HDAC, histone deacetylase; LDL, low density lipoproteins; LE/L, late endosomes/lysosomes; NPC, Niemann-Pick type C; SREBP, sterol response elementbinding protein 1

2 ABSTRACT Niemann-Pick type C (NPC) disease is a lysosomal storage disease in which endocytosed cholesterol becomes sequestered in late endosomes/lysosomes (LE/L) because of mutations in either the NPC1 or NPC2 gene. Mutations in either of these genes can impair the functions of the corresponding NPC1 or NPC2 proteins, and cause progressive neurodegeneration, as well as liver and lung disease. NPC1 is a polytopic protein of the LE/L limiting membrane whereas NPC2 is a soluble protein in the LE/L lumen. These two proteins act in tandem and promote the export of cholesterol from LE/L. Consequently, a defect in either NPC1 or NPC2 induces cholesterol accumulation in LE/L. In this review, we shall summarize the molecular mechanisms leading to NPC disease, particularly in the central nervous system. Recent exciting data on the mechanism by which the cholesterolsequestering agent cyclodextrin can bypass the functions of NPC1 and NPC2 in LE/L, and mobilize cholesterol from LE/L, will be highlighted. Moreover, the possible use of cyclodextrin as a valuable therapeutic agent for treatment of NPC patients will be considered. Keywords: neurodegeneration; lysosomal storage disease; Purkinje cells; gangliosides; neurons; astrocytes; microglia; cholesterol homeostasis; endoplasmic reticulum 2

3 1. Introduction Niemann-Pick type C (NPC) 1 disease is a progressive inherited disease in which unesterified cholesterol and other lipids accumulate in late endosomes/lysosomes (LE/L) of all cells and tissues. The underlying cause of NPC disease is distinct from that of Niemann-Pick disease types A/B that are caused by defects in the lysosomal acid sphingomyelinase gene (1). NPC disease is a recessive, autosomal disorder that affects approximately 1/150,000 live births and causes premature death due to relentless neurodegeneration as well as lung and liver dysfunction [reviewed in (2,3)]. Since neuropathological changes in NPC disease occur long before the onset of symptoms, early diagnosis of the disease is crucial. However, clinical diagnosis of NPC disease in childhood can be problematic as the early symptoms are typical of many other neurological disorders. Therapeutic intervention largely involves symptomatic treatment. Furthermore, access of therapeutic agents to the central nervous system is often restricted because the blood brain barrier is impermeable to many molecules and prevents their passage into the brain from the peripheral circulation. In this review we shall summarize the molecular mechanisms that lead to NPC disease, and discuss recent exciting data indicating that the cholesterol-sequestering agent, cyclodextrin, might be a valuable therapeutic agent for treatment of NPC disease. 2. Biochemical and Cell Biological Implications of NPC Deficiency In mammalian cells, cholesterol is a key component of cellular membranes and is a precursor of steroid hormones and bile acids. Cells acquire cholesterol from endogenous synthesis in the endoplasmic reticulum (ER) and from receptor-mediated uptake of exogenously-supplied lipoproteins, such as low density lipoproteins (LDL) via receptors of the LDL receptor family (4). The underlying cellular defect in NPC disease is an impaired egress of unesterified cholesterol from the LE/L, resulting in the accumulation of unesterified cholesterol in LE/L (5). This cellular defect was first revealed by 3

4 Pentchev and coworkers (6) who demonstrated that the esterification of LDL-derived cholesterol was profoundly diminished in fibroblasts from NPC patients. However, the activity of the cholesterol esterification enzyme, acyl-coa:cholesterol acyltransferase (ACAT), as well as the receptor-mediated uptake of LDL and the lysosomal hydrolysis of LDL-derived cholesteryl esters, were normal. Thus, these observations indicated that LDL-derived cholesterol became sequestered in the LE/L because the export of unesterified cholesterol from LE/L was disrupted. Consequently, the amount of cholesterol reaching the ER and plasma membrane from LE/L was reduced in these fibroblasts (6-9). In contrast, the transport of newly-synthesized cholesterol from the ER to the plasma membrane occurs normally in fibroblasts from NPC patients (8,10). Homozygous mutations in either the NPC1 gene (11,12) or the NPC2 gene (13,14) were subsequently identified as being causative for NPC disease. Most (~95%) known cases of NPC disease are due to mutations in the NPC1 gene whereas the remaining 5% of cases are caused by mutations in the NPC2 gene. The NPC1 gene encodes a LE/L membrane protein that contains 1254 amino acids, 13 presumed transmembrane domains, a leucine zipper, a lysosomal targeting motif and a sterol-sensing motif (12). The latter motif is similar to a sequence that has been identified in several proteins involved in cholesterol metabolism. The NPC2 gene (originally called HE1) encodes a 151-amino acid soluble protein of the LE/L lumen (14,15). The NPC1 and NPC2 proteins both bind cholesterol (16-19). The N- terminal domain of NPC1 is exposed to the LE/L lumen and binds cholesterol with high affinity in a 1:1 molar ratio (18-22). Fluorescence dequenching assays show that NPC2 rapidly transfers cholesterol, but not glycosphingolipids, ceramide, phospholipids or fatty acids (16,19,21), between phospholipid liposomes or membranes in vitro. This transfer is promoted by an acidic environment (such as that of the LE/L lumen) and is markedly stimulated by the presence of bis(monoacylglycerol) phosphate (16,23). This phospholipid is highly enriched in the lumenal multivesicular bodies of the LE/L, and accumulates in NPC-deficient cells (24). X-ray crystallographic studies showed that orientation of the 4

5 cholesterol molecule attached to NPC2 is opposite from that of cholesterol bound to the hydrophobic pocket of NPC1 (17,18,20-22). Importantly, homozygous mutations in either NPC1 or NPC2 cause essentially the same clinical and cellular phenotypes (25,26). Thus, a model was proposed for the concerted transfer of cholesterol from NPC2 to NPC1 within LE/L (Fig. 1). In this model, endocytosed cholesteryl esters are delivered from LDL to the LE/L, then hydrolyzed to unesterified cholesterol by lysosomal acid lipase. The unesterified cholesterol binds to NPC2 and is subsequently transferred via NPC2 from membranes of the internal vesicles of LE/L to NPC1 in the limiting membrane (19). An important feature of this model is that the hydrophobic cholesterol molecule can be exported without direct contact with the aqueous lumen of the LE/L. In support of this model, a direct interaction between NPC2 and a lumenal domain of NPC1 has been demonstrated (27). This protein-protein interaction occurs only at acidic ph as occurs in the LE/L lumen, and requires the binding of cholesterol to NPC2. The molecular mechanisms that mediate cholesterol export from NPC1 on the LE/L limiting membrane to other cellular organelles, such as the ER and plasma membrane, have not yet been elucidated although vesicular transport involving Rab proteins (28-30), transport via carrier proteins such as oxysterol binding proteins (31,32) and direct membrane contact have been proposed. Since the sequestration of cholesterol in the LE/L of NPC-deficient cells reduces the delivery of cholesterol to the ER (6-8), the amount of cholesterol in the ER, as a molar % of total ER lipids, can fall below a critical threshold (33). Thus, although NPC deficiency causes an accumulation of cholesterol in the LE/L, there is a deficit of cholesterol in the ER so that the sterol response elementbinding protein (SREBP) pathway (34) is stimulated, and the synthesis and uptake of cholesterol are increased (9,35). The impaired trafficking of cholesterol in NPC-deficient cells profoundly affects multiple cellular functions including the regulation of lysosomal calcium homeostasis (36), oxidative stress, (37,38) and vesicle trafficking pathways mediated by Rab proteins (28,29), as well as fusion of 5

6 late endosomes with lysosomes (39). NPC1 deficiency is particularly detrimental for functioning of LE/L in the brain (40). Intriguingly, however, cholesterol transport from LE/L to mitochondria is not defective in NPC1-deficient cells (41). Indeed, the cholesterol content of mitochondria isolated from brains is increased, not decreased, by NPC1 deficiency (41,42). Several properties of mitochondria such as ATP production, oxidative stress, and perhaps mitophagy, are altered by NPC1-deficiency (42-44), probably because of an increased mitochondrial cholesterol content. In contrast to NPC1 deficiency, however, NPC2 deficiency does inhibit the movement of endosomal cholesterol to mitochondria (45). Moreover, mutants of NPC2 that bind cholesterol, but cannot transfer cholesterol to NPC1, restore cholesterol trafficking to mitochondria in cells lacking functional NPC2 (45). Since NPC2 transfers cholesterol directly to membranes (23), a possible explanation for this difference between NPC1- and NPC2-deficient cells is that NPC2 transfers cholesterol from the LE/L lumen to the perimeter membrane of the LE/L, where the cholesterol would be available for transport by transmembrane proteins such as MLN64 and NPC1. 3. Clinical Manifestations of NPC deficiency The clinical manifestations of NPC disease usually become apparent in early childhood but the age of onset and rate of disease progression vary over a wide range (2,46,47). In both humans and mice, the most severe consequence of NPC deficiency is neurodegeneration, particularly the extensive death of Purkinje neurons in the cerebellum, resulting in progressive motor impairment (48,49). In other brain regions, neuronal abnormalities, such as axonal swelling and degeneration, and ectopic dendrite formation also occur and are often more prominent than neuronal death (50-53). These impairments contribute to the reduced weight gain, cognitive decline and premature death that characterize NPC disease. Severe liver disease is also prevalent in many NPC-deficient patients (2) and mice (54-57). Since the majority (~80%) of circulating LDL are cleared by the liver via receptor-mediated 6

7 endocytosis (58,59), cholesterol accumulation in the LE/L of livers of NPC1-deficient patients and mice is more pronounced than in any other tissue (59,60). The lung is another tissue whose function is impaired in NPC disease (59,61-63) and lipid-laden macrophages are abundant in lungs of NPCdeficient mice (64). Moreover, age-related retinal degeneration occurs in Npc1 -/- mice (65). Two recent studies have reported that levels of several mrnas and proteins involved in energy metabolism are altered by NPC deficiency and that lactate levels are increased in NPC1-deficient cerebella. Moreover, pyruvate oxidation is decreased, and anaerobic glycolysis is increased, during progression to the symptomatic stage of NPC disease (66,67). Thus, glucose metabolism appears to be affected at an early stage of NPC disease. Markers of oxidative stress are also increased in brains of human NPC patients and animal models (37,38,68). 4. Models Used for Studying NPC Disease Several cell and animal models of NPC disease are available and have provided key information on the mechanism by which a deficiency of NPC1 or NPC2 leads to the disease phenotype. The cell biological and biochemical defects in NPC-deficient cells were initially discovered using NPC1- deficient fibroblasts (mutant Chinese hamster ovary cells and fibroblasts from NPC patients) (6,7,10,35,69,70). The NPC defect has been mimicked in cultured cells by the class II amphiphilic amine, U18666, that induces cholesterol sequestration in LE/L. This amine has been widely used to mimic the NPC phenotype but exhibits side-effects including inhibition of cholesterol synthesis (71,72). The most commonly used animal model of NPC disease is the Balb/cNctr-Npc N/+ mouse that completely lacks NPC1 protein (73). These mice are asymptomatic at birth but develop ataxia and die within ~3 months. A more recently-derived mouse model carries the D1005G mutation in NPC1 (74); these mice have a more slowly progressing disease than that of NPC1-null mice. This mutation resides in the cysteine-rich loop of NPC1, the site of the most common mutations in human NPC patients. 7

8 NPC1 expression has also been specifically attenuated in mouse liver by antisense technology (75). In addition, hypomorphic NPC2-deficient mice (that express only 0.4% of the normal amount of NPC2 protein) (26) and NPC2-null mice (76) have been generated. The phenotype of these mice is similar to, but slightly less severe than, that of Npc1 -/- mice (26,76). More recently, NPC1 has been eliminated specifically in certain cell types of the brain (77) and, reciprocally, has been expressed only in specific cells of brains of Npc1 -/- mice (78). These studies have been particularly useful for understanding in which cells of the brain loss of NPC function is the most detrimental (Section 6). As an alternative to mouse models of NPC disease, a NPC1-deficient feline model has been established (79,80) and has provided valuable information relevant to human NPC disease. Other models that have been used to elucidate pathways defective in NPC disease are mutants of yeast (81) and Drosophila (82,83). An advantage of these non-mammalian models is that genetic manipulation is relatively straightforward. Recently, studies have been initiated in induced stem cells derived from fibroblasts of individual NPC patients, particularly stem cells that were differentiated into neuron-like cells (44,84-86). It is likely that these cells will be useful for evaluation of drug candidates for treatment of NPC disease. 5. Lipid Accumulation in the LE/L Cholesterol is not the only lipid that accumulates to abnormally high levels in LE/L of NPCdeficient cells. Sphingomyelin (87), gangliosides (particularly GM2 and GM3) (88-90), sphingosine (36) and the phospholipid bis(monoacylglycerol) phosphate (24,91) also accumulate. Nevertheless, compelling evidence indicates that the primary offending molecule is cholesterol and that the accumulation of the other lipids occurs secondarily to the sequestration of cholesterol. First, NPC1 and NPC2 each bind cholesterol, but not gangliosides or sphingosine, with high affinity (17,18,92). In addition, a mutation in the cholesterol-binding site of NPC1 abolishes the binding of cholesterol, and expression of this mutant protein in mice induces the typical NPC phenotype (93). Second, the storage 8

9 of gangliosides was genetically reduced in NPC1-deficient mice by crossing Npc1 -/- mice with mice that lacked the galactosyltransferase responsible for the synthesis of GM2 ganglioside, or with mice that lacked GM3 synthase (94). In these double mutant mice, ganglioside storage in LE/L was eliminated but cholesterol still accumulated, and the neurodegeneration and survival were not improved (94-96). Third, lysosomal acid sphingomyelinase activity is inhibited when the amount of cholesterol in LE/L membranes is increased. Thus, the accumulation of sphingomyelin in the LE/L of NPC-deficient cells is probably also secondary to the sequestration of cholesterol (87,97). Although sphingosine accumulation in LE/L was reported prior to cholesterol accumulation (36), these studies were not performed in NPC1-deficient cells but in cells treated with the amphiphile U18666A which also inhibits cholesterol synthesis. Fourth, 2-hydroxypropyl-β-cyclodextrin, which forms an inclusion complex with cholesterol [reviewed in (98)], dramatically reduces cholesterol storage in tissues of NPC-deficient mice, thereby attenuating the neurodegeneration and extending lifespan (99-101) (Sections 7 and 8). In combination, these data provide convincing evidence that cholesterol sequestration in LE/L of NPC-deficient cells is the primary event responsible for NPC disease. 6. Loss of NPC Function in Cells of the Brain Although NPC1 and NPC2 appear to be expressed in all cells and tissues, the most devastating consequences of NPC deficiency typically occur in the brain. Cholesterol metabolism in the brain is compartmentalized from that in the periphery by the blood-brain barrier (102,103). Thus, essentially all cholesterol in the brain is synthesized within the brain rather than being delivered from plasma lipoproteins. The most striking consequence of NPC1-deficiency in brains of mice and humans is a profound loss of Purkinje neurons in the cerebellum (49,104,105). In other regions of the brain, such as the cerebral cortex and thalamus, neuronal apoptosis also occurs but is less common than axonal 9

10 swelling and ectopic dendrite formation (52,53, ). The reason why Purkinje neurons are the neurons that are the most sensitive to NPC deficiency is not clear (40). Interestingly, although cholesterol accumulates in NPC1-deficient peripheral tissues, such as the liver, the cholesterol content of NPC-deficient brains does not increase, but even decreases, with age. The reason for this apparent anomaly is that myelin contains ~75% of the cholesterol in the brain, and extensive demyelination occurs in NPC-deficient brains (59,110), at least in part because genetic deletion of NPC1 in oligodendrocytes of mice reduces myelin formation (111,112). Cholesterol does, however, accumulate in the LE/L of NPC-deficient neurons, such as Npc1 -/- mouse sympathetic neurons (113), hippocampal neurons (Fig. 2) (114) and cerebellar granule neurons (115). In Npc1 -/- sympathetic neurons, cholesterol accumulates in LE/L of the cell bodies whereas the cholesterol content of distal axons is less than that of Npc1 +/+ neurons. In addition, cholesterol transport from cell bodies to distal axons is impaired by lack of NPC1 (116). Although the accumulation of cholesterol in cell bodies of NPC-deficient neurons is generally considered to be the major cause of the neurodegeneration, it is also possible that the deficiency of cholesterol in distal axons contributes in other ways to the neurological phenotype. The NPC1 protein is present in recycling endosomes of presynaptic nerve terminals and the morphology of synaptic vesicles isolated from brains of Npc1 -/- mice is aberrant (117). Moreover, defects in synaptic transmission have been observed in several brain areas and in experimental models of NPC1 deficiency. Synaptic membranes are relatively rich in cholesterol which is required at many levels for efficient synaptic transmission. For example, the activities of pre- and post-synaptic receptors are influenced by membrane cholesterol content [reviewed in (118,119)]. In addition, the formation of pre-synaptic SNARE complexes, the interaction between synaptobrevin and synaptophysin, the fusion of synaptic vesicles with pre-synaptic membranes, and the sorting of synaptic vesicles during endocytosis, all depend on an appropriate concentration of cholesterol ( ). Thus, extraction of 10

11 cholesterol from neurons, by brief incubation with cyclodextrin, impairs exocytosis and endocytosis of synaptic vesicles (114, ). Moreover, defects in exocytosis and mobilization of synaptic vesicles, as well as increased spontaneous neurotransmission, were observed in cultured hippocampal neurons from Npc1 -/- mice (114,126,129). The inhibitory GABAergic synapses were more severely affected than were the glutamatergic synapses, indicating an imbalance between inhibitory and excitatory neurotransmission (129). Studies in hippocampal slices also revealed hyper-excitability of the neuronal network in NPC1-deficient brains (114). Furthermore, NPC1-deficiency alters long-term synaptic plasticity in several brain regions ( ). Notably, the pre-synaptic defects in NPC1-deficient neurons are independent of the presence of glial cells (133). The mechanisms by which defective export of cholesterol from LE/L leads to neuropathology are poorly understood. Thus, an important question is: in which cell type(s) of the brain is NPC deficiency responsible for the neurological deficits? The three major types of cells in the brain are neurons, astrocytes, and microglia, all of which sequester cholesterol in LE/L in response to NPC deficiency (113,115,134,135). Microglia are resident immune cells of the central nervous system and proliferate and become activated in response to changes in their environment. Clearly, inflammation is widespread in NPC-deficient brains. Microglia participate in phagocytosis of injured/dying cells (136) and can generate a protective inflammatory response (137,138). On the other hand, chronic activation of microglia appears to induce neuro-inflammation and neuronal death in several neurodegenerative disorders [reviewed in (139)]. Microglial proliferation and activation are rampant in specific regions of the brains of NPC disease patients and Npc1 -/- mice prior to onset of neurological symptoms (51,53,135). Thus, the microglial activation that occurs in NPC disease could be either a protective mechanism induced in response to neuronal death, or could contribute directly to neuronal death. In mouse brains, NPC1 deficiency increased the expression of mrnas encoding pro-inflammatory cytokines, such as tumor necrosis factor (105,108,140). Moreover, treatment of Npc1 -/- mice with an 11

12 LXR agonist reduced microglial activation and neuro-inflammation, and prolonged survival of the mice (140). However, since LXR agonists alter the expression of genes involved in multiple pathways, one cannot conclude that the decrease in neuro-inflammation was directly linked to the increased lifespan. Indeed, the majority of recent data indicate that the inflammation in NPC-deficient brains is primarily a response to the neurodegeneration. For example, elimination of NPC1 only in neurons of mice caused neuropathology typical of that in mice in which NPC1 was eliminated globally (141). Moreover, expression of NPC1 only in neurons of Npc1 -/- mice prevented the neurodegeneration (78). In addition, in microglia-neuron co-cultures, the presence of activated Npc1 -/- microglia did not cause neuron death (135). Consistent with the idea that inflammation in NPC disease is secondary to the extensive neuronal death, deletion of the macrophage inflammatory gene Mip1a/Ccl3 in Npc1 -/- mice did not prevent the neurodegeneration (142), nor did deletion of the complement system in Npc1 -/- mice reduce neuronal death or prolong survival (143). In combination, these experiments imply that microglial activation in NPC-deficient brains is primarily a response to neuronal death, rather than a direct cause of the neurodegeneration. The proliferation and activation of astrocytes are also widespread in NPC1-deficient brains (51). However, most experiments indicate that this astrogliosis is not the primary cause of the neurodegeneration. For example, in a hybrid mouse model, Npc1 +/+ neurons were not killed by surrounding Npc1 -/- astrocytes/microglia (144). Furthermore, genetic deletion of NPC1 in mouse astrocytes alone did not cause the characteristic NPC neuropathology (141). Nor did Tet-induced expression of NPC1 in Npc1 -/- mouse astrocytes alone slow disease progression, except for a small delay in weight loss (78). On the other hand, neuron-specific deletion of NPC1 caused activation of astrocytes and microglia in murine brain (141,143). In contrast to these findings, one study has reported that cholesterol storage was decreased, and survival of Npc1 -/- mice was increased, by expression of NPC1 in astrocytes under control of the glial fibrillary acidic protein promoter. However, NPC1 12

13 expression in other cell types of the brain, such as neurons, was not completely excluded (145). Consequently, the astrogliosis and inflammation in NPC-deficient brains appear to be largely secondary to the effects of NPC deficiency in neurons. Overall, these observations in NPC-deficient cells of the brain strongly imply that the absence of NPC1 in neurons alone is responsible for the neuropathology. Consistent with this conclusion, in Npc1 -/- mice NPC1 expression in neurons alone largely prevented the neurodegeneration (78,146). Although Purkinje neurons are the neurons that are the most susceptible to degeneration caused by NPC1 deficiency, dysfunction of other populations of neurons contributes significantly to the overall NPC phenotype. In elegant studies in Npc1 -/- mice by Lopez et al. (78), NPC1 expression in either forebrain neurons or Purkinje neurons similarly improved the neurological deficits and survival of the mice. However, a nearly complete normalization of cognitive and motor functions, as well as lifespan, was achieved only when NPC1 was expressed in most neurons in the brain, under control of the Eno2 promoter (78). The contribution of neurons other than Purkinje neurons to the NPC phenotype was also indicated by studies in which NPC1 was eliminate specifically in Purkinje neurons. These mice exhibited ataxia and loss of Purkinje cells in an anterior-to-posterior gradient but did not suffer from weight loss or premature death (77). In another study, NPC1 was eliminated from neurons in nearly all brain areas, except the Purkinje cells, yet the mice exhibited the same neurodegenerative phenotype as that of NPC1-null mice (141). Thus, although the lack of NPC1 in Purkinje cells clearly contributes to Purkinje cell death and the neuropathology in NPC disease, lack of NPC1 in other types of neurons is clearly also detrimental. Overall, these observations demonstrate that NPC deficiency in neurons, rather than in astrocytes or microglia, is the primary cause of the neuropathology that characterizes NPC disease. 7. Cyclodextrin as a Therapy for NPC Disease 13

14 Potential therapies for NPC disease have focused on reduction of lipid storage in the LE/L. An important consideration when testing therapies for NPC disease patients is how disease progression can be quantitatively assessed. Thus, validation of biomarkers of NPC disease progression is an important goal ( ). Miglustat is an agent that inhibits glucosylceramide synthase, a key enzyme of glycosphingolipid synthesis [reviewed in (150)]. Consequently, since gangliosides accumulate in the LE/L of NPC-deficient cells, miglustat, which efficiently crosses the blood-brain barrier, has been used as a treatment for animal models of NPC disease, as well as NPC patients. For example, in the feline model of NPC disease, miglustat decreased GM2 ganglioside accumulation and slightly delayed the development of neurological symptoms (151). Similarly, in Npc1 -/- mice miglustat reduced ganglioside storage in LE/L and modestly extended lifespan (88). Miglustat also produced some neurological improvements in NPC patients ( ). However, the mechanism underlying these clinical improvements is not clear since a reduction of gangliosides in LE/L of NPC-deficient mice is not beneficial (94-96). Several other treatments for NPC disease are currently being investigated [reviewed in (157)]. One of the most promising is inhibition of histone deacetylases (HDACs); HDAC inhibitors have already been approved for treatment of certain cancers in humans. HDACs appear to play important roles in neuronal survival (158,159). The expression of several HDAC genes is increased by NPC deficiency in human fibroblasts (160,161). A clinically approved HDAC inhibitor decreased HDAC activity in NPC1-deficient cells and reduced cholesterol accumulation in the LE/L (160,161). Interestingly, however, treatment of NPC2-deficient human fibroblasts with an HDAC inhibitor did not reduce cholesterol storage in LE/L (161). Since HDAC inhibitors can increase the amount of NPC1 protein (162) these inhibitors might be useful in NPC patients in which residual NPC1 activity remains, or in which partially active NPC1 protein is mislocalized (163). However, validation of this approach for treatment of NPC patients awaits evidence that HDAC inhibitors are beneficial in NPC-deficient 14

15 mice and/or cats. The anti-apoptotic agent imatinib has also been suggested as an agent that might prevent neurodegeneration in NPC disease. Imatinib inhibits the c-abl pathway, the activity of which is increased in Npc1 -/- Purkinje cells (164). Imatinib treatment of Npc1 -/- mice partially prevented Purkinje cell death and modestly improved neurological parameters, but only slightly extended the lifespan of the mice (164). Perhaps the most exciting and promising approach for treatment of NPC disease is based on studies showing that the cholesterol-binding agent 2-hydroxypropyl-β-cyclodextrin (CYCLO) markedly delays the neurodegeneration and increases the lifespan of Npc1 -/- mice [(96,99,100); reviewed in (165)]. The use of CYCLO was prompted by studies in which injection of the neurosteroid allopregnanolone, dissolved in CYCLO, into 7-day-old Npc1 -/- mice delayed the neurodegeneration and approximately doubled the lifespan of these mice (166). Subsequent experiments revealed, however, that injection of allopregnanolone alone, without CYCLO, into 7-day-old Npc1 -/- mice did not produce the beneficial effects (99). On the other hand, a single intraperitoneal injection of CYCLO (4,000 mg/kg body weight), the carrier of the allopregnanolone (166), profoundly reduced the neurodegeneration and increased lifespan by ~50% (96,99). Thus, the reported beneficial effects of allopregnanolone in Npc1 -/- mice (166) appear to be attributable to CYCLO. Additional benefits accrue upon repeated administration of CYCLO. For example, subcutaneous injection of CYCLO (4,000 mg/kg) into Npc1 -/- mice once/week for 7 weeks reduced cholesterol accumulation in most tissues, markedly improved the neurodegeneration and liver functions, and approximately doubled lifespan (61); enigmatically, however, the lung disease was resistant to CYCLO treatment (61). In addition, when CYCLO (4,000 mg/kg) was subcutaneously injected into Npc1 -/- mice on alternate days, starting at either day 7 or day 21 after birth, the neuronal storage of cholesterol and gangliosides was reduced, neurodegeneration was delayed and lifespan was prolonged (100). Importantly, injection of CYCLO into NPC2-deficient mice is similarly beneficial (100,167). In careful balance studies on 15

16 quantification of the amount of cholesterol excreted into bile of Npc1 -/- mice, CYCLO reduced cholesterol storage in almost all tissues and promoted the excretion of cholesterol-derived bile acids (101). Importantly, although CYCLO injection mobilized LE/L cholesterol and reduced inflammatory parameters in 49-day-old Npc1 -/- mice (Fig. 3), their lifespan was not increased, probably because extensive tissue damage had already occurred (101). Moreover, CYCLO is cleared from the body 6 times more rapidly in mature mice than in 7-day-old Npc1 -/- pups, which could further contribute to the lower efficacy of CYCLO in older mice (101). The studies described above on the remarkable effects of subcutaneous injection of CYCLO into 7-day-old mice (Fig. 3) (96,99,100) imply that at least some CYCLO crosses the blood-brain barrier and enters the central nervous system of these mice. Nevertheless, a recent study has concluded that the amount of CYCLO that crosses the blood-brain barrier from plasma in mature mice is very small, and that the neurological benefits of peripherally-injected CYCLO are the result of the binding of CYCLO to the cerebral vascular endothelium (168). Other studies, however, indicate that a small percentage of the CYCLO in the peripheral circulation can cross the blood-brain barrier (169). It is also possible that in 7-day-old, but not in older, mice the blood-brain barrier is incompletely closed and is partially permeable to CYCLO. Thus, in light of the inefficient transport of CYCLO across the bloodbrain barrier, CYCLO was injected directly into the central nervous system of Npc1 -/- mice. The CYCLO diffused throughout all areas of the brain and the neurodegeneration was greatly attenuated with the benefits lasting more than a week (170). CYCLO was also infused continuously by intracerebroventricular delivery (35 mg/kg/day) into brains of Npc1 -/- mice for 4 weeks, starting at 3 weeks of age. When this treatment was combined with weekly subcutaneous injections of CYCLO (4,000 mg/kg) the histology of all regions of the brain was normal and the neurodegeneration was eliminated (170). Importantly, in Npc1 +/+ mice, cholesterol homeostasis was not altered when CYCLO was delivered peripherally or intracerebroventricularly (170). The ED 50 for the therapeutic effect of 16

17 CYCLO is 0.5 mg/kg (101,170) and the concentration of CYCLO required to mobilize cholesterol from the LE/L in either peripheral tissues or in the central nervous system of Npc1 -/- mice is less than 1 mm - probably ~0.1 mm (101,115,170). No toxic effects of these low doses of CYCLO were evident in Npc1 -/- mice. Indeed, CYCLO has been approved by the Federal Drug Administration (USA) as a drug delivery vehicle in humans. These encouraging studies suggest that the simultaneous delivery of CYCLO by subcutaneous injection into the periphery, and by direct infusion into the central nervous system, might represent a valid, if cumbersome, therapeutic approach for treatment of NPC patients. Indeed, a clinical trial in which CYCLO is being delivered intrathecally via osmotic mini-pumps to a few NPC patients was initiated at the National Institutes of Health (USA) in 2013; the outcome is anxiously awaited. The effectiveness of CYCLO was also evaluated in the feline NPC model. As in NPC-deficient mice, CYCLO (4,000 mg/kg) markedly attenuated the neurodegeneration and liver disease in the cats (80). However, an unexpected dose-dependent hearing defect developed in the CYCLO-treated cats suggesting that CYCLO had damaged the peripheral auditory pathway (80). This side-effect was further investigated by administration of a single dose of CYCLO subcutaneously (8,000 mg/kg), or intrathecally (120 mg), to Npc1 +/+ cats. The cats exhibited a significant increase in hearing threshold although the mechanism by which CYCLO caused this hearing defect has not yet been elucidated (80). Consequently, it will be important to determine whether or not CYCLO causes a similar deficit in hearing in NPC patients. 8. Mechanism by which CYCLO improves the NPC phenotype Cyclodextrins, such as CYCLO, are cyclic oligosaccharides consisting of multiple glycopyranose units [reviewed in (98)] and have been used for the delivery of hydrophobic drugs to humans. The cyclodextrins are water-soluble compounds but contain a hydrophobic pocket. 17

18 Cyclodextrins typically exist as hexamers (α-cyclodextrins), heptamers (β-cyclodextrins) or octamers (γ-cyclodextrins) that contain hydrophobic cavities of different sizes. The β-cyclodextrins have the highest affinity for cholesterol and are the most efficient in extracting cholesterol from cells. In contrast, α-cyclodextrins are the most effective at extracting phospholipids from cells: the hydrophobic cavity of α-cyclodextrins appears to be too small to accommodate a cholesterol molecule. The binding pocket of the γ-cyclodextrins is less hydrophobic than that of the β-cyclodextrins. The water-solubility of β-cyclodextrins can be increased by modification of the hydrophilic groups with moieties such as 2- hydroxypropyl groups. The methyl- and 2-hydroxypropyl derivatives of β-cyclodextrin are commonly used experimentally to deplete cells of cholesterol. For example, exposure of cells to 5 to 10 mm β- cyclodextrins for greater than 2 h removes 80-90% of cellular cholesterol (171). In addition, complexation of β-cyclodextrin with cholesterol can be used to deliver cholesterol to cells (172). Accumulating evidence indicates that the primary mechanism by which CYCLO mobilizes cholesterol from the LE/L of NPC-deficient cells depends on bulk-phase endocytosis (173) or clathrinmediated endocytosis (174) of CYCLO into the LE/L. Time course experiments have shown that cholesterol removal from the LE/L requires internalization of CYCLO into the cells, rather than extraction of cholesterol from the plasma membrane (173). For example, CYCLO was covalently linked to fluorescent dextran and trafficking of the fluorescent conjugate was visualized in NPCdeficient cells. The cholesterol was efficiently removed from the LE/L and the CYCLO conjugate was delivered to intracellular organelles (173). In addition, LE/L cholesterol was mobilized in Npc1 -/- fibroblasts even when the CYCLO was pre-loaded with cholesterol (173). Several studies have shown that the cholesterol that is released from the LE/L of NPC-deficient cells by CYCLO reaches the cytosolic compartment and is accessible to the ER. An indication that the cholesterol content of the ER was increased by CYCLO was that the production of cholesteryl esters (the storage form of cholesterol) 18

19 via the ER-localized enzyme acyl-coa:cholesterol acyltransferase (ACAT), was increased by up to 14- fold in various Npc1 -/- mouse tissues and cells after CYCLO treatment (99,115,170,173,175). Thus, the liberated cholesterol must have been accessible to ACAT on the ER. In cultured cerebellar neurons, astrocytes and microglia from Npc1 -/- mice, the cholesterol sequestered in LE/L was mobilized to the ER by low concentrations ( mm) of CYCLO (115). Another indicator of increased cholesterol availability at the ER is that the expression/processing of SREBP2, a key transcription factor that regulates cholesterol synthesis and uptake, is increased by NPC deficiency (34). Correspondingly, in Npc1 -/- mice CYCLO decreased expression of the SREBP2-responsive genes encoding the LDL receptor and 3-hydroxy-3-methylglutaryl-CoA reductase, as well as the rate of cholesterol synthesis in the brain and peripheral tissues (99,101,170). These observations clearly demonstrate that a low dose of CYCLO releases cholesterol stored in LE/L of Npc1 -/- cells so that the cholesterol is mobilized to the ER. In order to determine which cell types in the brain are responsive to CYCLO, and to investigate further the mechanism by which CYCLO mobilizes LE/L cholesterol in NPC-deficient brains, primary cultures of Npc1 -/- mouse neurons and astrocytes were exposed to 0.1 to 10 mm CYCLO for 24 h. Not surprisingly, since 5-10 mm CYCLO rapidly depletes cholesterol from the plasma membrane of cells, 10 mm CYCLO was profoundly toxic to neurons and markedly altered astrocyte morphology (115). In contrast, 0.1 and 1.0 mm CYCLO were not obviously toxic to Npc1 -/- neurons, astrocytes and microglia but essentially abolished cholesterol accumulation in the LE/L of these cells. Importantly, however, 0.1 mm CYCLO and 1.0 mm CYCLO exerted opposite effects on cholesterol homeostasis. Consistent with the hypothesis that CYCLO releases cholesterol from LE/L to the ER, 0.1 mm CYCLO reduced cholesterol synthesis in Npc1 -/- astrocytes and neurons, and profoundly enhanced cholesterol esterification in astrocytes (115). CYCLO did not stimulate cholesterol esterification in cerebellar neurons from Npc1 -/- mice, probably because ACAT activity and cholesteryl ester content are very low 19

20 in neurons compared to other cells (115). In marked contrast to 0.1 mm CYCLO, however, 1.0 mm CYCLO increased cholesterol synthesis and increased the expression of genes involved in cholesterol uptake and synthesis in these cells, while dramatically decreasing the rate of cholesterol esterification, suggesting that 1.0 mm CYCLO had depleted cholesterol from the ER. These data strongly support the concept that 0.1 mm CYCLO releases cholesterol trapped in the LE/L of NPC-deficient astrocytes and neurons, thereby delivering cholesterol to the ER. On the other hand, higher concentrations of CYCLO (1.0 mm and greater) appear to extract cholesterol from the plasma membrane, and eventually deplete cholesterol from the ER, and from the cells, in addition to removing cholesterol from the LE/L. These studies indicate that the concentration of CYCLO that might be beneficial in the brains of NPC patients is ~0.1 mm, consistent with the finding that in brains of Npc1 -/- mice in which CYCLO reversed the cholesterol transport defect, the concentration of CYCLO was ~ 0.1 mm (170). Thus, in the brain, and probably also in other tissues, concentrations of CYCLO that are 1 mm or higher would be expected to deplete cellular cholesterol and eventually impair neuronal survival and functions (115). Importantly, in Npc1 +/+ mice and cells, that do not sequester cholesterol in LE/L, CYCLO does not alter cholesterol homeostasis (115,176). Even CYCLO that is partially pre-loaded with cholesterol can mobilize cholesterol from LE/L without depleting cholesterol from the plasma membrane, suggesting that cholesterol-loaded CYCLO might be less toxic to the brain than CYCLO not loaded with cholesterol (173). A model for the action of CYCLO that is consistent with the above data is that CYCLO is taken up by cells via endocytosis and enters the LE/L. As discussed in Section 2, and depicted in Fig. 1, cholesterol transfer within the LE/L is normally mediated by the tandem action of NPC2 and NPC1 in a process that requires a physical association between NPC1 and NPC2 (27). However, in the presence of CYCLO, cholesterol that is stored in the LE/L can be transported to, and across, the limiting membrane of the LE/L, independent of either NPC1 or NPC2, and can subsequently be exported to the ER (by an 20

21 undefined mechanism). Since CYCLO reduces the storage of cholesterol in LE/L of either NPC1- or NPC2-deficient cells (100,173), CYCLO can apparently bypass the function of both proteins. In vitro studies have shown that CYCLO markedly accelerates the transfer of cholesterol between membranes via a collisional mechanism involving a direct interaction between CYCLO and membranes (177). The mechanism by which CYCLO mobilizes cholesterol from the LE/L was further investigated using two types of β-cyclodextrin: CYCLO (2-hydroxypropyl-β-cyclodextrin) and sulfobutylether-β-cyclodextrin (167,176). Previous studies have shown that 2-hydroxypropylβ-cyclodextrin (CYCLO), at concentrations of >1 mm, removes cholesterol from membranes into the aqueous medium by formation of a 2:1 molar complex of CYCLO:cholesterol. In contrast, cholesterol forms a 1:1 complex with sulfobutylether-β-cyclodextrin that cannnot solubilize cholesterol from membranes. At lower cyclodextrin concentrations (< 1 mm), however, both CYCLO and sulfobutylether-β-cyclodextrin can shuttle cholesterol between membranes (171). To determine if CYCLO removed cholesterol from the LE/L of NPC-deficient cells by solubilization or by a shuttle mechanism, Npc1 -/- mice were injected with either CYCLO or sulfobutylether-β-cyclodextrin (167,176). With both types of cyclodextrin, the mobilized cholesterol did not rapidly (at least within 12 h) appear in the circulation, either in the form of lipoproteins or as cholesterol:cyclo complexes. Nor was the released cholesterol excreted into the urine (176). However, both types of cyclodextrin reduced the expression of SREBP-responsive genes to a similar extent. Thus, the transfer of stored cholesterol out of the LE/L of Npc1 -/- cells did not depend on the ability of CYCLO to solubilize cholesterol into an intracellular or extracellular aqueous milieu. More likely, CYCLO shuttles cholesterol between vesicles within the LE/L lumen and the perimeter membrane of the LE/L, so that the cholesterol reaches the exit site on the LE/L limiting membrane without the participation of NPC1 or NPC2 (176). In an attempt to increase the efficiency of cholesterol clearance from the LE/L, supramolecular 21

22 complexes of cyclodextrin (polyrotaxanes) were designed, in which cyclodextrin molecules were threaded onto a bio-compatible, linear polymer that was capped by bulky end-groups that were amenable to enzymatic cleavage (178,179). These complexes were endocytosed by cells and high concentrations of cyclodextrin was released into the LE/L lumen upon enzymatic cleavage of the endcaps. Thus, treatment of NPC2-deficient fibroblasts with CYCLO-polyrotaxane complexes efficiently mobilized cholesterol from the LE/L (178,179). Moreover, the activity of cyclodextrin on the plasma membrane was avoided. Whether or not these complexes will also be effective in the CNS remains to be determined. Several other neurodegenerative disorders, such as Alzheimer s disease and Huntington s disease, are also associated with impaired cholesterol homeostasis in the brain [reviewed in (180)]. Some similarities have been noted between Alzheimer s disease and NPC disease, including alterations in lysosomal function (40,181), increased accumulation of β amyloid ( ) and the presence of neurofibrillary tangles in humans (185) [but not in Npc1 -/- mice (106)]. Interestingly, NPC1 expression is higher in the hippocampus and frontal cortex of Alzheimer patients than in control patients (186). Genetic over-expression of the amyloid precursor protein in Npc1 -/- mice significantly decreased the lifespan of the mice, whereas CYCLO prolonged survival (187). CYCLO also reduced cholesterol sequestration in the brains of mouse models of Alzheimer s disease and lowered the levels of β amyloid by reducing β-cleavage of the amyloid precursor protein and by increasing the expression of genes involved in the clearance of β-amyloid (188). These observations raise the possibility that CYCLO might be a useful therapeutic agent for treatment of Alzheimer s disease and, perhaps also, other neurodegenerative disorders in which cholesterol homoestasis is disrupted. 8. Conclusions 22

23 Identification of the defective genes in NPC disease has led to elucidation of the mechanism by which NPC1 and NPC2 proteins mediate the egress of cholesterol from LE/L. Currently, no effective treatment is available for NPC patients although the remarkable discovery that treatment of Npc1 -/- and Npc2 -/- mice with the cholesterol-sequestering agent, CYCLO, improves the neurodegeneration and prolongs life, are very encouraging. Nevertheless, because of the low permeability of the blood-brain barrier to CYCLO, it appears that administration of CYCLO directly into the central nervous system of NPC patients would be the most beneficial approach. Thus, a reasonable protocol for the treatment of NPC patients might be to inject CYCLO into the peripheral circulation and simultaneously administer CYCLO directly into the brain via an osmotic mini-pump. While such an approach would be cumbersome, both the liver disease and the neurodegeneration might be treatable. The most effective, non-toxic concentration of CYCLO in the brain must also be established for NPC patients but, based on the experiments in mice, this concentration is expected to be ~ 0.1 mm. An unresolved issue, however, is the lack of effectiveness of CYCLO against the lung disease in NPC-deficient mice. Another important requirement for clinical studies with CYCLO in NPC patients is that a reliable and convenient method must be developed for assessment of clinical progression of the disease. Some advances have been made on this front. For example, the concentrations of several oxidation products of cholesterol, including 25-hydroxycholesterol, 7-ketocholesterol and 7α,7β hydroxycholesterol, are higher in plasma of Npc1 -/- mice than in Npc1 +/+ mice. Moreover, levels of these oxysterols correlate with disease progression (68). Although CYCLO appears to be relatively non-toxic, the observation that CYCLO-treated NPC-deficient cats develop a hearing defect, raises some concerns about this possible side-effect in humans treated with CYCLO. Furthermore, although CYCLO is a very promising agent for prevention of the neurodegeneration in NPC disease, it is unlikely that CYCLO or any of the other proposed treatments would be able to replace neurons that had already been lost. Thus, 23

24 one of the greatest challenges for treatment for NPC patients is obtaining an early diagnosis of the disease. 24

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38 FIGURE LEGENDS Fig. 1. Proposed mechanism for the concerted action of NPC1/NPC2 in cholesterol export from LE/L. NPC2 (blue), a soluble protein in the LE/L lumen, binds cholesterol derived from endocytosed LDL (#1). Subsequently, NPC2 shuttles the cholesterol to the cholesterol-binding pocket of NPC1 (green), a polytopic protein in the LE/L limiting membrane, thereby avoiding direct contact of the hydrophobic cholesterol molecule with the aqueous LE/L lumen (#2). The mechanisms by which NPC1-associated cholesterol is exported from the outer LE/L membrane to the ER and plasma membrane (#3) have not been established. Reproduced from (165) with permission. Fig. 2. Cholesterol sequestration in LE/L of Npc1-deficient neurons. Primary hippocampal neurons from E17 Npc1 -/- (A-C) and Npc +/+ (D-F) mice were cultured for 14 days in serum-free medium, then stained for cholesterol (filipin) (A, D) and with antibodies directed against the lysosome-associated membrane protein-1, LAMP1 (B, E); merge (C, F). Scale bar = 20 µm. Fig. 3. CYCLO prevents Purkinje cell loss in Npc1 -/- mice. Npc1 -/- (B, C, E, F, H, I) and Npc +/+ (A, D, G) mice (7 days old) were given a single intraperitoneal injection of CYCLO (4,000 mg/kg body wt). At 49 days of age, sections of the cerebellum were stained with hematoxylin and eosin (H&E) (A-C), and also stained for calbindin (Purkinje cell marker) (D-F), and glial fibrillary acidic protein (marker of astrocyte activation) (G-I). Scale bar = 100 µm. Arrows indicate loss of Purkinje cells in a bandlike pattern (indicated by brackets) in untreated Npc1 -/- mice; *, necrotic Purkinje cells. Reproduced from (98) with permission.

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