Differential Effects of 2-Hydroxypropyl-Cyclodextrins on Lipid Accumulation in Npc1-Null Cells.
ABSTRACT: Niemann-Pick disease type C (NPC) is an autosomal recessive disorder characterized by abnormal accumulation of free cholesterol and sphingolipids in lysosomes. The iminosugar miglustat, which inhibits hexosylceramide synthesis, is used for NPC treatment, and 2-hydroxypropyl-?-cyclodextrin (HP-?-CD), a cyclic oligosaccharide derivative, is being developed to treat NPC. Moreover, therapeutic potential of 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) was shown in NPC models, although its mechanism of action remains unclear. Here, we investigated the effects of HP-?-CD, HP-?-CD, and their homolog 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) on lipid accumulation in Npc1-null Chinese hamster ovary (CHO) cells compared with those of miglustat. HP-?-CD and HP-?-CD, unlike HP-?-CD, reduced intracellular free cholesterol levels and normalized the lysosome changes in Npc1-null cells but not in wild-type CHO cells. In contrast, miglustat did not normalize intracellular free cholesterol accumulation or lysosome changes in Npc1-null cells. However, miglustat decreased the levels of hexosylceramide and tended to increase those of sphingomyelins in line with its action as a glucosylceramide synthase inhibitor in both Npc1-null and wild-type CHO cells. Interestingly, HP-?-CD and HP-?-CD, unlike HP-?-CD, reduced sphingomyelins in Npc1-null, but not wild-type, cells. In conclusion, HP-?-CD and HP-?-CD reduce the accumulation of sphingolipids, mainly sphingomyelins, and free cholesterol as well as lysosome changes in Npc1-null, but not in wild-type, CHO cells.
Project description:The drug 2-hydroxypropyl-?-cyclodextrin (HP?CD) reduces lysosomal cholesterol accumulation in Niemann-Pick disease, type C (NPC) and has been advanced to human clinical trials. However, its mechanism of action for reducing cholesterol accumulation in NPC cells is uncertain and its molecular target is unknown. We found that methyl-?-cyclodextrin (M?CD), a potent analog of HP?CD, restored impaired macroautophagy/autophagy flux in Niemann-Pick disease, type C1 (NPC1) cells. This effect was mediated by a direct activation of AMP-activated protein kinase (AMPK), an upstream kinase in the autophagy pathway, through M?CD binding to its ?-subunits. Knockdown of PRKAB1 or PRKAB2 (encoding the AMPK ?1 or ?2 subunit) expression and an AMPK inhibitor abolished M?CD-mediated reduction of cholesterol storage in NPC1 cells. The results demonstrate that AMPK is the molecular target of M?CD and its activation enhances autophagy flux, thereby mitigating cholesterol accumulation in NPC1 cells. The results identify AMPK as an attractive target for drug development to treat NPC.
Project description:Niemann-Pick type C (NPC) disease is a fatal hereditary neurodegenerative disorder characterized by a massive accumulation of cholesterol in lysosomes and late endosomes due to a defect in intracellular cholesterol trafficking. Dysfunction in intracellular cholesterol trafficking is responsible for about 50 rare inherited lysosomal storage disorders including NPC. The lysosomal proteins NPC1 and NPC2 play a crucial role in trafficking of cholesterol from late endosomes and lysosomes to other cellular compartments. However, the detailed mechanisms of cholesterol trafficking at the late endosomes/lysosomes (LE/LY) are poorly understood. Studies showed that 2-hydroxypropyl-?-cyclodextrin (HP?CD) alleviates the cholesterol accumulation defect in animal model and has been approved for a phase 2b/3 clinical trial for NPC. HP?CD is known to bind cholesterol; however, the mechanisms how HP?CD mediates the exit of cholesterol from the LE/LY compartments are still unknown. Further, another cyclodextrin (CD) derivative, 2-hydroxypropyl-?-cyclodextrin (HP?CD), was shown to reduce intracellular cholesterol accumulation in NPC patient cells and NPC mice model. Herein, we identified a number of candidate proteins differentially expressed in NPC patient-derived cells compared to cells derived from a healthy donor using a proteomic approach. Interestingly, both HP?CD and HP?CD treatments modulated the expression of most of these NPC-specific proteins. Data showed that treatment with both CDs induces the expression of the lysosome-associated membrane protein 1 (LAMP-1) in NPC patient-derived cells. Remarkably, LAMP-1 overexpression in HeLa cells rescued U18666A-induced cholesterol accumulation suggesting a role of LAMP-1 in cholesterol trafficking. We propose that HP?CD and HP?CD facilitate cholesterol export from the LE/LY compartments via the LAMP-1 protein, which may play a crucial role in cholesterol trafficking at the LE/LY compartments when there is no functional NPC1 protein. Together, this study uncovers new cellular mechanisms for cholesterol trafficking, which will contribute to development of novel therapeutic approaches for lysosomal storage diseases.
Project description:Niemann-Pick type C (NPC) disease is a fatal neurodegenerative disorder caused by mutations in NPC1 and NPC2 genes that result in an accumulation of cholesterol in lysosomes. The majority of children with NPC die in adolescence. Currently, no FDA-approved therapies exist for NPC and the mechanisms of NPC disease are not fully understood. Our recent study and the reports from other laboratories showed that 2-hydroxypropyl-?-cyclodextrin (HP?CD) alleviates cholesterol accumulation in NPC1-deficient cells in spite of its low binding affinity for cholesterol. In this study, we explored the cellular changes that are induced upon HP?CD treatment in NPC1 patient-derived fibroblasts. We show that HP?CD treatment increases lysosome-ER association and enhances autophagic activity. Our study indicates that HP?CD induces an activation of the transcription factor EB (TFEB), a master regulator of lysosomal functions and autophagy. Lysosome-ER association could potentially function as conduits for cholesterol transport from lysosomes to the ER. Accumulating evidence suggests a role for autophagy in rescuing the cholesterol accumulation in NPC and other degenerative diseases. Collectively, our findings suggest that HP?CD restores cellular homeostasis in NPC1-deficient cells via enhancing lysosomal dynamics and functions. Understanding the mechanisms of HP?CD-induced cellular pathways could contribute to effective NPC therapies.
Project description:Niemann-Pick type C disease (NPC) is a lysosomal storage disease that is characterized by a progressive accumulation of unesterified cholesterol in the lysosomes leading to organ damage from cell dysfunction. Hydroxypropyl-?-cyclodextrin (HP-?-CD) is an attractive drug candidate for treating NPC, as it diminishes cholesterol accumulation in NPC cells. Systemic HP-?-CD treatment, however, is limited by rapid renal clearance. We designed a new anionic HP-?-CD polyrotaxane to act as a slow release formulation based on a polyalkylene phosphate core to improve the pharmacokinetics. The polyalkylene phosphate comprises hydrophobic decamethylene spacers linked by biodegradable anionic phosphodiester bonds. HP-?-CD was threaded onto this polymer first and ?-CD afterwards to prevent burst release of the threaded HP-?-CD. Our findings show that HP-?-CD was slowly released from the watersoluble polyrotaxane over a 30 days period. The polyrotaxane provided persistently diminished cholesterol levels in NPC1 cells by 20% relative to untreated cells. These results demonstrate the therapeutic potential of this novel HP-?-CD polyrotaxane for the mobilization of aberrantly stored cholesterol in NPC1 cells.
Project description:Niemann-Pick disease type C (NPC) is a recessive hereditary disease caused by mutation of the <i>NPC1</i> or <i>NPC2</i> gene. It is characterized by abnormality of cellular cholesterol trafficking with severe neuronal and hepatic injury. In this study, we investigated the potential of glycoprotein nonmetastatic melanoma protein B (GPNMB) to act as a biomarker reflecting the therapeutic effect of 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) in an NPC mouse model. We measured serum, brain, and liver expression levels of GPNMB, and evaluated their therapeutic effects on NPC manifestations in the brain and liver after the intracerebroventricular administration of HP-?-CD in <i>Npc1</i> gene-deficient (<i>Npc1</i><sup>-/-</sup>) mice. Intracerebroventricular HP-?-CD inhibited cerebellar Purkinje cell damage in <i>Npc1</i><sup>-/-</sup> mice and significantly reduced serum and cerebellar GPNMB levels. Interestingly, we also observed that the intracerebral administration significantly reduced hepatic GPNMB expression and elevated serum ALT in <i>Npc1</i><sup>-/-</sup> mice. Repeated doses of intracerebroventricular HP-?-CD (30 mg/kg, started at 4 weeks of age and repeated every 2 weeks) drastically extended the lifespan of <i>Npc1</i><sup>-/-</sup> mice compared with saline treatment. In summary, our results suggest that GPNMB level in serum is a potential biomarker for evaluating the attenuation of NPC pathophysiology by intracerebroventricular HP-?-CD treatment.
Project description:Niemann-Pick type C (NPC) 1 disease is a rare, inherited, neurodegenerative disease. Clear evidence of the therapeutic efficacy of 2-hydroxypropyl-?-cyclodextrin (HP?CD) in animal models resulted in the initiation of a phase I/IIa clinical trial in 2013 and a phase IIb/III trial in 2015. With clinical trials ongoing, validation of a biomarker to track disease progression and serve as a supporting outcome measure of therapeutic efficacy has become compulsory. In this study, we evaluated calcium-binding protein calbindin D-28K (calbindin) concentrations in the cerebrospinal fluid (CSF) as a biomarker of NPC1 disease. In the naturally occurring feline model, CSF calbindin was significantly elevated at 3 weeks of age, prior to the onset of cerebellar dysfunction, and steadily increased to >10-fold over normal at end-stage disease. Biweekly intrathecal administration of HP?CD initiated prior to the onset of neurologic dysfunction completely normalized CSF calbindin in NPC1 cats at all time points analyzed when followed up to 78 weeks of age. Initiation of HP?CD after the onset of clinical signs (16 weeks of age) resulted in a delayed reduction of calbindin levels in the CSF. Evaluation of CSF from patients with NPC1 revealed that calbindin concentrations were significantly elevated compared with CSF samples collected from unaffected patients. Off-label treatment of patients with NPC1 with miglustat, an inhibitor of glycosphingolipid biosynthesis, significantly decreased CSF calbindin compared with pretreatment concentrations. These data suggest that the CSF calbindin concentration is a sensitive biomarker of NPC1 disease that could be instrumental as an outcome measure of therapeutic efficacy in ongoing clinical trials.
Project description:Niemann-Pick C1 (NPC1) disease is a rare, neurodegenerative lysosomal cholesterol storage disorder, typified by progressive cognitive and motor function impairment. Affected individuals usually succumb to the disease in adolescence. 2-Hydroxypropyl-?-cyclodextrin (HP-?-CD) has emerged as a promising intervention that reduces lipid storage and prolongs survival in NPC1 disease animal models. A barrier to the development of HP-?-CD and other treatments for NPC disease has been the lack of validated biochemical measures to evaluate efficacy. Here we explored whether cholesterol homeostatic responses resulting from HP-?-CD-mediated redistribution of sequestered lysosomal cholesterol could provide biomarkers to monitor treatment. Upon direct CNS delivery of HP-?-CD, we found increases in plasma 24(S)-HC in two independent NPC1 disease animal models, findings that were confirmed in human NPC1 subjects receiving HP-?-CD. Since circulating 24(S)-HC is almost exclusively CNS-derived, the increase in plasma 24(S)-HC provides a peripheral, non-invasive measure of the CNS effect of HP-?-CD. Our findings suggest that plasma 24(S)-HC, along with the other cholesterol-derived markers examined in this study, can serve as biomarkers that will accelerate development of therapeutics for NPC1 disease.
Project description:Niemann-Pick type C (NPC) disease is characterized by the lysosomal accumulation of cholesterols and impaired autophagic flux due to the inhibited fusion of autophagosomes to lysosomes. We have recently developed ?-cyclodextrin (?-CD)-threaded biocleavable polyrotaxanes (PRXs), which can release threaded ?-CDs in response to intracellular environments as a therapeutic for NPC disease. The biocleavable PRXs exhibited effective cholesterol reduction ability and negligible toxic effect compared with hydroxypropyl-?-CD (HP-?-CD). In this study, we investigated the effect of biocleavable PRX and HP-?-CD on the impaired autophagy in NPC disease. The NPC patient-derived fibroblasts (NPC1 fibroblasts) showed an increase in the number of LC3-positive puncta compared with normal fibroblasts, even in the basal conditions; the HP-?-CD treatment markedly increased the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts, indicating that autophagic flux was further perturbed. In sharp contrast, the biocleavable PRX reduced the number of LC3-positive puncta and the levels of p62 in NPC1 fibroblasts through an mTOR-independent mechanism. The mRFP-GFP-LC3 reporter gene expression experiments revealed that the biocleavable PRX facilitated the formation of autolysosomes to allow for autophagic protein degradation. Therefore, the ?-CD-threaded biocleavable PRXs may be promising therapeutics for ameliorating not only cholesterol accumulation but also autophagy impairment in NPC disease.
Project description:Niemann-Pick disease type C (NPC) is a rare neurodegenerative disorder caused by recessive mutations in the NPC1 or NPC2 gene that result in lysosomal accumulation of unesterified cholesterol in patient cells. Patient fibroblasts have been used for evaluation of compound efficacy, although neuronal degeneration is the hallmark of NPC disease. Here, we report the application of human NPC1 neural stem cells as a cell-based disease model to evaluate nine compounds that have been reported to be efficacious in the NPC1 fibroblasts and mouse models. These cells are differentiated from NPC1 induced pluripotent stem cells and exhibit a phenotype of lysosomal cholesterol accumulation. Treatment of these cells with hydroxypropyl-?-cyclodextrin, methyl-?-cyclodextrin, and ?-tocopherol significantly ameliorated the lysosomal cholesterol accumulation. Combined treatment with cyclodextrin and ?-tocopherol shows an additive or synergistic effect that otherwise requires 10-fold higher concentration of cyclodextrin alone. In addition, we found that hydroxypropyl-?-cyclodextrin is much more potent and efficacious in the NPC1 neural stem cells compared to the NPC1 fibroblasts. Miglustat, suberoylanilide hydroxamic acid, curcumin, lovastatin, pravastatin, and rapamycin did not, however, have significant effects in these cells. The results demonstrate that patient-derived NPC1 neural stem cells can be used as a model system for evaluation of drug efficacy and study of disease pathogenesis.
Project description:An injection of 2-hydroxypropyl-?-cyclodextrin (HP-?-CD) to mice lacking Niemann Pick type C (NPC) protein results in delayed neurodegeneration, decreased inflammation, and prolonged lifespan. Changes in sterol balance observed in Npc1(-/-) mice 24 h after HP-?-CD administration suggest that HP-?-CD facilitates the release of accumulated lysosomal cholesterol, the molecular hallmark of this genetic disorder. Current studies were performed to evaluate the time course of HP-?-CD effects. Within 3 h after HP-?-CD injection, decreases in cholesterol synthesis rates and increases in cholesteryl ester levels were detected in tissues of Npc1(-/-) mice. The levels of RNAs for target genes of sterol-sensing transcription factors were altered by 6 h in liver, spleen, and ileum. Despite the cholesterol-binding capacity of HP-?-CD, there was no evidence of increased cholesterol in plasma or urine of treated Npc1(-/-) mice, suggesting that HP-?-CD does not carry sterol from the lysosome into the bloodstream for ultimate urinary excretion. Similar changes in sterol balance were observed in cultured cells from Npc1(-/-) mice using HP-?-CD and sulfobutylether-?-CD, a variant that can interact with sterol but not facilitate its solubilization. Taken together, our results demonstrate that HP-?-CD works in cells of Npc1(-/-) mice by rapidly liberating lysosomal cholesterol for normal sterol processing within the cytosolic compartment.