?-Cyclodextrin-threaded biocleavable polyrotaxanes ameliorate impaired autophagic flux in Niemann-Pick type C disease.
ABSTRACT: 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 type C (NPC) disease is characterized by the accumulation of cholesterol in lysosomes. We have previously reported that biocleavable polyrotaxanes (PRXs) composed of ?-cyclodextrins (?-CDs) threaded onto a linear polymer capped with bulky stopper molecules via intracellularly cleavable linkers show remarkable cholesterol reducing effects in NPC disease patient-derived fibroblasts owing to the stimuli-responsive intracellular dissociation of PRXs and subsequent ?-CD release from the PRXs. Herein, we describe a series of novel acid-labile 2-(2-hydroxyethoxy)ethyl group-modified PRXs (HEE-PRXs) bearing terminal N-triphenylmethyl (N-Trt) groups as a cleavable component for the treatment of NPC disease. The N-Trt end groups of the HEE-PRXs underwent acidic pH-induced cleavage and led to the dissociation of their supramolecular structure. A kinetic study revealed that the number of HEE groups on the PRX did not affect the cleavage kinetics of the N-Trt end groups of the HEE-PRXs. The effect of the number of HEE groups of the HEE-PRXs, which was modified to impart water solubility to the PRXs, on cellular internalization efficiency, lysosomal localization efficiency, and cholesterol reduction ability in NPC disease-derived fibroblasts (NPC1 fibroblasts) was also investigated. The cellular uptake and lysosomal localization efficiency were almost equivalent for HEE-PRXs with different numbers of HEE groups. However, the cholesterol reducing ability of the HEE-PRXs in NPC1 fibroblasts was affected by the number of HEE groups, and HEE-PRXs with a high number of HEE groups were unable to reduce lysosomal cholesterol accumulation. This deficiency is most likely due to the cholesterol-solubilizing ability of HEE-modified ?-CDs released from the HEE-PRXs. We conclude that the N-Trt group acts as a cleavable component to induce the lysosomal dissociation of HEE-PRXs, and acid-labile HEE-PRXs with an optimal number of HEE groups (4.1 to 5.4 HEE groups per single ?-CD threaded onto the PRX) have great therapeutic potential for treating NPC disease.
Project description:Niemann-Pick type C (NPC) disease is an autosomal recessive lysosomal trafficking disorder, in which the cholesterols are abnormally accumulated in lysosomes. Recently, the ?-cyclodextrin (CD) derivatives are revealed to show therapeutic effect for NPC disease through the removal of accumulated cholesterols in lysosomes. Herein, to enhance the therapeutic effect and reduce the toxicity of ?-CD derivatives, biocleavable Pluronic/?-CD-based polyrotaxanes (PRXs) bearing terminal disulfide linkages that can release threaded ?-CDs in lysosomes were developed. The biocleavable PRXs show negligible interaction with the plasma membrane, leading to avoiding the toxicity of ?-CDs derived from their hydrophobic cavity. Additionally, lysosomal release of threaded ?-CDs from biocleavable PRXs by the intracellular cleavage of terminal disulfide linkages is found to achieve approximately 100-fold higher cholesterol removal ability from NPC disease-derived cells than ?-CD derivatives. Consequently, the biocleavable PRXs is considered to be a noninvasive and effective therapeutics for NPC disease.
Project description:To improve the therapeutic potential of β-cyclodextrin (β-CD)-threaded acid-degradable polyrotaxanes (β-CD PRXs) in cholesterol-related metabolic disorders, we investigated the effect of carboxylation of β-CD PRXs on intracellular uptake. In this study, we established a synthetic method for the modification of carboxylalkyl carbamates on β-CD PRXs without degradation and synthesized three series of carboxyalkyl carbamate group-modified β-CD PRXs with different alkyl spacer lengths. The modification of carboxymethyl carbamate (CMC), carboxyethyl carbamate (CEC), and carboxypropyl carbamate (CPC) on the β-CD PRXs slightly reduced the interaction of the PRXs with the lipid layer model compared with the modification of 2-(2-hydroxyethoxy)ethyl carbamate (HEE-PRX), which was used in our previous studies. However, all the carboxylated β-CD PRXs showed a significantly stronger interaction with a protein model compared with HEE-PRX. The carboxylated β-CD PRXs showed significantly high intracellular uptake, through macrophage scavenger receptor A (MSR-A)-mediated endocytosis, in MSR-A-positive RAW 264.7 cells compared with HEE-PRX. Interestingly, the carboxylated β-CD PRXs also showed significantly higher intracellular uptake even in MSR-A-negative cells compared with HEE-PRX. Carboxylated β-CD PRXs are considered to strongly interact with other membrane proteins, resulting in high intracellular uptake. The length of the alkyl spacer affected the intracellular uptake levels of carboxylated PRXs, however, this relationship was varied for different cell types. Furthermore, none of the carboxylated β-CD PRXs exhibited cytotoxicity in the RAW 264.7 and NIH/3T3 cells. Altogether, carboxylation of β-CD PRXs is a promising chemical modification approach for their therapeutic application because carboxylated β-CD PRXs exhibit high cellular internalization efficiency in MSR-A-negative cells and negligible toxicity.
Project description:Cyclodextrin (CD)-threaded polyrotaxanes (PRXs) with reactive functional groups at the terminals of the axle polymers are attractive candidates for the design of supramolecular materials. Herein, we describe a novel and simple synthetic method for end-reactive PRXs using bis(2-amino-3-phenylpropyl) poly(ethylene glycol) (PEG-Ph-NH2) as an axle polymer and commercially available 4-substituted benzoic acids as capping reagents. The terminal 2-amino-3-phenylpropyl groups of PEG-Ph-NH2 block the dethreading of the ?-CDs after capping with 4-substituted benzoic acids. By this method, two series of azide group-terminated polyrotaxanes (benzylazide: PRX-Bn-N3, phenylazide: PRX-Ph-N3,) were synthesized for functionalization via click reactions. The PRX-Bn-N3 and PRX-Ph-N3 reacted quickly and efficiently with p-(tert-butyl)phenylacetylene via copper-catalyzed click reactions. Additionally, the terminal azide groups of the PRX-Bn-N3 could be modified with dibenzylcyclooctyne (DBCO)-conjugated fluorescent molecules via a copper-free click reaction; this fluorescently labeled PRX was utilized for intracellular fluorescence imaging. The method of synthesizing end-reactive PRXs described herein is simple and versatile for the design of diverse functional PRXs and can be applied to the fabrication of PRX-based supramolecular biomaterials.
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 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 (NPCD) is a lysosomal storage disease (LSD) characterized by abnormal cholesterol accumulation in lysosomes, impaired autophagy flux, and lysosomal dysfunction. The activation of transcription factor EB (TFEB), a master lysosomal function regulator, reduces the accumulation of lysosomal substrates in LSDs where the degradative capacity of the cells is compromised. Genistein can pass the blood-brain barrier and activate TFEB. Hence, we investigated the effect of TFEB activation by genistein toward correcting the NPC phenotype. We show that genistein promotes TFEB translocation to the nucleus in HeLa TFEB-GFP, Huh7, and SHSY-5Y cells treated with U18666A and NPC1 patient fibroblasts. Genistein treatment improved lysosomal protein expression and autophagic flux, decreasing p62 levels and increasing those of the LC3-II in NPC1 patient fibroblasts. Genistein induced an increase in β-hexosaminidase activity in the culture media of NPC1 patient fibroblasts, suggesting an increase in lysosomal exocytosis, which correlated with a decrease in cholesterol accumulation after filipin staining, including cells treated with U18666A and NPC1 patient fibroblasts. These results support that genistein-mediated TFEB activation corrects pathological phenotypes in NPC models and substantiates the need for further studies on this isoflavonoid as a potential therapeutic agent to treat NPCD and other LSDs with neurological compromise.
Project description: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:Niemann-Pick disease Type C (NPC) is a rare lysosomal storage disease characterized by the dysfunction of intracellular cholesterol trafficking with progressive neurodegeneration and hepatomegaly. We evaluated the potential of 6-<i>O</i>-?-maltosyl-?-cyclodextrin (G2-?-CD) as a drug candidate against NPC. The physicochemical properties of G2-?-CD as an injectable agent were assessed, and molecular interactions between G2-?-CD and free cholesterol were studied by solubility analysis and two-dimensional proton nuclear magnetic resonance spectroscopy. The efficacy of G2-?-CD against NPC was evaluated using <i>Npc1</i> deficient Chinese hamster ovary (CHO) cells and <i>Npc1</i> deficient mice. G2-?-CD in aqueous solution showed relatively low viscosity and surface activity; characteristics suitable for developing injectable formulations. G2-?-CD formed higher-order inclusion complexes with free cholesterol. G2-?-CD attenuated dysfunction of intercellular cholesterol trafficking and lysosome volume in <i>Npc1</i> deficient CHO cells in a concentration dependent manner. Weekly subcutaneous injections of G2-?-CD (2.9 mmol/kg) ameliorated abnormal cholesterol metabolism, hepatocytomegaly, and elevated serum transaminases in <i>Npc1</i> deficient mice. In addition, a single cerebroventricular injection of G2-?-CD (21.4 ?mol/kg) prevented Purkinje cell loss in the cerebellum, body weight loss, and motor dysfunction in <i>Npc1</i> deficient mice. In summary, G2-?-CD possesses characteristics favorable for injectable formulations and has therapeutic potential against in vitro and in vivo NPC models.
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.