Lysosomal vitamin E accumulation in Niemann-Pick type C disease

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.     

Lysosomal vitamin E accumulation in Niemann-Pick type C disease

Niemann-Pick C disease (NPC) is a neuro-visceral lysosomal storage disorder mainly caused by genetic defects in the NPC1 gene. As a result of loss of NPC1 function large quantities of free cholesterol and other lipids accumulate within late endosomes and lysosomes. In NPC livers and brains, the buildup of lipids correlates with oxidative damage; however the molecular mechanisms that trigger it remain unknown. Here we study potential alterations in vitamin E (α-tocopherol, α-TOH), the most potent endogenous antioxidant, in liver tissue and neurons from NPC1 mice. We found increased levels of α-TOH in NPC cells. We observed accumulation and entrapment of α-TOH in NPC neurons, mainly in the late endocytic pathway. Accordingly, α-TOH levels were increased in cerebellum of NPC1 mice. Also, we found decreased mRNA levels of the α-TOH transporter, α-Tocopherol Transfer Protein (α-TTP), in the cerebellum of NPC1 mice. Finally, by subcellular fractionation studies we detected a significant increase in the hepatic α-TOH content in purified lysosomes from NPC1 mice. In conclusion, these results suggest that NPC cells cannot transport vitamin E correctly leading to α-TOH buildup in the endosomal/lysosomal system. This may result in a decreased bioavailability and impaired antioxidant function of vitamin E in NPC, contributing to the disease pathogenesis.     

Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders

The 20-fold increase of free sphingoid bases found in liver from a murine model of Niemann-Pick type C (NPC) combined to the NPC-like phenotype induced by addition of sphinganine to normal fibroblast cultures prompted us to investigate the potential involvement of these compounds in the human disease. The contents of sphingosine and sphinganine were measured in liver, spleen, brain and skin fibroblast cultures by a sensitive HPLC method. In liver and spleen from NPC patients, a 6- to 24-fold elevation of sphingosine and sphinganine already prominent at the fetal stage of the disease was observed, while no clear increase could be evidenced in brain tissue. A significant increase, not modulated by the intralysosomal content of free cholesterol, also occurred in skin fibroblast cultures. To investigate the specificity of these findings, other lysosomal storage disorders were studied. A striking accumulation was found in liver and spleen (24- to 36-fold) from patients with Niemann-Pick disease type A and B (sphingomyelinase-deficient forms), and in cerebral cortex of type A Niemann-Pick disease. A significant storage also occurred in Sandhoff disease, while several other sphingolipidoses showed a moderate elevation. In all cases but Sandhoff disease brain, the sphingosine/sphinganine ratio remained unchanged, suggesting that the accumulated free sphingoid bases derived from sphingolipid catabolism. Formation of complexes between sphingosine and the lipid material accumulated in lysosomes might be a general mechanism in lysosomal lipidoses. In NPC, however, an increase of free sphingoid bases disproportionate to the degree of lysosomal storage and a specific involvement of cultured fibroblasts suggested a more complex or combined mechanism.     

Phenanthridin-6-one derivatives as the first class of non-steroidal pharmacological chaperones for Niemann-Pick disease type C1 protein

Niemann-Pick disease type C is a fatal, progressive neurodegenerative disease mostly caused by mutations in Nieamnn-Pick type C1 (NPC1), a late endosomal membrane protein that is essential for intracellular cholesterol transport. The most prevalent mutation, I1061T (Ile to Thr), interferes with the protein folding process. Consequently, mutated but intrinsically functional NPC1 proteins are prematurely degraded via proteasome, leading to loss of NPC1 function. Previously, we reported sterol derivatives as pharmacological chaperones for NPC1, and showed that these derivatives can normalize folding-defective phenotypes of I1061T NPC1 mutant by directly binding to, and stabilizing, the protein. Here, we report a series of compounds containing a phenanthridin-6-one scaffold as the first class of non-steroidal pharmacological chaperones for NPC1. We also examined their structure-activity relationships.     

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder,Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.     

Restarting stalled autophagy a potential therapeutic approach for the lipid storage disorder,Niemann-Pick type C1 disease

Autophagy is essential for cellular homeostasis and its dysfunction in human diseases has been implicated in the accumulation of misfolded protein and in cellular toxicity. We have recently shown impairment in autophagic flux in the lipid storage disorder, Niemann-Pick type C1 (NPC1) disease associated with abnormal cholesterol sequestration, where maturation of autophagosomes is impaired due to defective amphisome formation caused by failure in SNARE machinery. Abrogation of autophagy also causes cholesterol accumulation, suggesting that defective autophagic flux in NPC1 disease may act as a primary causative factor not only by imparting its deleterious effects, but also by increasing cholesterol load. However, cholesterol depletion treatment with HP-β-cyclodextrin impedes autophagy, whereas pharmacologically stimulating autophagy restores its function independent of amphisome formation. Of potential therapeutic relevance is that a low dose of HP-β-cyclodextrin that does not perturb autophagy, coupled with an autophagy inducer, may rescue both the cholesterol and autophagy defects in NPC1 disease.     

Defective cholesterol traffic and neuronal differentiation in neural stem cells of Niemann-Pick type C disease improved by valproic acid, a histone deacetylase inhibitor

Niemann-Pick type C disease (NPC) is a neurodegenerative and lipid storage disorder for which no effective treatment is known. We previously reported that neural stem cells derived from NPC1 mice showed impaired self-renewal and differentiation. We examined whether valproic acid (VPA), a histone deacetylase inhibitor, could enhance neuronal differentiation and recover defective cholesterol metabolism in neural stem cells (NSCs) from NPC1-deficient mice (NPC1(-/-)). VPA could induce neuronal differentiation and restore impaired astrocytes in NSCs from NPC1(-/-) mice. Importantly, an increasing level of cholesterol within NSCs from NPC1(-/-) mice could be reduced by VPA. Moreover, essential neurotrophic genes (TrkB, BDNF, MnSoD, and NeuroD) were up-regulated through the repression of the REST/NRSF and HDAC complex by the VPA treatment. Up-regulated neurotrophic genes were able to enhance neural differentiation and cholesterol homeostasis in neural stem cells from NPC1(-/-) mice. In this study, we suggested that, along with cholesterol homeostasis, impaired neuronal differentiation and abnormal morphology of astrocytes could be rescued by the inhibition of HDAC and REST/NRSF activity induced by VPA treatment.     

Annexins in Niemann-Pick type C disease

Niemann-Pick disease is a genetic disorder, affecting approximately 1 to 150,000 living births per year; in Poland 1-5 cases. Usually diagnosed in the childhood, Niemann-Pick disease results in death in the teenage years. Niemann-Pick disease is defined as a lysosomal storage disorder and is related to impaired transport and/or accumulation of specific lipids inside the cell. In this report, we provide evidence about potential role of annexins, calcium- and membrane-binding proteins, in the formation and stabilization of cholesterol-rich microdomains and their possible function in organizing the membranes of early and late endosomes, organelles affected in the type C Niemann-Pick disease characterized by abnormal accumulation of cholesterol and glycosphingolipids in lysosomal like organelles.     

Liver findings in Niemann-Pick disease type C

Summary A complex analysis of liver from a series of eight cases of Niemann-Pick disease type C showed practically generalized storage of glycolipids and phosphoglycerides by chemical and histochemical techniques. In six of the eight cases the storage process was of low degree, barely recognizable by routine histology, but well recognizable by histochemistry and electronmicroscopy. In two cases it was marked and led to early functional impairment of the liver. Changes in various enzyme activities and in ultrastructural features of the storage process are described. Sphingomyelin was found to participate to a very low low degree and its accumulation was not proportional to the extent of overall storage. In two cases with prominent involvement of the liver normal levels of sphingomyelin were found. In other cases sphingomyelin was found, by lipid histochemistry, to be stored only in macrophages. To stress that the storage process in Niemann-Pick disease type C is qualitatively different a comparison was made with liver findings in sphingomyelinase-deficient patients. This feature is of practical as well as theoretical importance.     

Cholesterol as a factor regulating intracellular localization of annexin A6 in Niemann-Pick type C human skin fibroblasts

Lysosome-like storage organelles (LSOs) play a crucial role in excessive accumulation of cholesterol in the Niemann-Pick type C (NPC) disease characterized by altered vesicular traffic of lipids. Annexin A6 (AnxA6) is mainly present in cytosol but upon elevation of [Ca²⁺]_in binds to membranes. In addition, a pH or cholesterol-dependent mechanism of AnxA6 interaction with membranes was described. We found a several fold enrichment of AnxA6 in LSO compartment in fibroblasts isolated from NPC patients in comparison with fibroblasts from healthy individuals. We observed that AnxA6 relocates from cytosol to LSOs in a cholesterol-dependent manner. Cholesterol depletion caused reduction in the binding of AnxA6. Moreover, we found that in NPC cells AnxA6 translocates to the perinuclear region containing late endosomes (LE) loaded with cholesterol. We conclude that AnxA6 may participate in formation of cholesterol-rich platforms on LE and therefore may contribute to the pathology of the NPC disease.     

Regulation of intracellular cholesterol metabolism is defective in lymphoblasts from Niemann-Pick type C and type D patients

Regulation of intracellular cholesterol metabolism has been studied in Epstein-Barr virus-transformed lymphoblasts from patients with Niemann-Pick type C (NPC) and the Nova Scotia type D (NPD) disease. Addition of LDL to normal lymphoblasts cultured in lipoprotein-deficient medium increased cholesterol esterification 10-fold (to a maximum of 1.0 nmol/h/mg protein at 15 h), while little stimulation was seen in NPC cells. The response by NPD lymphoblasts was intermediate, reaching approximately half of normal values by 12–24 h. Lymphoblasts from both NPC and NPD obligate heterozygotes exhibited 50% of normal LDL-stimulated cholesterol esterification at 6 h, when activity was $\text{s}\text{1}\text{?}\text{0\%}$ of normal values in patient cells. Fluorescence staining with filipin indicated excessive intracellular accumulation of LDL-derived cholesterol in both NPC and NPD lymphoblasts. Downregulation of LDL receptor mRNA levels by LDL, measured by S1 nuclease protection assay, was also impaired in NP lymphoblasts and fibroblasts (NPC > NPD), although a similar rate of receptor protein down-regulation by LDL ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 10–15}\text{h}$) was observed in normal and NP lymphoblasts. In contrast, LDL down-regulation of 3-hydroxy-3-methylglutaryl-CoA reductase mRNA did not appear to be affected in NP cells: LDL produced a 3-fold (lymphoblasts) of > 10-fold (fibroblasts) decrease by 12 h in both normal and affected cells. Thus, NPC and NPD lymphoblasts exhibit distinct defects in cholesterol esterification and storage, similar to those observed in mutant fibroblasts. Other regulatory responses are also impaired in NPC lymphoblasts but appear to be less affected in NPD cells. Lymphoblasts should provide a valuable immortalized cell line model for study of defective regulation of cholesterol esterification and transfort in Niemann-Pick type II disease, and may also suitable for diagnosis and carrier detection.     

Computational screening of disease associated mutations on NPC1 gene and its structural consequence in Niemann-Pick type-C1

Niemann-Pick disease type C1 （NPC1）, caused by mutations of NPC1 gene, is an inherited lysosomal lipid storage disorder. Loss of functional NPC1 causes the accumulation of free cholesterol （FC） in endocytic organelles that comprised the characteristics of late endosomes and/or lysosomes. In this study we analyzed the pathogenic effect of 103 nsSNPs reported in NPC1 using computational methods. Rl186C, S940L, R958Q and I1061T mutations were predicted as most deleterious and disease associated with NPC1 using SIFT, Polyphen 2.0, PANTHER, PhD-SNP, Pmut and MUTPred tools which were also endorsed with previous in vivo experimental studies. To understand the atomic arrangement in 3D space, the native and disease associated mutant （Rl186C, S940L, R958Q and I1061T） structures were modeled. Quantitative structural and flexibility analysis was conceded to observe the structural consequence of prioritized disease associated mutations （R1186C, S940L, R958Q and I1061T）. Accessible surface area （ASA）, free folding energy （FFE） and hydrogen bond （NH bond） showed more flexibility in 3D space in mutant structures. Based on the quantitative assessment and flexibility analysis of NPC1 variants, I1061T showed the most deleterious effect. Our analysis provides a clear clue to wet laboratory scientists to understand the structural and functional effect of NPCI gene upon mutation.     

Cellular pathology of Niemann-Pick type C disease

Niemann-Pick type C (NPC) is a lysosomal storage disorder that results in the accumulation of cholesterol and sphingolipids. Mutations in the NPC1 or NPC2 gene are responsible for the disease but the precise functions of the encoded proteins remain unresolved. Recent observations have challenged the traditional concept of NPC as a primary cholesterol transport defect. This review updates the recent NPC literature, summarizing the increasing insight into the cholesterol trafficking circuits and also addressing the contribution of other lipids in the cellular pathogenesis. The importance of NPC as a model for subcellular lipid imbalance in studying more common diseases, such as Alzheimer's and cardiovascular diseases, is discussed.     

Therapy of Niemann-Pick disease, type C

Niemann-Pick disease, type C (NPC) is a progressive autosomal recessive neurodegenerative disease, characterized by late endosomal-lysosomal accumulation of multiple lipid molecules in association with abnormal tubulovesicular trafficking. The major gene product, NPC1 protein, is not suitable for transduction therapies, and gene replacement or repair is not yet practicable for NPC and related disorders. Attempts at therapy to date have focused on reduction of the accumulating molecules that are presumed to have direct or indirect toxic effects. More recent insights into the pathophysiology of NPC raise the possibility of small molecule therapies to interdict pathways triggering apoptosis and related routes to cell death and dysfunction.     

Lipid Changes in Niemann-Pick Disease Type C Brain: Personal Experience and Review of the Literature

Niemann-Pick disease type C (NPC) is a neurovisceral disorder characterized by lysosomal sequestration of endocytosed LDL-cholesterol, premature and abnormal enrichment of cholesterol in trans Golgi cisternae and accompanying anomalies in intracellular sterol trafficking. In addition to cholesterol, the NPC lesion has also been shown to impact the metabolism of sphingolipids. Lipids, more particularly glycolipids, were studied in brain tissue from eight cases with proven NPC, ranging from 21 fetal weeks to 19 years of age (one case with rapidly fatal neonatal cholestatic icterus, three cases with infantile neurological onset, one late infantile and two juvenile neurological cases). In gray matter, the concentrations of total cholesterol, sphingomyelin and total gangliosides were within the normal range in all cases. In white matter, a severe loss of galactosylceramide and other myelin lipids (including cholesterol) was prominent in patients with the neurological severe infantile form (levels similar to those in 6–8 month-old infants) or the late infantile form of the disease, but only a slight decrease was observed in patients with a juvenile neurological onset. Analysis of the ganglioside profiles and study of minor neutral glycolipids revealed striking abnormalities, although not present at the fetal stage. In cerebral cortex, gangliosides GM3 and GM2 showed a significant increase, 10–15 fold and 3–5-fold the normal level, respectively, with already some abnormalities in a 3-month-old patient. Except in the latter patient, a prominent storage of glucosylceramide, lactosylceramide and gangliotriaosylceramide (asialo-GM2) was observed, with 10–50-fold increases from the normal concentration. The fatty acid composition of these glycolipids suggests that they have a neuronal origin. A slight increase of globotriaosyl- and globotetraosyl-ceramide and of more complex neutral glycolipids also occurred. While ganglioside changes were essentially similar in gray and white matter, changes of the neutral glycolipids were only minimal in the latter. Our data are in good accordance with previous studies and provide additional information. They emphasize that, apart a varying demyelinating process (most pronounced in children with a severe infantile neurological form) brain lipids abnormalities are essentially located to the gray matter. They confirm and give more precise information on the glycolipid nature of the neuronal storage, and establish that a similar type of changes occurs in the different neurological forms of the disease. Yet, our study indicates that glycolipid changes in brain do not occur before a few months after birth, possibly at a period concomitant with the onset of neurological symptoms, in contrast to the very early glycolipid abnormalities observed in non-neural organs. Glycolipid changes rather similar to those seen in NPC brain, in particular for gangliosides, have been described for other lysosomal disorders such as Niemann-Pick type A and mucopolysaccharidoses. The glucosyl-and lactosylceramide accumulation, however, is more striking in NPC, especially taking into account that there is no other known storage in NPC brain. Some neuropathological changes, such as ectopic neurites, could be related to the glycolipid changes. Metabolic studies in cultured fibroblasts combined to the observation that no lipids other than glycolipids accumulate in brain suggest that the NPC gene products possibly participate in intracellular transport or regulate metabolism of glycolipids.