Type C Niemann-Pick disease: dimethyl sulfoxide moderates abnormal LDL-cholesterol processing in mutant fibroblasts

Biochemical and cytochemical studies have revealed that abnormal processing of low-density-lipoprotein (LDL) cholesterol can be reversed in mutant Niemann-Pick C (NP-C) fibroblasts when 2% dimethyl sulfoxide (DMSO) is added to the culture medium. Both the excessive lysosomal accumulation of LDL cholesterol and the delayed induction of cellular homeostatic responses associated with the uptake of LDL by the mutant cells were substantially reversed by DMSO. DMSO appears to accelerate the intracellular mobilization of LDL-derived cholesterol through effects that may reflect enhanced membrane permeability or cholesterol solubilization.     

Niemann-Pick type C disease: NPC1 mutations associated with severe and mild cellular cholesterol trafficking alterations

Niemann-Pick type C disease (NPC) is a rare neurodegenerative disorder characterised by lysosomal/late endosomal accumulation of endocytosed unesterified cholesterol and delayed induction of cholesterol homeostatic reactions. The large majority of mutations in the NPC1 gene described thus far have been associated with severe cellular cholesterol trafficking impairment (classic biochemical phenotype, present in about 85% of NPC patients). In our population of 13 unrelated NP-C1 patients, among which 12 were of Portuguese extraction, we observed an unusually large proportion of families presenting mild alterations of intracellular cholesterol transport (variant biochemical phenotype), without strict correlation between the biochemical phenotype and the clinical expression of the disease. Mutational studies were carried out to compare molecular lesions associated with severe and mild cholesterol traffic impairment. Levels of NPC1 protein were studied by Western blot in cultured fibroblasts of four patients with homozygous mutant alleles. Ten novel mutations were identified (Q92R, C177Y, R518W, W942C, R978C, A1035V, 2129delA, 3662delT, IVS23+1 G>A and IVS16-82 G>A). The mutational profile appeared to be correlated with the biochemical phenotype. Splicing mutations, I1061T and A1035V, corresponded to "classic" alleles, while three missense mutations, C177Y, R978C and P1007A, could be defined as "variant" alleles. All "variant" mutations described so far appear to be clustered within the cysteine-rich luminal loop between TM 8 and 9, with the remarkable exception of C177Y. The latter mutant allele, at variance with P1007A, was correlated to a decreased level of NPC1 protein and a severe course of the disease, and disclosed a new location for "variant" mutations, the luminal loop located at the N-terminal end of the protein.     

Neurodegeneration in heterozygous Niemann-Pick type C1 (NPC1) mouse: implication of heterozygous NPC1 mutations being a risk for tauopathy

Niemann-Pick type C1 (NPC1) disease is an autosomal recessive, fatal disorder characterized by a defect in cholesterol trafficking and progressive neurodegeneration. The disease is predominantly caused by mutations in the NPC1 gene; however, it has been assumed that heterozygous NPC1 mutations do not cause any symptoms. Here we demonstrate that cholesterol accumulation does not occur in young mouse brains; however, it does in aged (104-106-week-old) NPC1+/- mouse brains. In addition, Purkinje cell loss was observed in aged NPC1+/- mouse cerebellums. Immunoblot analysis using anti-phospho-tau antibodies (AT-8, AT-100, AT-180, AT-270, PHF-1, and SMI-31) demonstrates the site-specific phosphorylation of tau at Ser-199, Ser-202, Ser-212, and Thr-214 in the brains of aged NPC1+/- mice. Mitogen-activated protein kinase, a potential serine kinase known to phosphorylate tau, was activated, whereas other serine kinases, including glycogen synthase kinase 3beta, cyclin-dependent kinase 5, or stress-activated protein kinase/c-Jun N-terminal kinase were not activated. Cholesterol level in the lipid raft isolated from the cerebral cortices, ATP level, and ATP synthase activity in the cerebral cortices significantly decreased in the aged NPC1+/- brains compared with those in the NPC1+/+ brains. All of these changes observed in NPC1+/- brains were determined to be associated with aging and were not observed in the age-matched NPC1+/+ brains. These results clearly demonstrate that heterozygous NPC1 impairs neuronal functions and causes neurodegeneration in aged mouse brains, suggesting that human heterozygous NPC1 mutations may be a risk factor for neurodegenerative disorders, such as tauopathy, in the aged population.     

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.     

Impaired dynamics of the late endosome/lysosome compartment in human Niemann-Pick type C skin fibroblasts carrying mutation in NPC1 gene

The Niemann-Pick type C (NPC) disease is characterized by accumulation of lipids within the late endosome/lysosome (LE/LY) compartment as a result of dysfunctions of the NPC1 or NPC2 proteins and an altered distribution and/or functioning of proteins involved in the regulation of membrane dynamics. In our previous report we isolated membranes of the LE/LY compartment from NPC L1 skin fibroblasts with a mutation in the NPC1 gene (exon 8, R348X) and showed that annexin A6 (AnxA6) may contribute to the impaired dynamics of these membranes in a cholesterol-dependent manner and therefore to the overnormative storage of cholesterol. In this report we show that the LE/LY fraction isolated from NPC L1 cells is characterized by a 4-fold enrichment in cholesterol, 2.5-fold in sphingomyelin and 2-fold in saturated fatty acids. As a result, the fluidity of LE/LY membranes isolated from NPC L1 cells is greatly reduced in comparison to control ones. We conclude that modified lipid composition and properties of this compartment may affect distribution and function of proteins implicated in cellular membrane dynamics. As a consequence, the backward vesicular transport of cholesterol from the LE/LY compartment to the Golgi apparatus, endoplasmic reticulum and finally to plasma membrane is impaired.     

Tau normal function influences Niemann-Pick type C disease pathogenesis in mice and modulates autophagy in NPC1-deficient cells

The tauopathies are a diverse class of devastating neurodegenerative disorders, characterized by the hyperphosphorylation and aggregation of the microtubule binding protein tau. Niemann-Pick type C disease (NPC) is a tauopathy that affects children, and is caused by mutations in intracellular lipid and cholesterol trafficking proteins. Loss-of-function mutations in the NPC1 gene are responsible for 95 percent of all NPC cases, and lead to progressive neurodegeneration and early death. To assess the extent to which tau affects NPC pathology, we generated mice that lack both NPC1 and tau. NPC1/tau double-null mutants exhibit an exacerbated NPC phenotype, including severe systemic manifestations, and die significantly earlier than NPC1 single-null mutants. Since autophagy has been previously implicated in NPC pathogenesis, we investigated the impact of tau deletion on this pathway. Acute reductions of tau in NPC1-deficient fibroblasts significantly decrease autophagic induction and flux, while having no effect on the autophagic pathway in control cells. Here we propose a model in which tau’s normal function is critical to the induction of autophagy in NPC1 deficiency, and suggest that this novel mechanism contributes to cellular dysfunction in the tauopathies.     

Intracellular transport of cholesterol in type C Niemann-Pick fibroblasts

The purpose of this study was to determine the capacity of Niemann-Pick type C (NPC) fibroblasts to transport cholesterol from the cell surface to intracellular membranes. This is relevant in light of the observations that NPC cells display a sluggish metabolism of LDL-derived cholesterol, a phenomenon which could be explained by a defective intracellular transport of cholesterol. Treatment of NPC cells for 4 h with 0.1 mg/ml of LDL failed to increase the incorporation of [14C]oleic acid into cholesterol [14C]oleate, an observation consistent with previous reports on this cell type (Pentchev et al. (1985) Proc. Natl. Acad. Sci. USA 82, 8247). Normal fibroblasts, however, displayed the classical upregulation (6-fold over control) of the endogenous esterification reaction in response to LDL exposure. Incubation of normal or NPC fibroblasts with sphingomyelinase (100 mU/ml; Staphylococcus aureus) led to a rapid and marked increase (9- and 10-fold for normal and NPC fibroblasts, respectively, after 4 h) in the esterification of plasma-membrane-derived [3H]cholesterol suggesting that sphingomyelin degradation forced a net transfer of cholesterol from the cell surface to the endoplasmic reticulum. The similar response in normal and mutant fibroblasts to the degradation of sphingomyelin suggests that plasma membrane cholesterol can be transported into the substrate pool of ACAT to about the same extent in these two cell types. Degradation of cell sphingomyelin in NPC fibroblasts also resulted in the movement of 20-25% of the cellular cholesterol from a cholesterol oxidase susceptible pool into oxidase-resistant pools, implying that a substantial amount of plasma membrane cholesterol was internalized after sphingomyelin degradation. This cholesterol internalization was not accompanied by an increased rate of membrane internalization, as measured by [3H]sucrose uptake. Although NPC cells showed a relative accumulation of unesterified cholesterol and a sluggish esterification of LDL-derived cholesterol when exposed to LDL, these cells responded like normal fibroblasts with regard to their capacity to transport cholesterol from the cell surface into intracellular sites in response to sphingomyelin degradation. It therefore appears that NPC cells, in contrast to the impaired intracellular movement of lipoprotein-derived cholesterol, do not display a general impairment of cholesterol transport between the cell surface and the intracellular regulatory pool of cholesterol.     

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.     

Progesterone metabolism in human fibroblasts is independent of P-glycoprotein levels and Niemann-Pick type C disease

Progesterone inhibits intracellular transport of lysosomal cholesterol in cultured cells, and thus at least in part mimics the biochemical phenotype of Niemann–Pick type C disease (NPC) in human fibroblasts. The goal of this study was to determine whether metabolism of progesterone to other steroids is affected by the NPC mutation or by P-glycoprotein (a known progesterone target). We found that human fibroblasts metabolize progesterone in three steps: rapid conversion to 5-pregnane-3,20-dione, which is then reduced to 5-pregnane-3β()-ol-20-one with subsequent 6-hydroxylation. The pattern and rates of progesterone metabolism were not significantly different in a variety of fibroblasts from normal individuals, NPC patients, and obligate heterozygotes. Inhibition of steroid 5-reductase with finasteride completely blocked metabolism of progesterone but had no effect on inhibition of LDL-stimulated cholesterol esterification (IC₅₀=10 μM). Progesterone also partially inhibited 25-hydroxycholesterol-induced cholesterol esterification, with similar dose-dependence in normal and NPC fibroblasts. P-glycoprotein levels varied significantly among the various fibroblasts tested, but no correlation with NPC phenotype or rate of progesterone metabolism was noted, and P-glycoprotein inhibitors did not affect conversion of progesterone to products. These results indicate that metabolism of progesterone in human fibroblasts is largely independent of its ability to interfere with cholesterol traffic and P-glycoprotein function.     

Olfactory Deficits in Niemann-Pick Type C1 (NPC1) Disease

Background

Niemann-Pick type C disease (NPC) is a rare autosomal recessive lipid storage disease characterized by progressive neurodegeneration. As only a few studies have been conducted on the impact of NPC on sensory systems, we used a mutant mouse model (NPC1^−/−) to examine the effects of this disorder to morphologically distinct regions of the olfactory system, namely the olfactory epithelium (OE) and olfactory bulb (OB).

Methodology/Principal findings

For structural and functional analysis immunohistochemistry, electron microscopy, western blotting, and electrophysiology have been applied. For histochemistry and western blotting, we used antibodies against a series of neuronal and glia marker proteins, as well as macrophage markers.NPC1^−/− animals present myelin-like lysosomal deposits in virtually all types of cells of the peripheral and central olfactory system. Especially supporting cells of the OE and central glia cells are affected, resulting in pronounced astrocytosis and microgliosis in the OB and other olfactory cortices. Up-regulation of Galectin-3, Cathepsin D and GFAP in the cortical layers of the OB underlines the critical role and location of the OB as a possible entrance gate for noxious substances. Unmyelinated olfactory afferents of the lamina propria seem less affected than ensheathing cells. Supporting the structural findings, electro-olfactometry of the olfactory mucosa suggests that NPC1^−/− animals exhibit olfactory and trigeminal deficits.

Conclusions/Significance

Our data demonstrate a pronounced neurodegeneration and glia activation in the olfactory system of NPC1^−/−, which is accompanied by sensory deficits.     

Niemann-Pick C1 disease: correlations between NPC1 mutations, levels of NPC1 protein, and phenotypes emphasize the functional significance of the putative sterol-sensing domain and of the cysteine-rich luminal loop

To obtain more information of the functional domains of the NPC1 protein, the mutational spectrum and the level of immunoreactive protein were investigated in skin fibroblasts from 30 unrelated patients with Niemann-Pick C1 disease. Nine of them were characterized by mild alterations of cellular cholesterol transport (the "variant" biochemical phenotype). The mutations showed a wide distribution to nearly all NPC1 domains, with a cluster (11/32) in a conserved NPC1 cysteine-rich luminal loop. Homozygous mutations in 14 patients and a phenotypically defined allele, combined with a new mutation, in a further 10 patients allowed genotype/phenotype correlations. Premature-termination-codon mutations, the three missense mutations in the sterol-sensing domain (SSD), and A1054T in the cysteine-rich luminal loop all occurred in patients with infantile neurological onset and "classic" (severe) cholesterol-trafficking alterations. By western blot, NPC1 protein was undetectable in the SSD missense mutations studied (L724P and Q775P) and essentially was absent in the A1054T missense allele. Our results thus enhance the functional significance of the SSD and demonstrate a correlation between the absence of NPC1 protein and the most severe neurological form. In the remaining missense mutations studied, corresponding to other disease presentations (including two adults with nonneurological disease), NPC1 protein was present in significant amounts of normal size, without clear-cut correlation with either the clinical phenotype or the "classic"/"variant" biochemical phenotype. Missense mutations in the cysteine-rich luminal loop resulted in a wide array of clinical and biochemical phenotypes. Remarkably, all five mutant alleles (I943M, V950M, G986S, G992R, and the recurrent P1007A) definitively correlated with the "variant" phenotype clustered within this loop, providing new insight on the functional complexity of the latter domain.     

Type C Niemann-Pick disease: documentation of abnormal LDL processing in lymphocytes

Type C Niemann-Pick disease (NPC) is an autosomal recessive neurovisceral storage disorder in which defective intracellular cholesterol processing has been demonstrated in fibroblasts from NPC patients and obligate heterozygotes. In the present paper, the ability to esterify LDL-cholesterol was examined in cultured lymphocytes from 8 NPC patients, 8 obligate heterozygotes and 8 controls. Cholesteryl ester synthesis was 8% (+/- 5%) and 45% (+/- 16%) of controls in homozygous and heterozygous cell lines, respectively. Histochemical and electron microscopic examinations confirmed that this biochemical lesion was associated with abnormal intracellular accumulation of unesterified cholesterol in mutant lymphocytes. These results demonstrate that measurement of cholesterol esterification in cultured lymphocytes offers a quick and reliable means of confirming the diagnosis of NPC and that these cells may be useful for probing the primary molecular lesion of NPC.     

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.     

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.     

Niemann-Pick C variant detection by altered sphingolipid trafficking and correlation with mutations within a specific domain of NPC1

Niemann-Pick disease type C (NPC) is a fatal, autosomal recessive lipidosis characterized by lysosomal accumulation of unesterified cholesterol and multiple neurological symptoms, such as vertical supranuclear ophthalmoplegia, progressive ataxia, and dementia. More than 90% of cases of NPC are due to a defect in Niemann-Pick C1 (NPC1), a late endosomal, integral membrane protein that plays a role in cholesterol transport or homeostasis. Biochemical diagnosis of NPC has relied on the use of patient skin fibroblasts in an assay to demonstrate delayed low-density lipoprotein (LDL)-derived cholesterol esterification and a cytological technique-filipin staining-to demonstrate the intracellular accumulation of cholesterol. A small percentage of patients, referred to as "NPC variants," present with clinical symptoms of NPC but show near-normal results of these biochemical tests, making laboratory confirmation of NPC disease problematic. Here, we demonstrate that NPC-variant fibroblast samples can be detected as sphingolipid storage disease cells, using a fluorescent sphingolipid analog, BODIPY-lactosylceramide. This lipid accumulated in endosomes/lysosomes in variant cells preincubated with LDL cholesterol but targeted to the Golgi complex in normal cells under these conditions. The reproducibility of this technique was validated in a blinded study. In addition, we performed mutation analysis of the NPC1 gene in NPC variant and "classical" NPC cell samples and found a high incidence of specific mutations within the cysteine-rich region of NPC1 in variants. We also found that 5 of the 12 variant cell samples had no apparent defect in NPC1 but were otherwise indistinguishable from other variant cells. This is a surprising result, since, in general, approximately 90% of patients with NPC possess defects in NPC1. Our findings should be useful for the detection of NPC variants and also may provide significant new insight regarding NPC1 genotype/phenotype correlations.     

Dynamics of lysosomal cholesterol in Niemann-Pick type C and normal human fibroblasts

The dynamics of endolysosomal cholesterol were investigated in Niemann-Pick type C (NPC) cells and in human fibroblasts treated with class 2 amphiphiles to mimic NPC cells. We showed through new approaches that the massive pools of endolysosomal cholesterol in these cells are not trapped but, rather, circulate to the cell surface at about the normal rate. This flux spared NPC and amphiphile-treated cells from disruption by the extraction of their plasma membrane cholesterol with cyclodextrin. Nocodazole, a microtubule-depolymerizing agent, reversed the resistance of NPC and U18666A-treated cells to cholesterol depletion, apparently by reducing the flux of endolysosomal cholesterol to the plasma membrane. Neither nocodazole nor bafilomycin A1 (an inhibitor of the vacuolar proton pump) acted in the same way as the NPC mutation or class 2 amphiphiles: both agents decreased plasma membrane cholesterol at the expense of the endolysosomal pool and both blocked the actions of the amphiphile, U18666A. Finally, the resistance of NPC cells to lysis by amphotericin B was shown not to reflect a reduction in plasma membrane cholesterol arising from a block in lysosomal cholesterol export but rather the diversion of the amphotericin B to cholesterol-rich endolysosomes. We conclude that the large pool of endolysosomal cholesterol in NPC and amphiphile-treated fibroblasts is dynamic and that its turnover, as in normal cells, is dependent on microtubules.