Sphingosylphosphorylcholine in Niemann-Pick Disease Brain: Accumulation in Type A But Not in Type B

A study of brain lipids in patients with the sphingomyelinase-deficient types of Niemann-Pick disease demonstrated that abnormal accumulation of sphingomyelin occurs only in the brain of neuronopathic type A patients but not in the non-neuronopathic type B. Additional lipid abnormalities were present in the type A brain. In contrast, the brain lipid profile was normal in type B patients. Since lysosphingolipids have been implicated in the biochemical pathogenesis of other genetic lysosomal sphingolipidoses, the occurrence of Sphingosylphosphorylcholine (lysosphingomyelin) was specifically investigated in brain and extraneural tissues, using an HPLC method with fluorescent detection of orthophtalaldehyde derivatives. Levels close to or below the limit of detection (10 pmol/mg tissue protein) were observed in normal and pathological controls. A striking accumulation was observed in brain of two Niemann-Pick type A patients (830 and 430 pmol/mg protein in 27-and 16-month-old children with severe and milder neurological course, respectively), which was not present at the fetal stage of the disease. No significant increase was found in brain tissue from a 3.5 year-old type B patient. In liver and spleen, abnormally high Sphingosylphosphorylcholine levels were observed in both types of the disease, with indication of a progressive increase during development. This study establishes the integrity of brain tissue in Niemann-Pick disease type B and suggests that the lysocompound Sphingosylphosphorylcholine could play a role in the pathophysiology of brain dysfunction in the neuronopathic type A.     

B型尼曼-匹克病一例附文献复习

目的:报道一例B型尼曼-匹克病患者的病例资料,提高对该病的认识。方法:观察1例B型尼曼-匹克病患者的临床表现、骨髓涂片及骨髓活检结果,并进行相关的文献复习。结果:B型尼曼-匹克为自幼发病,无神经系统受损表现,伴有肝脾肿大、外周血三系降低,骨髓涂片及活检结果可见尼曼-匹克细胞。结论:尼曼-匹克病是一种罕见的鞘磷脂沉积性遗传性疾病,临床表现多样,骨髓、肝脾淋巴结病理及基因检测是确诊的关键方法,此病预后差,无特效治疗药物。     

The Role of Sphingolipids in Neuronal Development: Lessons from Models of Sphingolipid Storage Diseases

The study of sphingolipids has undergone a renaissance over the past decade due to the realization that these lipids are involved in a variety a biological processes, such as differentiation, apoptosis, cell growth, and cell migration. In the nervous system, sphingolipids, particularly gangliosides, have attracted particular attention as they occur at high levels and their levels change in a developmentally regulated program. Despite the fact that a large body of data has accumulated on the expression and metabolism of individual gangliosides within specific brain regions, the role of individual gangliosides in neuronal development is still poorly understood, and their specific functions are only now beginning to be elucidated. In the present article, we discuss various aspects of our current knowledge concerning the involvement of sphingolipids and gangliosides in neuronal development, and then discuss some recent findings that shed light on the role of sphingolipids and gangliosides obtained with animal models of sphingolipid and other lysosomal storage diseases.     

A fluorescence-based,high-throughput sphingomyelin assay for the analysis of Niemann-Pick disease and other disorders of sphingomyelin metabolism

Sphingomyelin is an important lipid component of cell membranes and lipoproteins that can be hydrolyzed by sphingomyelinases into ceramide and phosphorylcholine. The Type A and B forms of Niemann-Pick disease (NPD) are lipid storage disorders due to the deficient activity of the enzyme acid sphingomyelinase and the resultant accumulation of sphingomyelin in cells, tissues, and fluids. In this paper we report a new, enzymatic method to quantify the levels of sphingomyelin in plasma, urine, or tissues from NPD patients and mice. In this assay, bacterial sphingomyelinase is first used to hydrolyze sphingomyelin to phosphorylcholine and ceramide. Alkaline phosphatase then generates choline from the phosphorylcholine, and the newly formed choline is then used to generate hydrogen peroxide in a reaction catalyzed by choline oxidase. Finally, with peroxidase as a catalyst, hydrogen peroxide reacts with the Amplex Red reagent to generate a highly fluorescent product, resorufin. These enzymatic reactions are carried out simultaneously in a single 100-microl reaction mixture for 20 min. Use of a 96-well microtiter plate permits automated and sensitive quantification using a plate reader and fluorescence detector. This procedure allowed quantification of sphingomyelin over a broad range from 0.02 to 10 nmol, similar in sensitivity to a recently described radioactive method using diacylglycerol kinase and 50 times more sensitive than a colorimetric, aminoantipyrine/phenol-based assay. To validate this new assay method, we quantified sphingomyelin in plasma, urine, and tissues from normal individuals and from NPD mice and patients. The sphingomyelin content in adult homozygous or heterozygous NPD mouse plasma and urine was significantly elevated compared to that of normal mice. Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 15 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several Type B NPD patients also was significantly elevated compared to normal individuals of the same age. Based on these results, we propose that this new, fluorescence-based procedure can provide simple, fast, sensitive, and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could also be a useful tool for the study of other sphingomyelin-related diseases and in a variety of research settings where sphingomyelin quantification is required.     

Defective calcium homeostasis in the cerebellum in a mouse model of Niemann-Pick A disease

We recently demonstrated that calcium homeostasis is altered in mouse models of two sphingolipid storage diseases, Gaucher and Sandhoff diseases, owing to modulation of the activities of a calcium-release channel (the ryanodine receptor) and of the sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) respectively, by the accumulating sphingolipids. We now demonstrate that calcium homeostasis is also altered in a mouse model of Niemann-Pick A disease, the acid sphingomyelinase (A-SMase)-deficient mouse (ASM-/-), with reduced rates of calcium uptake via SERCA in the cerebellum of 6-7-month-old mice. However, the mechanism responsible for defective calcium homeostasis is completely different from that observed in the other two disease models. Thus, levels of SERCA expression are significantly reduced in the ASM-/- cerebellum by 6-7 months of age, immediately before death of the mice, as are levels of the inositol 1,4,5-triphosphate receptor (IP3R), the major calcium-release channel in the cerebellum. Systematic analyses of the time course of loss of SERCA and IP3R expression revealed that loss of the IP3R preceeded that of SERCA, with essentially no IP3R remaining by 4 months of age, whereas SERCA was still present even after 6 months. Expression of zebrin II (aldolase C), a protein found in about half of the Purkinje cells in the adult mouse cerebellum, was essentially unchanged during development. We discuss possible pathological mechanisms related to calcium dysfunction that may cause Purkinje cell degeneration, and as a result, the onset of neuropathology in Niemann-Pick A disease.     

Cellular Cholesterol Storage in the Niemann-Pick Disease Type C Mouse Is Associated with Increased Expression and Defective Processing of Apolipoprotein D

Abstract: Apolipoprotein D (apoD), a member of the lipocalin superfamily of ligand transporters, has been implicated in the transport of several small hydrophobic molecules including sterols and steroid hormones. We have previously established that apoD is a secreted protein from cultured mouse astrocytes and that treatment with the oxysterol 25-hydroxycholesterol markedly stimulates apoD release. Here, we have investigated expression and cellular processing of apoD in the Niemann-Pick type C (NPC) mouse, an animal model of human NPC, which is a genetic disorder affecting cellular cholesterol transport. NPC is phenotypically characterized by symptoms of chronic progressive neurodegeneration. ApoD gene expression was up-regulated in cultured NPC astrocytes and in NPC brain. ApoD protein levels were also increased in NPC brain with up to 30-fold higher apoD content in the NPC cerebellum compared with control mice. Subcellular fractionation of NPC brain homogenates revealed that most of the apoD was associated with the myelin fraction. ApoD was found to be a secreted protein from cultured normal astrocytes and treatment with the oxysterol, 25-hydroxycholesterol, markedly stimulated apoD release (by five- to 10-fold). By contrast, secretion of apoD from NPC astrocytes was markedly reduced and could not be stimulated by oxysterol treatment. Secretion of apoE, another apolipoprotein normally produced by astrocytes, was similar in NPC and control cells. Furthermore, apoE secretion was not potentiated by oxysterol treatment in either cell type. Plasma levels of apoD were sixfold higher in NPC, whereas hepatic levels were substantially reduced compared with controls, possibly reflecting reduced hepatic clearance of the circulating protein. These results reveal hitherto unrecognized defects in apoD metabolism in NPC that appear to be linked to the known defects in cholesterol homeostasis in this disorder.     

A comparative study of sphingolipids in transplanted melanomas with high and low metastatic activity

The content of sphingolipids in M3 and B16/F10 melanomas with a high metastatic potential and in Claudmanís and B16/F1 melanomas with a low metastatic potential was studied. It was shown that the content of total lipid-bound sialic acids and ganglioside GM3 in melanomas with a high metastatic potential is considerably higher than that in melanomas with a low metastatic potential. On the other hand, the ceramide to glucosylceramide molar ratio is higher in melanomas with a low metastatic potential.     

Effect of Long-Term Feeding with Acetyl-L-Carnitine on the Age-Related Changes in Rat Brain Lipid Composition: A Study by 31P NMR Spectroscopy

Changes in brain lipid composition have been determined in 24 months-old Fischer rats with respect to 6 months-old ones. The cerebral levels of sphingomyelin and cholesterol were found to be significantly increased in aged rats, whereas the amount of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, and phosphatidic acid appear to be unaffected by aging. Long-term feeding with acetyl-L-carnitine was able to reduce the age-dependent increase of both sphingomyelin and cholesterol cerebral levels with no effect on the other measured phospholipids. These findings shown that changes in membrane lipid metabolism and/or composition represent one of the alterations occurring in rat brain with aging, and that long-term feeding with acetyl-L-carnitine can be useful in normalizing these age-dependent disturbances.     

Niemann-Pick Type II fibroblasts exhibit impaired cholesterol esterification in response to shingomyelin hydrolysis

Fibroblasts from patients with Niemann-Pick Type II disease, including the panethnic type C (NPC) and Nova Scotia Acadian type D (NPD) forms, exhibit reduced or delayed stimulation of cholesterol esterification by low density lipoprotein (LDL). Based on recent evidence that cholesterol esterification can also be stimulated by cell surface sphingomyelin hydrolysis, we have compared the response of normal, NPC and NPD fibroblasts to treatment with exogenous sphingomyelinase (SMase). Staphylococcus aureus SMase (> 0.05 U/ml) hydrolyzed over 90% of endogenous sphingomyelin within 1 h and increased incorporation of [³H]oleic acid into cholesterol-[³H]oleate after an initial lag in all three cell types. However, normal levels of cholesterol esterification were not observed for NP Type II fibroblasts: four NPD cell lines exhibited an average of 32% of normal response while cholesterol esterification was only 20% in two well-characterized NPC lines. A third NPC line exhibited normal response to SMase despite greater than 90% impairment of LDL-stimuated cholesterol esterification. Incubation of fibroblasts with LDL followed by SMase produced a synergistic response, particularly in NPC cells where there was little response to either treatment alone. Chloroquinone abolished LDL-stimulated cholesterol esterification in normal fibroblasts but had no effect on the response to SMase, indicating that lysosomal enzymes may not be involved in SMase-mediated cholesterol esterification. These results suggest that intracellular processing of cholesterol derived from either LDL or release from the plasma membrane (by sphingomyelin hydrolysis) is affected in Niemann-Pick Type II cells and that these pathways can complement one another in the stimulation of cholesterol esterification.     

Change in Contents of Biologically Active Sphingolipids Modulating Cell Growth and Survival in Hepatoma 27 Compared to Rat Liver

The contents of bioactive sphingolipids (sphingomyelin, ceramide, glucosyl- and lactosylceramides, gangliosides) were studied in rat hepatoma 27 and rat liver. The amounts of sphingomyelin, ceramide, and glucosyl- and lactosylceramides were about twofold and that of gangliosides was about 3.5-fold increased in the tumor compared to normal tissue. Since sphingomyelin promotes angiogenesis, glucosyl- and lactosylceramides stimulate proliferation, gangliosides inhibit apoptosis, but ceramides suppress proliferation and stimulate apoptosis, it is obvious that the balance of these effectors in hepatoma 27 moves with the tumor growth.     

Lipid Trafficking Defects Increase Beclin-1 and Activate Autophagy in Niemann-Pick Type C Disease

Niemann-Pick type C disease (NPC) is a sphingolipid storage disorder characterized by progressive neurodegeneration that typically shows juvenile onset. Mutations in the Npc1 gene cause ~95% of NPC cases. NPC1 is a multipass transmembrane protein involved in lipid and cholesterol trafficking. Loss of function mutations in Npc1 lead to the accumulation of sphingolipids and cholesterol in late endosomes and lysosomes. In our study, we demonstrated that NPC1 deficiency results in increased basal autophagy in human fibroblasts and in mice. We further demonstrated that NPC1 deficiency activates basal autophagy through increased expression of Beclin-1, a highly conserved member of the class III PI3K complex that is critical for the formation of autophagosomes. In contrast, enhanced basal autophagy was not associated with activation of the Akt–mTOR–p70 S6K signaling pathway. Increased Beclin-1 levels and elevated autophagy were also observed in other sphingolipid storage diseases characterized by disrupted cholesterol and sphingolipid trafficking. We propose a model in which the disordered cholesterol trafficking that occurs in many sphingolipid storages diseases results in upregulation of Beclin-1 and enhanced levels of autophagy.

Addendum to:

Autophagy in Niemann-Pick Type C is Beclin-1 Dependent and Responsive to Lipid Trafficking Defects

C.D. Pacheco, R. Kunkle and A.P. Lieberman

Human Mol Genet 2007; 16:1495-503     

A sensitive and specific LC-MS/MS method for rapid diagnosis of Niemann-Pick C1 disease from human plasma

Niemann-Pick type C1 (NPC1) disease is a rare, progressively fatal neurodegenerative disease for which there are no FDA-approved therapies. A major barrier to developing new therapies for this disorder has been the lack of a sensitive and noninvasive diagnostic test. Recently, we demonstrated that two cholesterol oxidation products, specifically cholestane-3β,5α,6β-triol (3β,5α,6β-triol) and 7-ketocholesterol (7-KC), were markedly increased in the plasma of human NPC1 subjects, suggesting a role for these oxysterols in diagnosis of NPC1 disease and evaluation of therapeutics in clinical trials. In the present study, we describe the development of a sensitive and specific LC-MS/MS method for quantifying 3β,5α,6β-triol and 7-KC human plasma after derivatization with N,N-dimethylglycine. We show that dimethylglycine derivatization successfully enhanced the ionization and fragmentation of 3β,5α,6β-triol and 7-KC for mass spectrometric detection of the oxysterol species in human plasma. The oxysterol dimethylglycinates were resolved with high sensitivity and selectivity, and enabled accurate quantification of 3β,5α,6β-triol and 7-KC concentrations in human plasma. The LC-MS/MS assay was able to discriminate with high sensitivity and specificity between control and NPC1 subjects, and offers for the first time a noninvasive, rapid, and highly sensitive method for diagnosis of 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.     

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.     

Accurate Differentiation of Neuronopathic and Nonneuronopathic Forms of Niemann-Pick Disease by Evaluation of the Effective Residual Lysosomal Sphingomyelinase Activity in Intact Cells

Abstract: Niemann-Pick disease types A and B are two clinical forms of an inherited lysosomal storage disorder characterized by accumulation of sphingomyelin due to deficient activity of the lysosomal enzyme, acid sphingomyelinase. Patients with both types have hepatosplenomegaly, but only those with type A have nervous system involvement leading to death in early infancy. The residual activities of lysosomal sphingomyelinase in types A and B have never been well characterized because of limitations in both in vitro enzymatic assays and loading tests on intact cells. To evaluate the effective level of sphingomyelinase activity, intact, living cultured Epstein-Barr virus-transformed lymphoid cells were incubated with a radiolabeled sphingomyelin that was first associated to human low-density lipoproteins. This lipoprotein-associated sphingomyelin was targeted to lysosomes, thereby permitting selective hydrolysis by the lysosomal sphingomyelinase. Short-term pulse-chase experiments allowed the determination of the initial rates of degradation; in normal cells, the half-time of sphingomyelin degradation averaged 4.5 h. Whereas cells from the severe neuronopathic type A form of Niemann-Pick disease exhibited about 0.15% residual sphingomyelinase activity, cells from patients with the visceral type B form exhibited about 4%, i.e., 27 times more. Cells from heterozygous Niemann-Pick subjects showed about 70% residual activity. These results provide the first approach to measuring the effective activity of a lysosomal enzyme and represent an accurate method for the differential diagnosis of Niemann-Pick disease types A and B. They also support the hypothesis of relationships among the effective in situ residual enzyme activity, the amount of stored substrate, and the severity of the ensuing lysosomal storage disorder.     

Saccadic Eye Movement Characteristics in Adult Niemann-Pick Type C Disease: Relationships with Disease Severity and Brain Structural Measures

Niemann-Pick Type C disease (NPC) is a rare genetic disorder of lipid metabolism. A parameter related to horizontal saccadic peak velocity was one of the primary outcome measures in the clinical trial assessing miglustat as a treatment for NPC. Neuropathology is widespread in NPC, however, and could be expected to affect other saccadic parameters. We compared horizontal saccadic velocity, latency, gain, antisaccade error percentage and self-paced saccade generation in 9 adult NPC patients to data from 10 age-matched controls. These saccadic measures were correlated with appropriate MRI-derived brain structural measures (e.g., dorsolateral prefrontal cortex, frontal eye fields, supplemental eye fields, parietal eye fields, pons, midbrain and cerebellar vermis) and with measures of disease severity and duration. The best discriminators between groups were reflexive saccade gain and the two volitional saccade measures. Gain was also the strongest correlate with disease severity and duration. Most of the saccadic measures showed strongly significant correlations with neurophysiologically appropriate brain regions. While our patient sample is small, the apparent specificity of these relationships suggests that as new diagnostic methods and treatments become available for NPC, a broader range of saccadic measures may be useful tools for the assessment of disease progression and treatment efficacy.     

Multiple Surface Regions on the Niemann-Pick C2 Protein Facilitate Intracellular Cholesterol Transport

The cholesterol storage disorder Niemann-Pick type C (NPC) disease is caused by defects in either of two late endosomal/lysosomal proteins, NPC1 and NPC2. NPC2 is a 16-kDa soluble protein that binds cholesterol in a 1:1 stoichiometry and can transfer cholesterol between membranes by a mechanism that involves protein-membrane interactions. To examine the structural basis of NPC2 function in cholesterol trafficking, a series of point mutations were generated across the surface of the protein. Several NPC2 mutants exhibited deficient sterol transport properties in a set of fluorescence-based assays. Notably, these mutants were also unable to promote egress of accumulated intracellular cholesterol from npc2^−/− fibroblasts. The mutations mapped to several regions on the protein surface, suggesting that NPC2 can bind to more than one membrane simultaneously. Indeed, we have previously demonstrated that WT NPC2 promotes vesicle-vesicle interactions. These interactions were abrogated, however, by mutations causing defective sterol transfer properties. Molecular modeling shows that NPC2 is highly plastic, with several intense positively charged regions across the surface that could interact favorably with negatively charged membrane phospholipids. The point mutations generated in this study caused changes in NPC2 surface charge distribution with minimal conformational changes. The plasticity, coupled with membrane flexibility, probably allows for multiple cholesterol transfer routes. Thus, we hypothesize that, in part, NPC2 rapidly traffics cholesterol between closely appositioned membranes within the multilamellar interior of late endosomal/lysosomal proteins, ultimately effecting cholesterol egress from this compartment.     

Defective mitophagy in human Niemann-Pick Type C1 neurons is due to abnormal autophagy activation

Although traditionally regarded as a cellular adaptive process triggered by nutrient deprivation, autophagy in neurons appears to provide an important neuroprotective mechanism. Neurons in the brain are protected from starvation, and neuronal autophagy serves a critical role in the turnover of abnormal proteins and damaged organelles. As post-mitotic, highly polarized cells with active protein trafficking, neurons rely heavily on an efficient autophagic pathway. Using human embryonic stem cell-derived neurons engineered to mimic the cholesterol lysosomal storage disease Niemann Pick type C1 (NPC1), we have shown that excessive activation and impaired progression of the autophagic pathway conspire to cause abnormal mitochondrial clearance. Defective mitophagy is exceptionally severe in human NPC1 neurons, as compared with patient fibroblasts, and may explain the selective neuronal failure observed in NPC1 and related neurodegenerative disorders.