Xingxuan He

Niemann-Pick disease type C1 is a sphingosine storage disease that causes deregulation of lysosomal calcium.

Niemann-Pick type C1 (NPC1) disease is a neurodegenerative lysosomal storage disorder caused by mutations in the acidic compartment (which we define as the late endosome and the lysosome) protein, NPC1. The function of NPC1 is unknown, but when it is dysfunctional, sphingosine, glycosphingolipids, sphingomyelin and cholesterol accumulate. We have found that NPC1-mutant cells have a large reduction in the acidic compartment calcium store compared to wild-type cells. Chelating luminal endocytic calcium in normal cells with high-affinity Rhod-dextran induced an NPC disease cellular phenotype. In a drug-induced NPC disease cellular model, sphingosine storage in the acidic compartment led to calcium depletion in these organelles, which then resulted in cholesterol, sphingomyelin and glycosphingolipid storage in these compartments. Sphingosine storage is therefore an initiating factor in NPC1 disease pathogenesis that causes altered calcium homeostasis, leading to the secondary storage of sphingolipids and cholesterol. This unique calcium phenotype represents a new target for therapeutic intervention, as elevation of cytosolic calcium with curcumin normalized NPC1 disease cellular phenotypes and prolonged survival of the NPC1 mouse.

Deregulation of sphingolipid metabolism in Alzheimer's disease

Abnormal sphingolipid metabolism has been previously reported in Alzheimers disease (AD). To extend these findings, several sphingolipids and sphingolipid hydrolases were analyzed in brain samples from AD patients and age-matched normal individuals. We found a pattern of elevated acid sphingomyelinase (ASM) and acid ceramidase (AC) expression in AD, leading to a reduction in sphingomyelin and elevation of ceramide. More sphingosine also was found in the AD brains, although sphingosine-1-phosphate (S1P) levels were reduced. Notably, significant correlations were observed between the brain ASM and S1P levels and the levels of amyloid beta (Abeta) peptide and hyperphosphorylated tau protein. Based on these findings, neuronal cell cultures were treated with Abeta oligomers, which were found to activate ASM, increase ceramide, and induce apoptosis. Pre-treatment of the neurons with purified, recombinant AC prevented the cells from undergoing Abeta-induced apoptosis. We propose that ASM activation is an important pathological event leading to AD, perhaps due to Abeta deposition. The downstream consequences of ASM activation are elevated ceramide, activation of ceramidases, and production of sphingosine. The reduced levels of S1P in the AD brain, together with elevated ceramide, likely contribute to the disease pathogenesis.

Exocytosis of acid sphingomyelinase by wounded cells promotes endocytosis and plasma membrane repair

Lysosomal enzyme acid sphingomyelinase is released extracellularly when cells are wounded, converting sphingomyelin to ceramide and inducing endosome formation to internalize membrane lesions.

The sphingosine 1-phosphate receptor 1 causes tissue retention by inhibiting the entry of peripheral tissue T lymphocytes into afferent lymphatics

Although much is known about the migration of T cells from blood to lymph nodes, less is known about the mechanisms regulating the migration of T cells from tissues into lymph nodes through afferent lymphatics. Here we investigated T cell egress from nonlymphoid tissues into afferent lymph in vivo and developed an experimental model to recapitulate this process in vitro. Agonism of sphingosine 1-phosphate receptor 1 inhibited the entry of tissue T cells into afferent lymphatics in homeostatic and inflammatory conditions and caused the arrest, mediated at least partially by interactions of the integrin LFA-1 with its ligand ICAM-1 and of the integrin VLA-4 with its ligand VCAM-1, of polarized T cells at the basal surface of lymphatic but not blood vessel endothelium. Thus, the increased sphingosine 1-phosphate present in inflamed peripheral tissues may induce T cell retention and suppress T cell egress.

Infusion of recombinant human acid sphingomyelinase into Niemann-Pick disease mice leads to visceral, but not neurological, correction of the pathophysiology

An inherited deficiency of acid sphingomyelinase (ASM) activity results in the Type A and B forms of Niemann‐Pick disease (NPD). Using the ASM‐deficient mouse model (ASMKO) of NPD, we evaluated the efficacy of enzyme replacement therapy (ERT) for the treatment of this disorder. Recombinant human ASM (rhASM) was purified from the media of overexpressing Chinese Hamster ovary cells and i.v. injected into 16 five‐month‐old ASMKO mice at doses of 0.3, 1, 3, or 10 mg/kg every other day for 14 days (7 injections). On day 16, the animals were killed and the tissues were analyzed for their sphingomyelin (SPM) content. Notably, the SPM levels were markedly reduced in the hearts, livers, and spleens of these animals, and to a lesser degree in the lungs. Little or no substrate depletion was found in the kidneys or brains. Based on these results, three additional 5‐month‐old ASMKO ani‐mals were injected every other day with 5 mg/kg for 8 days (4 injections) and killed on day 10 for histological analysis. Consistent with the biochemical results, marked histological improvements were observed in the livers, spleens, and lungs, indicating a reversal of the disease pathology. A group of 10 ASMKO mice were then i.v. injected once a week with 1 mg/kg rhASM for 15 wk, starting at 3 wk of age. Although anti‐rhASM antibodies were produced in these mice, the antibodies were not neutralizing and no adverse effects were observed from this treatment. Weight gain and rota‐rod performance were slightly improved in the treated animals as compared with ASMKO control animals, but significant neurological deficits were still observed and their life span was not extended by ERT. In contrast with these CNS results, striking histological and biochemical improvements were found in the reticuloendothelial system organs (livers, spleens, and lungs). These studies indicate that ERT should be an effective therapeutic approach for Type B NPD, but is unlikely to prevent the severe neurodegeneration associated with Type A NPD.—Miranda, S. R. P., He, X., Simonaro, C. M., Gatt, S., Dagan, A., Desnick, R. J., Schuchman, E. H. Infusion of recombinant human acid sphingomyelinase into Niemann‐Pick disease mice leads to visceral, but not neurological, correction of the pathophysiology. FASEB J. 14, 1988–1995 (2000)

Involvement of the Toll-like receptor 4 pathway and use of TNF-α antagonists for treatment of the mucopolysaccharidoses

Enzyme replacement therapy is currently available for three of the mucopolysaccharidoses (MPSs) but has limited effects on the skeletal lesions. We investigated the involvement of the Toll-like receptor 4 (TLR4) signaling pathway in the pathogenesis of MPS bone and joint disease, and the use of the anti-TNF-α drug, Remicade (Centocor, Inc.), for treatment. TLR4 KO (TLR4(lps−/−)) mice were interbred with MPS VII mice to produce double-KO (DKO) animals. The DKO mice had longer and thinner faces and longer femora as revealed by micro-computed tomography analysis compared with MPS VII mice. Histological analyses also revealed more organized and thinner growth plates. The serum levels of TNF-α were normalized in the DKO animals, and the levels of phosphorylated STAT1 and STAT3 in articular chondrocytes were corrected. These findings led us to evaluate the effects of Remicade in MPS VI rats. When initiated at 1 month of age, i.v. treatment prevented the elevation of TNF-α, receptor activator of NF-κB, and other inflammatory molecules not only in the blood but in articular chondrocytes and fibroblast-like synoviocytes (FLSs). Treatment of 6-month-old animals also reduced the levels of these molecules to normal. The number of apoptotic articular chondrocytes in MPS VI rats was similarly reduced, with less infiltration of synovial tissue into the underlying bone. These studies revealed the important role of TLR4 signaling in MPS bone and joint disease and suggest that targeting TNF-α may have positive therapeutic effects.

Characterization of human acid sphingomyelinase purified from the media of overexpressing Chinese hamster ovary cells.

A rapid purification method was developed to isolate milligram quantities of human acid sphingomyelinase from the media of overexpressing Chinese hamster ovary cells. The purified, recombinant enzyme (rhASM) had physical and kinetic characteristics that were consistent with those reported for the non-recombinant enzyme, including an acidic pH optimum and sensitivity to sulfhydryl reducing reagents and the zinc specific chelator, 1, 10-phenanthroline. A novel assay using fluorescently conjugated sphingomyelin was developed to explore the substrate binding properties of rhASM. Substrate binding required a fatty acid chain length of at least six carbons and the presence of the phosphocholine headgroup on sphingomyelin. Substrate binding also required an acidic pH, and was inhibited by pretreatment of the enzyme with sulfhydral reducing reagents or 1,10-phenanthroline. rhASM was rapidly internalized by cultured skin fibroblasts from Niemann-Pick disease (NPD) patients, and approximately 50% of this uptake was dependent on the mannose 6-phosphate receptor system. Studies using FITC-labeled rhASM revealed that by 1 h the internalized enzyme was localized to acidic compartments and could degrade sphingomyelin, the first demonstration that a lysosomal sphingolipid hydrolase can be fluorescently labeled and retain its biological activity. Intravenous injection of rhASM into ASM knock-out mice showed that the t(1/2) in the plasma was less than 5 min, and that the majority of the injected enzyme was taken up by the liver, followed by the spleen. Thus, these studies lay the foundation for future structure/function investigations of ASM, further investigations into this enzymes role in ceramide mediated signal transduction, and the evaluation of enzyme replacement therapy for NPD using the mouse model.

Acid ceramidase is a novel factor required for early embryo survival

Recent studies suggest that the lipid, ceramide, induces the default apoptosis process in eggs. Yet, it is obscure how newly formed embryos overcome this fate. Acid ceramidase (AC) is a key regulatory enzyme involved in ceramide metabolism, and mutations in the AC gene (Asah1) result in Farber Lipogranulomatosis, a fatal human genetic disorder. Our previous studies revealed that AC knockout (Asah1—/—) mice had a lethal phenotype, and herein we reveal the mechanism underlying this observation. A single‐cell, polymerase chain reaction (PCR) genotyping method was developed to analyze individual embryos from Asah1 ± intercrosses. Combined with Annexin V staining, this genotype analysis demonstrated that Asah1— /— embryos could not survive beyond the 2‐cell stage, and underwent apoptotic death. Notably, sphingosine‐1‐phosphate (S1P) treatment of early 2‐cell embryos from the Asah1 ± intercrosses rescued Asah1— /— embryos, and enabled their progression from the 2‐cell to 4‐8‐cell stage. Quantitative PCR also revealed that expression of the Asah1 gene in healthy embryos was initiated at the 2‐cell stage, coincident with embryonic genome activation (EGA). AC activity and Western blot analyses further demonstrated high expression and activity of the enzyme in normal, unfertilized eggs, which likely provide the protein to newly formed embryos prior to EGA. Based on these observations, we suggest that AC is an essential factor required for embryo survival that functions by removing ceramide from the newly formed embryos, thus inhibiting the default apoptosis pathway.—Eliyahu, E., Park, J.‐H., Shtraizent, N., He, X., Schuchman, E. H. Acid ceramidase is a novel factor required for early embryo survival. FASEB J. 21, 1403–1409 (2007)

Purification and Characterization of Recombinant, Human Acid Ceramidase CATALYTIC REACTIONS AND INTERACTIONS WITH ACID SPHINGOMYELINASE

Human acid ceramidase was overexpressed in Chinese hamster ovary cells by amplification of the transfected, full-length cDNA. The majority of the overexpressed enzyme was secreted into the culture media and purified to apparent homogeneity. The purified protein contained the same 13-(α) and 40 (β)–kDa subunits as human acid ceramidase from natural sources, had an acidic pH optimum (4.5), and followed normal Michaelis-Menten kinetics using 14C- and BODIPY-labeled C12-ceramide as substrates. Deglycosylation studies showed that the recombinant enzyme contained mostly “high mannose” type oligosaccharides and that two distinct β-subunits were present. Amino acid sequencing of these subunit polypeptides revealed a single N terminus, suggesting that the ∼2–4-kDa molecular mass difference was likely due to C-terminal processing. The purified enzyme also catalyzed ceramide synthesis in vitro using 14C-labeled C12 fatty acid and sphingosine as substrates. Surprisingly, we found that media from the overexpressing hamster cells had increased acid sphingomyelinase activity and that this activity could be co-precipitated with acid ceramidase using anti-ceramidase antibodies. Overexpression of acid ceramidase in normal human skin fibroblasts also led to enhanced acid sphingomyelinase secretion, but this was not observed in Niemann-Pick disease cells. RNA studies showed that this increased activity was not due to overexpression of the endogenous acid sphingomyelinase gene. Uptake studies using mouse macrophages revealed rapid internalization of the acid ceramidase activity from the hamster cell media but not acid sphingomyelinase. These studies provide new insights into acid ceramidase and the related lipid hydrolase, acid sphingomyelinase.

Analysis of the lung pathology and alveolar macrophage function in the acid sphingomyelinase--deficient mouse model of Niemann-Pick disease.

Types A and B Niemann-Pick disease (NPD) are lipid storage diseases caused by the deficient activity of the lysosomal enzyme, acid sphingomyelinase (ASM). Type B NPD is associated with progressive pulmonary function decline and frequent respiratory infections. ASM knock-out (ASMKO) mice are available as a model for NPD, but the lung pathology in these mice has not been adequately characterized. This study shows that by 10 weeks of age ASMKO mice have a significantly higher number of cells in their pulmonary airspaces than normal mice, consisting primarily of enlarged and often multinucleated macrophages. These mice also have much higher levels of sphingomyelin in their airspaces at 10 weeks of age, and both cell numbers and sphingomyelin concentrations remain elevated until 26 weeks of age. In these older mice an increased number of neutrophils is also seen. The alveolar cell population in the ASMKO mice produces less superoxide when stimulated, but this can be corrected by providing recombinant ASM to the culture media. Elevated levels of the chemokines macrophage inflammatory protein-2 and macrophage inflammatory protein-1α were also present in the bronchoalveolar lavage fluid of ASMKO mice, and this correlated with increased production of these chemokines by cultured macrophages and enhanced immunostaining in situ. Also, lung histology showed increased cellularity in the alveolar walls of ASMKO mice, but no evidence of fibrosis. Ultrastructural analysis of the lungs showed that the ASMKO mice have similar pathologic features to human NPD patients, with variable lipid storage evident in type I pneumocytes, endothelial cells, and airway ciliated epithelia. The alveolar macrophage, however, was the most dramatically affected cell type in both mice and humans. These studies indicate that the ASMKO mice can be used as a model to study the lung pathology associated with NPD, and demonstrate that the cellular and biochemical analysis of pulmonary airspaces may be a useful approach to monitoring disease progression and/or treatment.