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Acid sphingomyelinase deficiency (ASMD) is a lysosomal storage disorder caused by mutations in the SMPD1 gene encoding for the acid sphingomyelinase (ASM). While intravenous infusion of recombinant ASM is an effective treatment for the peripheral disease, the neurological complications of ASMD remain unaddressed. It has been shown that aberrantly high level of total brain sphingomyelin (SM) is a key pathological event leading to neurodegeneration. Using mice lacking ASM (ASMko), which mimic the disease, we here demonstrate that among the SM species, SM16:0 shows the highest accumulation and toxicity in ASMko neurons. By targeting lysosomes, SM16:0 causes permeabilization and exocytosis of these organelles and induces oxidative stress and cell death. We also show that genetic silencing of Ceramide Synthase 5, which is involved in SM16:0 synthesis and overexpressed in the ASMko brain, prevents disease phenotypes in ASMko cultured neurons and mice. The levels of SM16:0 in plasma also show a strong correlation with those in brain that is higher than in liver, even at early stages of the disease. These results identify SM16:0 both as a novel therapeutic target and potential biomarker of brain pathology in ASMD.

Acid sphingomyelinase (ASM) has been implicated in neurodegenerative disease pathology, including Alzheimer’s disease (AD). However, the specific role of plasma ASM in promoting these pathologies is poorly understood. Herein, we explore plasma ASM as a circulating factor that accelerates neuropathological features in AD by exposing young APP/PS1 mice to the blood of mice overexpressing ASM, through parabiotic surgery. Elevated plasma ASM was found to enhance several neuropathological features in the young APP/PS1 mice by mediating the differentiation of blood-derived, pathogenic Th17 cells. Antibody-based immunotherapy targeting plasma ASM showed efficient inhibition of ASM activity in the blood of APP/PS1 mice and, interestingly, led to prophylactic effects on neuropathological features by suppressing pathogenic Th17 cells. Our data reveals insights into the potential pathogenic mechanisms underlying AD and highlights ASM-targeting immunotherapy as a potential strategy for further investigation.

Although many reports have revealed the importance of defective microglia-mediated amyloid β phagocytosis in Alzheimer’s disease (AD), the underlying mechanism remains to be explored. Here we demonstrate that neurons in the brains of patients with AD and AD mice show reduction of sphingosine kinase1 (SphK1), leading to defective microglial phagocytosis and dysfunction of inflammation resolution due to decreased secretion of specialized proresolving mediators (SPMs). Elevation of SphK1 increased SPMs secretion, especially 15-R-Lipoxin A4, by promoting acetylation of serine residue 565 (S565) of cyclooxygenase2 (COX2) using acetyl-CoA, resulting in improvement of AD-like pathology in APP/PS1 mice. In contrast, conditional SphK1 deficiency in neurons reduced SPMs secretion and abnormal phagocytosis similar to AD. Together, these results uncover a novel mechanism of SphK1 pathogenesis in AD, in which impaired SPMs secretion leads to defective microglial phagocytosis, and suggests that SphK1 in neurons has acetyl-CoA-dependent cytoplasmic acetyltransferase activity towards COX2., Sphingosine kinase (SphK) converts sphingosine into lipids, and is implicated in inflammation. Here the authors show that SphK1 functions as an acetyltransferase, regulates microglial phagocytosis and is reduced in a model of Alzheimer’s Disease, such that its restoration ameliorates pathology

Autologous endothelial progenitor cell (EPC) transplantation has been suggested as a potential therapeutic approach in diabetic neuropathy (DN). However, such treatment might be limited by safety concerns regarding possible unwanted proliferation or differentiation of the transplanted stem cells. An alternative approach is the stimulation of endogenous bone-marrow-derived EPC (BM-EPC) recruitment into ischemic lesions by the administration of stem cell mobilization agents or chemokines. We first tested the EPC mobilization effect of vascular endothelial growth factor (VEGF) and AMD3100 in a mouse model of diabetes and found that AMD3100 was effective as an EPC mobilization agent, whereas VEGF did not increase circulating EPCs in these animals. Because recent studies have suggested that deceased local expression of stromal-cell-derived factor (SDF)-1α in diabetes is the main cause of defective EPC migration, AMD3100 was administrated systemically to stimulate EPC mobilization and SDF-1α was injected locally to enhance its migration into the streptozotocin-induced DN mice model. This combined therapy increased local expression levels of vasculogenesis-associated factors and newly formed endothelial cells in the sciatic nerve, resulting in the restoration of the sciatic vasa nervorum. The treatment also improved the impaired conduction velocity of the sciatic nerve in DN mice. Thus, AMD3100 might be an effective EPC mobilization agent in diabetes, with local SDF-1α injection synergistically increasing vascularity in diabetic nerves. This represents a novel potential therapeutic option for DN patients.

In several neurodegenerative diseases, sphingolipid metabolism is deeply deregulated, leading to the expression of abnormal membrane sphingolipid patterns and altered plasma membrane organization. In this paper, we review the potential importance of these alterations to the pathogenesis of these diseases and focus the reader's attention on some secondary alterations of sphingolipid metabolism that have been sporadically reported in the literature. Moreover, we present a detailed analysis of the lipid composition of different central nervous system and extraneural tissues from the acid sphingomyelinase-deficient mouse, the animal model for Niemann-Pick disease type A, characterized by the accumulation of sphingomyelin. Our data show an unexpected, tissue specific selection of the accumulated molecular species of sphingomyelin, and an accumulation of GM3 and GM2 gangliosides in both neural and extraneural tissues, that cannot be solely explained by the lack of acid sphingomyelinase.

Types A and B Niemann-Pick disease (NPD) result from the deficient activity of acid sphingomyelinase (ASM), due to mutations in the sphingomyelin phosphodiesterase 1 (SMPD1) gene. Here we report the identification, characterization and genotype/phenotype correlations of eight novel mutations in six unrelated NPD patients. These mutations included seven missense mutations: c.631T > C (p.W211R), c.757G > C (p.D253H), c.940G > A (p.V314M), c.1280A > G (p.H427R), c.1564A > G (p.N522S), c.1575G > C (p.Q525H) and c.1729A > G (p.H577R), and a novel frameshift mutation, c.1657delACCGCCT (fsT553). Each missense mutation was expressed in 293T or COS-7 cells; mutant enzymes p.W211R, p.D253H, p.H427R and p.H577R had G mutation obliterated a known N-glycosylation site and its p.N522S mutant enzyme had ~10% of expressed wild-type activity. Western blot analysis revealed that each mutant protein was expressed at near wild-type amounts, despite their differences in residual activity. The novel seven-base deletion occurred at codon 553, leading to a premature truncation after residue 609. The expression studies predicted the clinical phenotypes of the six patients: two type A patients had genotypes with only type A alleles [c.631T > C (p.W211R), c.757G > C (p.D253H) and c.1729A > G (p.H577R)], and the other four type B disease patients had at least one neuroprotective mutant type B allele [c.940G > A (p.V314M), c.1280A > G (p.H427R), c.1564A > G (p.N522S) and c.1575G > C (p.Q525H)] that expressed >5% residual ASM activity. Thus, these new mutations provide novel genotype/phenotype correlations and further document the genetic heterogeneity in types A and B NPD.

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.

The mucopolysaccharidoses (MPS) are inherited metabolic disorders resulting from the defective catabolism of glycosaminoglycans. In this report, we find that the stimulation of MPS connective tissue cells by the inflammatory cytokines causes enhanced secretion of several matrix-degrading metalloproteinases (MMPs). In addition, expression of tissue inhibitor of metalloproteinase-1 was elevated, consistent with the enhanced MMP activity. These findings were not restricted to one particular MPS disorder or species, and are consistent with previous observations in animal models with chemically induced arthritis. Bromodeoxyuridine incorporation studies also revealed that MPS chondrocytes proliferated up to 5-fold faster than normal chondrocytes, and released elevated levels of transforming growth factor-beta, presumably to counteract the marked chondrocyte apoptosis and matrix degradation associated with MMP expression. Despite this compensatory mechanism, studies of endochondral ossification revealed a reduction in chondro-differentiation in the growth plates. Thus, although MPS chondrocytes grew faster, most of the newly formed cells were immature and could not mineralize into bone. Our studies suggest that altered MMP expression, most likely stimulated by inflammatory cytokines and nitric oxide, is an important feature of the MPS disorders. These data also identify several proinflammatory cytokines, nitric oxide, and MMPs as novel therapeutic targets and/or biomarkers of MPS joint and bone disease. This information should aid in the evaluation of existing therapies for these disorders, such as enzyme replacement therapy and bone marrow transplantation, and may lead to the development of new therapeutic approaches.

The past decade has witnessed remarkable advances in our ability to treat inherited metabolic disorders, especially the lysosomal storage diseases, a group of more than 40 disorders, each of which is caused by the deficiency of a lysosomal enzyme or protein. During the past few years, both enzyme replacement and enhancement therapies have been developed to treat these disorders. This review discusses the successes and shortcomings of these therapeutic strategies, and the contributions that they have made to treating lysosomal storage diseases.

The time at which ovarian failure (menopause) occurs in females is determined by the size of the oocyte reserve provided at birth, as well as by the rate at which this endowment is depleted throughout post-natal life. Here we show that disruption of the gene for acid sphingomyelinase in female mice suppressed the normal apoptotic deletion of fetal oocytes, leading to neonatal ovarian hyperplasia. Ex vivo, oocytes lacking the gene for acid sphingomyelinase or wild-type oocytes treated with sphingosine-1-phosphate resisted developmental apoptosis and apoptosis induced by anti-cancer therapy, confirming cell autonomy of the death defect. Moreover, radiation-induced oocyte loss in adult wild-type female mice, the event that drives premature ovarian failure and infertility in female cancer patients, was completely prevented by in vivo therapy with sphingosine-1-phosphate. Thus, the sphingomyelin pathway regulates developmental death of oocytes, and sphingosine-1-phosphate provides a new approach to preserve ovarian function in vivo.

Stroke is a major cause of long-term disability, the severity of which is directly related to the numbers of neurons that succumb to the ischemic insult. The signaling cascades activated by cerebral ischemia that may either promote or protect against neuronal death are not well understood. One injury-responsive signaling pathway that has recently been characterized in studies of non-neural cells involves cleavage of membrane sphingomyelin by acidic and/or neutral sphingomyelinase (ASMase) resulting in generation of the second messenger ceramide. We now report that transient focal cerebral ischemia induces large increases in ASMase activity, ceramide levels, and production of inflammatory cytokines in wild-type mice, but not in mice lacking ASMase. The extent of brain tissue damage is decreased and behavioral outcome improved in mice lacking ASMase. Neurons lacking ASMase exhibit decreased vulnerability to excitotoxicity and hypoxia, which is associated with decreased levels of intracellular calcium and oxyradicals. Treatment of mice with a drug that inhibits ASMase activity and ceramide production reduces ischemic neuronal injury and improves behavioral outcome, suggesting that drugs that inhibit this signaling pathway may prove beneficial in stroke patients.

Types A and B Niemann-Pick disease (NPD) result from the deficient activity of acid sphingomyelinase (ASM). An animal model of NPD has been created by gene targeting. In affected animals, the disease followed a severe, neurodegenerative course and death occurred by eight months of age. Analysis of these animals showed their tissues had no detectable ASM activity, the blood cholesterol levels and sphingomyelin in the liver and brain were elevated, and atrophy of the cerebellum and marked deficiency of Purkinje cells was evident. Microscopic analysis revealed 'NPD cells' in reticuloendothelial organs and characteristic NPD lesions in the brain. Thus, the ASM deficient mice should be of great value for studying the pathogenesis and treatment of NPD, and for investigations into the role of ASM in signal transduction and apoptosis.

Nature Reviews Genetics 3, 954–966 (2002). The entry for Niemann–Pick Type C disease in Table 1 was incorrect. It should have read as follows: Table 1 The entries for Gaucher type 1 and MPSII in Table 2 were incorrect. They should have read as follows: Table 2 In addition, the second sentence in thesection 'Enzyme replacement therapy: clinical trials' contained an error and should have read: Preclinical studies in knockout mice or other naturally occurring animal models (such as the dog model of mucopolysacch aridosis I) are under way for several other disorders, including type B Niemann–Pick disease42, galactosialidosis86, Wolman disease (cholesteryl ester storage disease)59 and mucopolysaccharidosis VII87,88.

The mucopolysaccharidoses (MPS) are a family of glycosaminoglycan (GAG) storage diseases caused by the deficient activities of specific lysosomal enzymes. A major feature of the MPS disorders is abnormal cartilage and bone development leading to short stature, dysostosis multiplex, and degenerative joint disease. Ultrastructurally, the cytoplasm of affected MPS chondrocytes is filled with membrane bound vacuoles containing undegraded GAGs. Clusters of hypertrophic chondrocytes form in these disorders, leading to the disruption of the normal cartilage organization. To further investigate the cellular pathology of the MPS disorders, primary cultured chondrocytes and articular cartilage were examined from cats and rats with MPS typeVI (Maroteaux-Lamy disease). Markedly increased numbers of apoptotic chondrocytes were identified by TUNEL staining and immunohistochemically with anti-poly (ADP-ribose) polymerase antibodies in the MPS animals as compared to age-matched controls. The prevalence of apoptotic cells was correlated with increased levels of nitrite and tumor necrosis factor-α. Marked proteoglycan depletion also was observed in the MPS epiphyses by Safranin-O staining, and the presence of excess collagen type II was detected by immunostaining. A model of MPS endochondryl bone pathology is proposed in which an inflammatory response to mechanical trauma is caused by the deformed and irregular subchondral bone contours, leading to increased nitric oxide (NO) production in response to TNF-α, thereby inducing chondrocyte cell death. Novel treatment strategies for these disorders might, therefore, be considered to prevent inflammation or inhibit NO production as an alternative to providing the missing enzymatic activities.

Twenty patients with Niemann-Pick disease (NPD) type B (4 to 42 yrs) from 18 different families who had each been diagnosed by the demonstration of reduced acid sphingomyelinase (ASM) activity in peripheral leukocytes were serially evaluated to: 1) characterize the clinical phenotype; 2) determine the plasma lipid levels and 3) determine the ASM genotype. Molecular analysis for the common NPD type A (R496L, L302P and fsP33O) and B (delR608) mutations was performed using genomic DNA from each patient. No patient had any of the type A mutations, whereas four patients were heterozygous for the common type B mutation, and three were delR608 homozygotes. Sequencing of unknown alleles identified five new mutations (P323A, C431F, Q292K, H575L, and H425R), two of which occurred in delR608 compound heterozygotes. Clinical evaluation demonstrated hepatosplenomegaly in all patients, with the exception of two adults who had undergone splenectomy and only had hepatomegaly. Fourteen patients were under the age of 18, and 12 had growth retardation (height and weight < 5th%). Liver function, assessed by SGPT and SGOT levels, was minimally impaired in several patients. Ophthalmologic examination revealed a macular halo or red spot in 10 patients, all of whom had mild neurologic findings (e.g. peripheral neuropathy, learning disabilities etc.). No ophthalmologic or neurologic features were present in any of the seven patients with one or more copies of delR608 suggesting a protective effect for this mutation. Determination of lipid levels revealed that all patients had abnormal profiles, regardless of genotype, which were characterized by total cholesterol and triglyceride levels above the 95th percentile for age, HDL fractions less than the 5th percentile, markedly decreased ApoA1 levels and increased ApoB levels. Echocardiography, stress tests and cardiac evaluation in five adult patients revealed mild mitral valve regurgitation in three, but no other abnormalites. Treatment of the hyperlipidemia in two adult patients by diet modification resulted in significant decreases in the lipid levels, but not into the normal range. In the pediatric patients, diet modification also reduced lipid levels, but had an adverse impact on growth. Efforts are now being directed to achieve diets that provide high caloric intake while restricting cholesterol to determine the efficacy of this approach. Thus, these studies provide the first genotype-phenotype correlations in NPD type B, particularly with regard to the ophthalmologic and neurologic features.

Both human and f MPS VI result from deficient ASB activity (~ 6% of normal) and accumulation of dermatan sulfate (DS). Normal human, normal f and f MPS VI hepatic ASB were purified (spec. act. 55, 106, 2.7 umol/h/mg prot., resp.) and molecular weights (MW) were determined by PAGE (~ 80,000, 160,000, 78,000, resp.) and Sephadex G-200 chromatography (~ 45,000, 110,000, 53,000, resp.). These data were consistent with human ASB and rASB being monomers and the normal f ASB a dimer. When the rASB was incubated with 1 mM DTT, the MW was 160,000 (PAGE) and 100,000 (Sephadex), suggesting that DTT caused dimerization of the rASB. The DTT-treated purified rASB was increased 5-fold and had markedly increased cryostability. Normal f and f MPS VI buffered whole bloods were incubated ± 10 mM DTT, leukocytes isolated, ASA and ASB separated by DEAE-cellulose chromatography, and DS determined by cellogel electrophoresis. The WBC rASB activity was increased 6-fold with DTT (spec. act. 0.025 0.164) and the DS was markedly decreased (0.37 < 0.02 ug/ug prot.); DTT had no effect on ASA, normal ASB, or normal DS levels. These findings suggest that the mutation in f MPS VI may involve a cysteine residue which hinders dimerization and that -SH reduction results in dimerization with enhanced rASB activity. Thiol-induced subunit association provides a novel approach for enzyme manipulation therapy which can be tested in vivo using the animal model.