Acid ceramidase treatment enhances the outcome of autologous chondrocyte implantation in a rat osteochondral defect model.

OBJECTIVE: The overall aim of this study was to evaluate how supplementation of chondrocyte media with recombinant acid ceramidase (rhAC) influenced cartilage repair in a rat osteochondral defect model. METHODS: Primary chondrocytes were grown as monolayers in polystyrene culture dishes with and without rhAC (added once at the time of cell plating) for 7 days, and then seeded onto Bio-Gide® collagen scaffolds and grown for an additional 3 days. The scaffolds were then introduced into osteochondral defects created in Sprague-Dawley rat trochlea by a microdrilling procedure. Analysis was performed 6 weeks post-surgery macroscopically, by micro-CT, histologically, and by immunohistochemistry. RESULTS: Treatment with rhAC led to increased cell numbers and glycosaminoglycan (GAG) production (∼2 and 3-fold, respectively) following 7 days of expansion in vitro. Gene expression of collagen 2, aggrecan and Sox-9 also was significantly elevated. After seeding onto Bio-Gide®, more rhAC treated cells were evident within 4 h. At 6 weeks post-surgery, defects containing rhAC-treated cells exhibited more soft tissue formation at the articular surface, as evidenced by microCT, as well as histological evidence of enhanced cartilage repair. Notably, collagen 2 immunostaining revealed greater surface expression in animals receiving rhAC treated cells as well. Collagen 10 staining was not enhanced. CONCLUSION: The results further demonstrate the positive effects of rhAC treatment on chondrocyte growth and phenotype in vitro, and reveal for the first time the in vivo effects of the treated cells on cartilage repair.

Enzyme replacement therapy for lysosomal diseases: lessons from 20 years of experience and remaining challenges.

In 1964, Christian de Duve first suggested that enzyme replacement might prove therapeutic for lysosomal storage diseases (LSDs). Early efforts identified the major obstacles, including the inability to produce large quantities of the normal enzymes, the lack of animal models for proof-of-concept studies, and the potentially harmful immune responses to the "foreign" normal enzymes. Subsequently, the identification of receptor-mediated targeting of lysosomal enzymes, the cloning and overexpression of human lysosomal genes, and the generation of murine models markedly facilitated the development of enzyme replacement therapy (ERT). However, ERT did not become a reality until the early 1990s, when its safety and effectiveness were demonstrated for the treatment of type 1 Gaucher disease. Today, ERT is approved for six LSDs, and clinical trials with recombinant human enzymes are ongoing in several others. Here, we review the lessons learned from 20 years of experience, with an emphasis on the general principles for effective ERT and the remaining challenges.

The pathogenesis and treatment of acid sphingomyelinase-deficient Niemann-Pick disease.

Patients with Niemann-Pick disease (NPD) Types A and B have an inherited deficiency of acid sphingomyelinase (ASM) activity. The clinical spectrum of this disorder ranges from the infantile neurological form that results in death by 3 years of age (NPD Type A) to the non-neurological form that is compatible with survival into adulthood (NPD Type B). Intermediate cases have also been reported, and the disease is best thought of as a single entity with a spectrum of phenotypes. ASM deficiency is panethnic, but appears to be more frequent in individuals of Middle Eastern and North African descent. Current estimates of the disease incidence range from 0.5 to 1 per 100,000 births, although these approximations are thought to underestimate the true frequency of the disorder. The gene encoding ASM--SMPD1--has been studied extensively, and over 100 mutations in SMPD1 have been found to cause ASM-deficient NPD. Based on these findings, DNA-based carrier screening has been implemented in the Ashkenazi Jewish community. ASM-knockout mouse models also have been generated and used to investigate disease pathogenesis and treatment with stem cell transplantation, gene therapy and enzyme replacement therapy (ERT). Based on these studies, clinical trials of ERT are underway in patients with non-neurological ASM-deficient NPD.

Acid sphingomyelinase-deficient Niemann-Pick disease: novel findings in a Greek child.

Niemann-Pick Disease (NPD) is a heterogeneous group of autosomal recessive disorders characterized by progressive accumulation of sphingomyelin and cholesterol in lysosomes. Six types of NPD have been described based on clinical presentation and involved organs. The primary defect in NPD types A and B is a deficiency of lysosomal acid sphingomyelinase (ASM). We present a case of a 5-year-old boy with type B NPD who had severe clinical manifestations, including heart involvement. He was first admitted to the hospital at 2 months because of vomiting, refusal to feed, lethargy, hepatomegaly and mild transaminasaemia. Liver biopsy at 12 months showed lipid accumulation and fibrosis. Investigations for lysosomal storage disorders revealed increased plasma chitotriosidase (549 nmol/h per ml, normal value 0-150). At 18 months, no detectable ASM activity was observed in cultured fibroblasts (normal range 23-226 nmol/h per mg protein) confirming NPD B. Pulmonary involvement was detected with high-resolution computerized tomography which revealed reticulonodular infiltrations and thickening of the interlobular septa. At 2 years growth retardation and kyphosis were noted. At 2.5 years he manifested neurodevelopment regression, indicating CNS involvement. Cardiac involvement (grade III mitral valve insufficiency) developed at 4 years and heart failure at 5 years. Genetic analysis revealed two mutations: a H421Y mutation that is common in Saudi Arabian and Turkish patients, and a W32X mutation, which has been found in other Mediterranean patients.

The pathogenesis and treatment of acid sphingomyelinase-deficient Niemann-Pick disease.

Patients with types A and B Niemann-Pick disease (NPD) have an inherited deficiency of acid sphingomyelinase (ASM) activity. The clinical spectrum of this disorder ranges from the infantile, neurological form that results in death by 3 years of age (type A NPD) to the non-neurological form (type B NPD) that is compatible with survival into adulthood. Intermediate cases also have been reported, and the disease is best thought of as a single entity with a spectrum of phenotypes. ASM deficiency is panethnic, but appears to be more frequent in individuals of Middle Eastern and North African descent. Current estimates of the disease incidence range from approximately 0.5 to 1 per 100,000 births. However, these approximations likely under estimate the true frequency of the disorder since they are based solely on cases referred to biochemical testing laboratories for enzymatic confirmation. The gene encoding ASM (SMPD1) has been studied extensively; it resides within an imprinted region on chromosome 11, and is preferentially expressed from the maternal chromosome. Over 100 SMPD1 mutations causing ASM-deficient NPD have been described, and some useful genotype-phenotype correlations have been made. Based on these findings, DNA-based carrier screening has been implemented in the Ashkenazi Jewish community. ASM 'knockout' mouse models also have been constructed and used to investigate disease pathogenesis and treatment. Based on these studies in the mouse model, an enzyme replacement therapy clinical trial has recently begun in adult patients with non-neurological ASM-deficient NPD.

Preimplantation diagnosis of a lysosomal storage disorder by in situ enzymatic activity: 'proof of principle' in acid sphingomyelinase-deficient mice.

Genetic diagnosis of preimplantation embryos (PGD) can substantially reduce the chance that at-risk couples have children afflicted with inherited diseases. However, PGD requires DNA,which is usually obtained from single cells following embryo biopsy. In addition, PGD requires that the genetic defect(s) causing the disorder be known. We have therefore developed an alternative to PGD, which we term preimplantation enzymatic diagnosis (PED). PED has several advantages over PGD, including the facts that it does not require embryo biopsy and that the gene defect(s) causing the disorder need not be known. We have demonstrated 'proof of principle' for this approach using embryos obtained from a mouse model (ASMKO mice) of acid sphingomyelinase (ASM)-deficient Niemann-Pick disease, an inherited lysosomal storage disorder. For this technique, fluorescently (BODIPY)-conjugated sphingomyelin was used to detect ASM activity in situ. Wild-type, preimplantation embryos degraded the substrate following a short 'pulse-chase' period, resulting in markedly reduced fluorescence compared to ASMKO embryos, which retained the fluorescent substrate. Thus, the two embryo types could be easily distinguished by fluorescent microscopy. The fluorescent sphingomyelin was not toxic to the embryos, and the entire procedure could be accomplished within 48 h without embryo biopsy. We suggest that PED may be useful for the preimplantation diagnosis of lysosomal storage disorders, and perhaps other enzymatic defects where similar in situ assay methods are available.

Niemann-Pick disease: sixteen-year follow-up of allogeneic bone marrow transplantation in a type B variant.

Allogenic bone marrow transplantation (BMT) was carried out on a 3-year-old white caucasian girl with Niemann-Pick disease (NPD) type B. The donor was her unaffected brother. The patient was neurologically normal at the time of transplantation. Engraftment was based on cytogenetic studies and increased leukocyte acid sphingomyelinase (ASM) activity. However, liver biopsies taken up to 33 months post transplantation showed only a moderate reduction in stored sphingomyelin and no significant increase in ASM activity. The post-transplantation period was complicated by severe graft-versus-host disease and a respiratory arrest. By 6 years of age, neurological involvement was observed, including bilateral cherry red spots. The proband is now severely mentally and physically disabled. Liver cirrhosis has continued to progress despite the BMT, and haematemesis due to portal hypertension occurred at 17 years of age. However, pulmonary infiltration regressed after BMT and there has been no clinical evidence of pulmonary insufficiency.

Articular chondrocytes from animals with a dermatan sulfate storage disease undergo a high rate of apoptosis and release nitric oxide and inflammatory cytokines: a possible mechanism underlying degenerative joint disease in the mucopolysaccharidoses.

Mucopolysaccharidosis (MPS) Type VI (Maroteaux-Lamy Disease) is the lysosomal storage disease characterized by deficient arylsulfatase B activity and the resultant accumulation of dermatan sulfate-containing glycosaminoglycans (GAGs). A major feature of this and other MPS disorders is abnormal cartilage and bone development leading to short stature, dysostosis multiplex, and degenerative joint disease. To investigate the underlying cause(s) of degenerative joint disease in the MPS disorders, articular cartilage and cultured articular chondrocytes were examined from rats and cats with MPS VI. An age-progressive increase in the number of apoptotic chondrocytes was identified in the MPS animals by terminal transferase nick-end translation (TUNEL) staining and by immunohistochemical staining with anti-poly (ADP-ribose) polymerase (PARP) antibodies. Articular chondrocytes grown from these animals also released more nitric oxide (NO) and tumor necrosis factor alpha (TNF-alpha) into the culture media than did control chondrocytes. Notably, dermatan sulfate, the GAG that accumulates in MPS VI cells, induced NO release from normal chondrocytes, suggesting that GAG accumulation was responsible, in part, for the enhanced cell death in the MPS cells. Coculture of normal chondrocytes with MPS VI cells reduced the amount of NO release, presumably because of the release of arylsulfatase B by the normal cells and reuptake by the mutant cells. As a result of the enhanced chondrocyte death, marked proteoglycan and collagen depletion was observed in the MPS articular cartilage matrix. These results demonstrate that MPS VI articular chondrocytes undergo cell death at a higher rate than normal cells, because of either increased levels of dermatan sulfate and/or the presence of inflammatory cytokines in the MPS joints. In turn, this leads to abnormal cartilage matrix homeostasis in the MPS individuals, which further exacerbates the joint deformities characteristic of these disorders.

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-1alpha 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.

Human acid ceramidase: processing, glycosylation, and lysosomal targeting.

The biosynthesis of human acid ceramidase (hAC) starts with the expression of a single precursor polypeptide of approximately 53-55 kDa, which is subsequently processed to the mature, heterodimeric enzyme (40 + 13 kDa) in the endosomes/lysosomes. Secretion of hAC by either fibroblasts or acid ceramidase cDNA-transfected COS cells is extraordinarily low. Both lysosomal targeting and endocytosis critically depend on a functional mannose 6-phosphate receptor as judged by the following criteria: (i) hAC-precursor secretion by NH(4)Cl-treated fibroblasts and I-cell disease fibroblasts, (ii) inhibition of the formation of mature heterodimeric hAC in NH(4)Cl-treated fibroblasts or in I-cell disease fibroblasts, and (iii) blocked endocytosis of hAC precursor by mannose 6-phosphate receptor-deficient fibroblasts or the addition of mannose 6-phosphate. The influence of the six individual potential N-glycosylation sites of human acid ceramidase on targeting, processing, and catalytic activity was determined by site-directed mutagenesis. Five glycosylation sites (sites 1-5 from the N terminus) are used. The elimination of sites 2, 4, and 6 has no influence on lysosomal processing or enzymatic activity of recombinant ceramidase. The removal of sites 1, 3, and 5 inhibits the formation of the heterodimeric enzyme form. None of the mutant ceramidases gave rise to an increased rate of secretion, suggesting that lysosomal targeting does not depend on one single carbohydrate chain.

An enzymatic assay for quantifying sphingomyelin in tissues and plasma from humans and mice with Niemann-Pick disease.

Sphingomyelin is an important lipid component of cell membranes and lipoproteins which 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 and tissues. In this paper we report a new, enzyme-based method to quantify the levels of sphingomyelin in tissues and plasma of normal individuals and NPD patients. The method utilizes sphingomyelinase from Bacillus cereus to completely hydrolyze the sphingomyelin into ceramide. Quantification of the sphingomyelin-derived ceramide is accomplished using Escherichia coli diacylglycerol (DAG) kinase and [gamma-(32)P]ATP. The resulting [(32)P]ceramide is quantified using a phosphor-imager system following TLC separation. This procedure allowed quantification of sphingomyelin over a broad range from 10 pmol to 1 nmol. To validate this assay we quantified sphingomyelin in plasma and tissues obtained from normal and NPD mice and humans. The sphingomyelin content in adult homozygous (-/-) or heterozygous (+/-) NPD mouse plasma was significantly elevated compared to that of normal mice (up to twofold). Moreover, the accumulated sphingomyelin in the tissues of NPD mice was 4 to 40 times higher than that in normal mice depending on the tissue analyzed. The sphingomyelin levels in plasma from several type B NPD patients also were significantly elevated compared to normal individuals of the same age. Based on these results we propose that this new, enzyme-based procedure can provide sensitive and reproducible sphingomyelin quantification in tissues and fluids from normal individuals and NPD patients. It could be a useful tool for the diagnosis of NPD and the evaluation of NPD treatment protocols, as well as for the study of ceramide-mediated apoptosis since the method provides the simultaneous determination of sphingomyelin and ceramide in the same lipid extract.

Patterned cerebellar Purkinje cell death in a transgenic mouse model of Niemann Pick type A/B disease.

Niemann Pick disease is a family of autosomal recessive disorders characterized by cholesterol accumulation. The most common type is Niemann Pick type A/B (NPA/B), resulting from deficient acid sphingomyelinase activity, which leads to sphingomyelin and cholesterol accumulation. The neuropathology of NPA/B includes widespread neuronal degeneration. An acid sphingomyelinase knockout mouse model of NPA/B (ASMKO) has been developed by the targeted deletion of the acid sphingomyelinase gene. When cerebellar morphology was examined in the ASMKO mouse at postnatal day 60 (P60), a dramatic pattern of longitudinal stripes was revealed in which roughly half the Purkinje cells had died, leaving a highly stereotyped, bilaterally symmetrical array of stripes. Antizebrin II immunocytochemistry revealed that the absent Purkinje cells corresponded exactly to the zebrin II-negative subset, leaving the zebrin II-positive subset apparently intact. By P120, some of the zebrin II-positive Purkinje cells had also been eliminated from the posterior vermis and hemispheres. By P180, all Purkinje cells had been lost from the anterior lobe. Finally at P240, almost all Purkinje cells had disappeared to leave a stereotyped distribution in lobules VI, IX-X and the flocculus and paraflocculus. The temporal pattern of Purkinje cell death demonstrates differential susceptibility of morphologically identical cells that appear to be linked to their molecular phenotypes.

Molecular analysis of acid ceramidase deficiency in patients with Farber disease.

Farber disease is a rare, autosomal recessively inherited sphingolipid storage disorder due to the deficient activity of lysosomal acid ceramidase, leading to the accumulation of ceramide in cells and tissues. Here we report the identification of six novel mutations in the acid ceramidase gene causing Farber disease: three point mutations resulting in single amino acid substitutions, one intronic splice site mutation resulting in exon skipping, and two point mutations also leading to occasional or complete exon skipping. Of interest, these latter two mutations occurred in adjacent nucleotides and led to abnormal splicing of the same exon. Expression of the mutated acid ceramidase cDNAs in COS-1 cells and subsequent determination of acid ceramidase residual enzyme activity demonstrated that each of these mutations was the direct cause of the acid ceramidase deficiency in the respective patients. In contrast, two known polymorphisms had no effect on acid ceramidase activity. Metabolic labeling studies in fibroblasts of four patients showed that even though acid ceramidase precursor protein was synthesized in these individuals, rapid proteolysis of the mutated, mature acid ceramidase occurred within the lysosome.

Hematopoietic stem cell gene therapy leads to marked visceral organ improvements and a delayed onset of neurological abnormalities in the acid sphingomyelinase deficient mouse model of Niemann-Pick disease.

Types A and B Niemann-Pick disease (NPD) result from the deficient activity of acid sphingomyelinase (ASM). Currently, no treatment is available for either form of NPD. Using the ASM knockout (ASMKO) mouse model, we evaluated the effects of ex vivo hematopoietic stem cell gene therapy on the NPD phenotype. Thirty-two newborn ASMKO mice were preconditioned with low dose radiation (200 cGy) and transplanted with ASMKO bone marrow cells which had been transduced with an ecotropic retroviral vector encoding human ASM. Engraftment of donor-derived cells ranged from 15 to 60% based on Y-chromosome in situ hybridization analysis of peripheral white blood cells, and was achieved in 92% of the transplanted animals. High levels of ASM activity (up to five-fold above normal) were found in the engrafted animals for up to 10 months after transplantation, and their life-span was extended from a mean of 5 to 9 months by the gene therapy procedure. Biochemical and histological analysis of tissues obtained 4-5 months after transplantation indicated that the ASM activities were increased and the sphingomyelin storage was significantly reduced in the spleens, livers and lungs of the treated mice, major sites of pathology in type B NPD. The presence of Purkinje cell neurons was also markedly increased in the treatment group as compared with non-treated animals at 5 months after transplantation, and a reduction of storage in spinal cord neurons was observed. However, all of the transplanted mice eventually developed ataxia and died earlier than normal mice. Overall, these results indicated that hematopoietic stem cell gene therapy should be effective for the treatment of non-neurological type B NPD, but improved techniques for targeting the transplanted cells and/or expressed enzyme to specific sites of pathology in the central nervous system must be developed in order to achieve effective treatment for type A NPD.

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 animals 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.

Oocyte apoptosis is suppressed by disruption of the acid sphingomyelinase gene or by sphingosine-1-phosphate therapy.

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.

Creation of a mouse model for non-neurological (type B) Niemann-Pick disease by stable, low level expression of lysosomal sphingomyelinase in the absence of secretory sphingomyelinase: relationship between brain intra-lysosomal enzyme activity and central nervous system function.

Most lysosomal storage diseases result in neurodegeneration, but deficiencies in the same enzymes can also lead to syndromes without neurologic manifestations. The hypothesis that low levels of residual, intra-lysosomal enzymatic activities in the central nervous system (CNS) are protective has been difficult to prove because of inconsistencies in assays of tissue samples. Experimental correction of lysosomal enzyme deficiencies in animal models suggests that low-level enzymatic activity may reduce CNS pathology, but these results are difficult to interpret owing to the partial and transient nature of the improvements, the presence of secretory hydrolases, and other confounding factors. Using a novel transgenic/knockout strategy to manipulate the intracellular targeting of a hydrolase, we created a mouse that stably expresses low levels of lysosomal sphingomyelinase (L-SMase) in the complete absence of secretory sphingomyelinase (S-SMase). The brains of these mice exhibited 11.5-18.2% of wild-type L-SMase activity, but the cerebellar Purkinje cell layer, which is lost by 4 months of age in mice completely lacking L- and S-SMase, was preserved for at least 8 months. The L-SMase activities in other organs were 1-14% of wild-type levels, and by 8 months of age all peripheral organs had accumulated sphingomyelin and demonstrated pathological intracellular inclusions. Most importantly, L-SMase-expressing mice showed no signs of the severe neurologic disease observed in completely deficient mice, and their life span and general health were essentially normal. These findings show that stable, continuous, low-level expression of intra-lysosomal enzyme activity in the brain can preserve CNS function in the absence of secretory enzyme or other confounding factors.

Role of acidic sphingomyelinase in Fas/CD95-mediated cell death.

Engagement of the Fas receptor has been reported to induce ceramide generation via activation of acidic sphingomyelinase (aSMase). However, the role of aSMase in Fas-mediated cell death is controversial. Using genetically engineered mice deficient in the aSMase gene (aSMase(-/-)), we found that thymocytes, concanavalin A-activated T cells, and lipopolysaccharide-activated B cells derived from both aSMase(-/-) and aSMase(+/+) mice were equally sensitive to Fas-mediated cell death, triggered by either anti-Fas antibody or Fas ligand in vitro. Similarly, activation-induced apoptosis of T lymphocytes was unaffected by the status of aSMase, and aSMase(-/-) mice failed to show immunological symptoms seen in animals with defects in Fas function. In vivo, intravenous injection of 3 microg/25 g mouse body weight of anti-Fas Jo2 antibody into aSMase(-/-) mice failed to affect hepatocyte apoptosis or mortality, whereas massive hepatocyte apoptosis and animal death occurred in wild type littermates. Animals heterozygous for aSMase deficiency were also significantly protected. Susceptibility of aSMase(-/-) mice to anti-Fas antibody was demonstrated with higher antibody doses (>/=4 microg/25 g mouse). These data indicate a role for aSMase in Fas-mediated cell death in some but not all tissues.