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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.

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An overexpression system was recently developed to produce and purify recombinant, human acid ceramidase. In addition to ceramide hydrolysis, the purified enzyme was able to catalyze ceramide synthesis using [ 14 C]lauric acid and sphingosine as substrates. Herein we report detailed characterization of this acid ceramidase-associated "reverse activity" and provide evidence that this reaction occurs in situ as well as in vitro. The pH optimum of the reverse reaction was ϳ5.5, as compared with ϳ4.5 for the hydrolysis reaction. Non-ionic detergents and zinc cations inhibited the activity, whereas most other cations were stimulatory. Of note, sphingomyelin also was very inhibitory toward this reaction, whereas the anionic lipids, phosphatidic acid and phosphatidylserine, were stimulatory. Of various sphingosine stereoisomers tested in the reverse reaction, only the natural, D-erythro form could efficiently serve as a substrate. Using D-erythro-sphingosine and lauric acid as substrates, the reaction followed normal Michaelis-Menten kinetics. The K m and V max values toward sphingosine were 23.75 M and 208.3 pmol/g/h, respectively, whereas for lauric acid they were 73.76 M and 232.5 pmol/g/h, respectively. Importantly, the reverse activity was reduced in cell lysates from a Farber disease patient to the same extent as the acid ceramidase activity. Furthermore, when 12-(N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)) (NBD)-conjugated lauric acid and sphingosine were added to cultured lymphoblasts from a Farber disease patient in the presence of fumonisin B (1), the conversion to NBD-ceramide was reduced ϳ30% when compared with normal cells. These data provide important new information on human acid ceramidase and further document its central role in sphingolipid metabolism.

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from the deficient activity of acid sphingomyelinase (ASM). In this manuscript we report the pathobiology of male gonadal tissue and sperm in a knockout mouse model of NPD and demonstrate the importance of ASM for normal sperm maturation and function. Characteristic lipid-filled vacuoles were evident in light micrographs of testis' seminiferous tubules and epithelial cells lining the epididymis of ؊/؊ mice. Electron micrographs extended these findings and revealed storage vesicles within Sertoli cells of the seminiferous tubules. Mature spermatozoa from ؊/؊ mice showed marked ASM deficiency and elevated levels of sphingomyelin and cholesterol. Flow cytometric analysis revealed that affected spermatozoa had disrupted plasma and acrosome membranes, and mitochondrial membrane depolarization. They also did not undergo proper capacitation. Morphological abnormalities such as kinks and bends at the midpieceprinciple piece junction were evident in spermatozoa from affected mice, with consequent deficits in motility. Notably, the mutant sperm regained normal morphology on incubation in mild detergent, demonstrating that the bending defects were a direct consequence of membrane lipid accumulation. A mechanism for these abnormalities is proposed that suggests lipid accumulation in the gonads results in regulatory volume decrease defects within the developing sperm, and that regulatory volume decrease defects, in turn, lead to the observed abnormalities in sperm morphology and function. These results provide in vivo evidence that ASM activity plays a critical role in sperm maturation and function, and a basis for similar studies in sexually mature, male NPD patients. 1 Type A NPD is a severe, neurodegenerative form of the disorder that generally leads to death by ϳ3 years of age. In contrast, patients with type B NPD have little or no neurological involvement and may survive into adolescence or adulthood. ASM belongs to a family of sphingomyelinases that catabolize sphingomyelin (SPM) to ceramide and phosphorylcholine. 2,3 It is presumed that the pathophysiology in NPD is primarily because of the accumulation of SPM and other metabolically related lipids (eg, cholesterol) within the cells and tissues of affected patients, although signaling through the ceramide pathway may also be disrupted.