Genetic deficiencies of individual saposins (A, B, C and D) lead to lysosomal storage diseases (9
). About 10 cases of saposin B deficiency states have been described in humans (31
). To understand the in vivo
functions of saposin B, mice were generated by introducing a point mutation into the saposin B domain of the prosaposin gene. This mutation disrupted a conserved disulfide bond that led to an unstable/undetectable saposin B protein, but preserved prosaposin, and saposin A, C and D processing and function. This strategy was also used to generate the saposin A, D and CD deficient mice (14
), and facilitated the in vivo
assessment of isolated saposin deficiency states in various organs.
The B−/− mice mimicked the biochemistry and phenotype of the human disease. Similar to human patients (31
), B−/− mice developed neurological impairment including ataxia, head tremor and impaired neuromotor coordination. The early onset severe and late onset variants of saposin B deficiency have phenotypes resembling MLD with normal ASA activity (31
). Furthermore, the point substitution and aberrant splicing mutations described in humans lead to a deficiency of mature saposin B with no effect on precursor transport and processing (36
), but manifest excesses of Gb3 (TriCer), LacCer and GM3 in cultured fibroblasts from affected patients (31
). Urine from these patients also contains elevated levels of sulfatide, Gb3 and digalactosylceramide (37
). These findings are similar to those of the B−/− mouse. Electron microscopic findings in B−/− mice showed similar inclusion bodies as reported in humans (32
). However, the disease course in mice was more slowly progressive than humans, suggesting substrate turnover differences between mice and humans as has been observed in Gaucher disease point mutation models in mice (38
The predominant function of saposin B was in sulfatide degradation. However, the accumulation of LacCer and Gb3 (~10–20%) in B−/− mice demonstrated that saposin B also is involved in degradation of these lipids. This finding indicates that saposin B does have in vivo
functions in assisting α-galactosidase A and the β-galactosidase(s) involved in Gb3 and LacCer degradation, respectively, but these roles are minor in most tissues. LacCer is increased >2-fold and is the major lipid accumulated in the PS−/− mice (23
), and is likely a major contributor to the pathogenesis of the prosaposin deficiency. The effects of the increases in LacCer and Gb3 in the B−/− mice are unknown, but may be minor, as suggested by the increases in Gb3 in the Fabry mouse model (39
). The lipid transport functions of saposin B could be compensated by saposin C that also is involved in the degradation of the LacCer (27
). In fact, slight increases in LacCer were detected in our newly generated saposin C deficient mice (Y. Sun et al
., unpublished data). Such mouse models demonstrate overlapping functions of saposins in GSL metabolism in vivo
B−/− mice had progressive accumulation of multiple GSLs, but predominantly sulfatide, in the CNS and PNS. In the brain and spinal cord, sulfatide was detected in microglial cells, oligodendrocytes and neuronal processes. A proinflammatory response was demonstrated by the presence of activated microglial cells or astrogliosis in tissues that accumulated sulfatide. This suggests that the initiating event for proinflammation in these tissues was the presence of excess sulfatide. The oligodendrocytes were the major cell types in B−/− mice brains filled with storage materials although no apparent changes were observed in myelin sheets. Both NFA and HFA sulfatide accumulated in B−/− mice. The ratio of NFA/HFA galactosylceramide has been linked to developmental programming (41
) and myelin stability (42
). However, there was no effect of sulfatide accumulation on myelin integrity in B−/− mice. The normal myelin structure in B−/− mice could be due to maintenance of unchanged NFA/HFA ratios despite an increase in total sulfatide.
The neuronal inclusions were observed in both ASA deficient and B−/− mice. In particular, storage inclusions were found in their acoustic neurons. Neuronal storage of sulfatide has been reported to cause hyperexcitability and axonal degeneration in mice (43
). The acoustic startle response was tested in the B−/− mice, but neither WT nor B−/− mice showed adequate responses in the C57BL/6J strain. This is due to the development of deafness in this stain of mice by the age of testing. The C57BL/6J background strain is homozygous for the age-related hearing loss mutation Cdh23ahl
that causes progressive hearing loss with onset after 10 months of age (JAX mice data base).
Saposin B participates in the degradation of sulfatide by enhancing ASA activity (37
). ASA null mice have slow accumulation of sulfatide and develop CNS abnormalities (33
). The B−/− mouse phenotype closely resembled this phenotype and biochemistry. Both models exhibit head tremor caused by accumulation of sulfatide in CNS and normal myelin ultrastructure. Like ASA null mice, B−/− kidney had remarkable sulfatide storage in the tubule epithelial cells that was detectable as early as 7 weeks. Lung had only a slight increase in sulfatide. Other visceral organs showed no sulfatide accumulation. A unique feature of Sap B−/− mice compared with ASA deficient mice was the accumulation of LacCer and Gb3. In both mouse models, the disease is much slower developing than the human analogues. Alternative sulfatide metabolism in mice may account for these differences between humans and mice (44
). Differences between mouse models and the human diseases have been observed for several lysosomal storage diseases, including acid β-glucosidase knock-out mice (45
), acid β-glucosidase N370S mutant mice (38
) and β-hexosaminidase A (Tay-Sach disease) mice (46
Saposins are essential lysosomal proteins in the GSLs degradation pathway. The saposin B−/− mouse model, as well as those for other saposins (saposin A, D and CD), should provide useful tools for investigating the pathogenesis of sphingolipid storage diseases and for understanding how saposins and their cognate enzymes interact and to maintain the homeostasis of GSLs in the cell/tissues specific manner.