To understand the functions of saposin C in vivo
, a saposin C-deficient mouse was created in the presence of near-normal amounts of saposins A, B and D that are derived from prosaposin. The primary manifestations in the saposin C−/− mouse were of neurodegeneration that was slow in onset and slow to progress. Within the CNS, the spinal cord and cerebellum were the primary organs involved and in particular showed axonal ballooning, neuronal loss and axonal deterioration, particularly in the spinal cord. In the cerebellum, granule cell loss was followed by Purkinje cell loss that progressed from lobules III to X, with resultant decreases in cerebellar size. Within the cerebellum, astrogliosis and some infiltration by CD68+ microglial cells indicated a significant pro-inflammatory reaction. Importantly, at both the light microscopic and the ultrastructural levels, the cerebral cortical neurons and hippocampal neurons were unchanged compared with saposin C+/− littermates, yet hippocampal LTP in Schaefer collaterals was severely reduced. Biochemically, minor storage of GC and GS was present in the spinal cord, and some increases in LacCer and LacSph were present in the cerebellum, with minor changes in LacSph in the spinal cord. No histological storage was evident in visceral tissues, and the levels of GSL in the visceral tissues were normal. The deficiency of saposin C as well as the resultant histological and biochemical changes in the CNS and in the viscera, were accompanied by decreases in GCase activity and protein, thus verifying the effect of saposin C as a proteolytic protector for GCase within the lysosome (7
). These findings provide insight into the functions of saposin C independent of its function in the degradation of GC, and highlight potential previously unrecognized functions related to LacSph. Because of the ultrastructural inclusions evident in axons of the saposin C−/− mice, the current findings indicate cellular effects on axonal transport and secondary retrograde neuronal degeneration. Interestingly, the previously observed increase in multivesicular body formation in prosaposin-deficient mouse models (25
) was not seen in the isolated saposin C-deficient mice described here.
The data presented demonstrate that saposin C is a non-essential enhancer for GC degradation in vivo
at the levels produced in these mice in vivo
, i.e. GCase residual activity is present, whereas with an essential activator, a total deficiency of GCase function would exist. The total absence of GCase is incompatible with extra uterine survival in the mouse. However, saposin C optimizes the hydrolysis of GC by GCase (12
), and probably also of GS. Importantly, saposin C is a stabilizer of GCase in lysosomes, protecting it from proteolytic digestion (7
). When saposin C is absent in the presence of a detrimental mutation in GCase, the activity of GCase drops below a threshold level and enhances the accumulation of GC and GS in various tissues (Sun et al
., in review). Thus, saposin C appears to be an optimizer of GCase activity: it interacts with GCase, whether mutant or WT, and protects these enzymes against proteolytic digestion by lysosomal proteases.
Although total deficiency did not lead to massive increases in GC or GS in all tissues, excluding the spinal cord, saposin C could facilitate the hydrolysis of other GSLs in vivo
. Specifically, in vitro
saposin C is involved in the metabolism of LacCer and ceramide (26
). Also, both saposin C and B enhance the degradation of LacCer (10
). The unchanged levels of LacCer in the saposin C deficient mice in the visceral tissues, cerebral cortex and spinal cord, were not unexpected since saposin B can compensate for the deficiency of saposin C. In addition, the normal levels of ceramide in the saposin C deficient mice indicate a compensatory effect of saposins D and C in the degradation of ceramide by acid ceramidase (27
Of interest were the significant increases in LacCer and LacSph in the cerebellum of the saposin C deficient mice. Such accumulations, particularly of LacCer, were also found in prosaposin deficiency and in Niemann-Pick C mice (29
). LacCer is a bioactive lipid that has been shown to participate in osteoclastogenesis, angiogenesis and neural inflammation (32
). The biological activities of LacSph have not been well studied, but in analogy to other lysophinoglipids, GS and galactosylsphingosine, this lysolipid probably has significant toxicity to sensitive tissues, including neurons. Indeed, LacSph mobilizes calcium in isolated brain microsomes (35
), suggesting its participation in a pathway that could lead to neuropathological consequences and neuronal inflammation. Similar to several other lysosomal storage diseases (36
), saposin C deficient mice showed sensitivity of Purkinje cells to the disease state. Why this should be true for Purkinje cells, and secondarily granule cells that show specific degeneration in particular lysosomal storage diseases and in the saposin C deficient mice, is unknown, but this sensitivity has now been observed in several models, including saposin D, Niemann-Pick C, Niemann-Pick A, Chediak-Higashi syndrome, neuronal ceroid lipofucinoses and lysosomal acid phosphatase (36
), indicating vulnerability of these cells to metabolic abnormalities including GSL.
In prosaposin-deficient mice and humans, fibroblasts and other tissues have been shown to have increased numbers of multivesicular bodies (42
). This has led to the suggestion that prosaposin and its function in GSL metabolism may be critical to the formation of multivesicular bodies, endosomes and lysosomes (42
). However, in the CNS of the saposin C deficient mice no increase in multivesicular bodies was observed by EM studies. These studies were applied to both cortical neurons and the Purkinje cells. Consequently, multivesicular bodies formation does not appear to be a mechanism involved in pathologic degeneration in the cortex, cerebellum or spinal cord. Cell death caused by apoptosis was unlikely in these mice as suggested by negative TUNEL assay. However, saposin C may have additional functions in cellular metabolism beyond that for the degradation of GSLs, particularly GC, GS, LacCer and LacSph. As is evident in the spinal cord, retrograde axonal degeneration was present as evidenced by the large deposits in the dorsal horn of the spinal cord. This correlated with the presence of inclusion bodies within the axons and suggests a mechanism of retrograde degeneration caused by saposin C deficiency. The precise role in this transport process and potential release at synapses remains to be elucidated, but it seems to be fundamental to the pathological processes within the CNS.
The clinical and pathological phenotype presented in saposin C−/− mice was primarily related to neuronal dysfunction. Massive Purkinje cell death occurred in cerebellum. The spheroids that contained inclusion bodies were found in axons. These pathological defects correlated with the phenotype of ataxia and decreased coordination in saposin C−/− mice. Axonal spheroids have been noted in several lysosomal storage diseases, including Niemann-Pick C, GM2 gangliosidoses, α-N
-acetylgalactosaminidase and α-mannosidosis (45
). Early findings on these lysosomal disease animal models suggest that the loss of Purkinje cells is secondary to axonal dystrophy (48
). The axonal dystrophy may affect anterograde or retrograde transport, which can cause axonal degeneration and cell death, as suggested in Alzheimer's disease and other neurodegenerative disorders (49
). Deterioration of neuromotor function and impaired hippocampal LTP could be the result of such axonal dysfunction. Saposin C is a lysosomal protein, and lysosomes in the neuron are located in the soma. Further studies will be required to define how the deficiency of saposin C leads to axonal spheroids.