Neuronopathic Gaucher disease variants present with brainstem, cerebellar, thalamic, cerebral cortical and substantia nigra involvement, but the degrees of involvement and affected regions vary among the patients (26
). The neuropathological involvement has been correlated with the accumulation of GS and GC (6
) with increased GS mostly associated with the widespread neuron loss in Gaucher disease type 2 (7
). This neuronal loss is also associated with astrogliosis and microglial nodules (6
). Additionally, GS suppresses neuritogenesis (31
). In vitro
GC addition to microsomes from type 1, 2 or 3 Gaucher patients stimulated agonist-induced Ca2+
release and this alteration correlated with the level of GC accumulation (32
). GS also directly augments the Ca2+
release in an agonist-independent manner in such microsomes (32
). This Ca2+
release, presumably from endoplasmic reticulum, has been speculated to be responsible for neuron cell death due to GC or GS increases (34
). Furthermore, hippocampal CA2-4 regions are significantly involved by neuronal injury in neuronopathic Gaucher disease variants (30
). Here, the neuronal deficit in 4L;C* mice was likely caused by excess levels of GS and GC as well as the striking inclusions in axonal and neuronal processes. Furthermore, 4L;C* mice showed impaired hippocampal LTP that correlated with substrate accumulation and abnormal neuronal processes. Negative Tunnel assays in 4L;C* CNS tissues indicate that apoptosis was not the cause of neurodegeneration. The accumulation of p62, an autophagic receptor that mediates autophagic degradation of ubiquitinated proteins (25
) and enhanced Lamp2 expression suggest an impairment of protein degradation in the autophagosome and lysosome system that could be the cause of neurodegeneration in this model.
Comparisons of the saposin C−/− mice and the 4L;C* mice provide insights into the function of saposin C and GCase. Importantly, saposin C provides a proteolytic protective effect for GCase within the lysosome and that its presence is required for the full activity of the enzyme. Here, the singular absence of saposin C led to further decreases in V394L mutant GCase activity as measured in vitro. This GCase protein level was also decreased indicating an increased instability or susceptibility of the mutant protein to proteolysis. Because of this decrease in GCase activity from ~15 to ~6% of WT levels, a new phenotype emerged with primarily central nervous system disease and with biochemical evidence of GC and GS storage in the viscera. Furthermore, the reduced V394L GCase activity in the brain was below a threshold to prevent the development of central nervous system disease, since neither the V394L homozygote nor the saposin C−/− mice themselves develop progressive central nervous system disease in the time frame of these studies.
A curious finding was that the level of GC in the brain had only minor changes, unlike the level of GS that showed major increases. GS is known to be highly toxic to cells, particularly neurons (31
), and the elevated GS levels led to selective deterioration of the central nervous system in the 4L;C* mice. Although, the levels of GS in visceral tissues, on a fold basis, were similar to or greater than those in the CNS, the visceral organs do not show any major histological abnormalities. The lipid abnormalities were detectable only using MS. Thus, it appears that the visceral tissues are relatively resistant or insensitive to the toxic effects of GS. There is a much greater toxicity to similarly raised levels of GS for CNS cells. This difference in sensitivity may relate to the fact that neurons and glial cells in central nervous system are more vulnerable to GSLs accumulation than the cells in the liver and some other visceral tissues. Within the central nervous system, this disease process is also selective with specific involvement of neurons and some cells within the spinal cord while leaving much of the white matter uninvolved.
It is also clear that saposin C interacts functionally with both WT and mutant GCases. In both the saposin C−/− mouse with WT or with V394L GCases, the amount of enzyme protein present in the central nervous system is diminished compared with saposin C+/+ animals. The amount of WT GCase in saposin C−/− mice appears to be sufficient for the maintenance of central nervous system function in a normal state for periods >12 months and that the eventual phenotype is not clearly related to the accumulation of GC or GS within the CNS. Thus, the axonal degeneration and other histological abnormalities seen in the saposin C−/− mouse must relate to other intrinsic functions of saposin C, unrelated to its activity in promoting optimal GCase function (23
). In contrast, the additional decrease in V394L activity caused by the absence of saposin C is below a threshold necessary for the protection of the central nervous system from GS toxicity. The early demise of the 4L;C* mice (48 days) compared with the longer lifespan of the saposin C−/− mice (>20 months), suggests that the toxic effects may be attributed to GS.
An interesting question is the differential accumulation of GS compared with GC in the brains of 4L;C* mice. Possible explanations include either functions related to saposin C or to the mutant enzyme V394L or both. Saposin C could affect substrate specificity and enhance the activity of GCase to a greater degree toward the deacylated analog, GS, compared with GC. This will be investigated in future studies. An alternative explanation is that the V394L has a preference for GS or GC substrates. GS has an affinity (as measured by Ki
) for V394L that is ~4- to 5-fold decreased compared with WT, and that GC has a normal binding constant for this enzyme (Liou and Grabowski, unpublished data). This suggests a potentially pure kinetic effect of a diminished enzymatic activity in which, once below a threshold, GS, because of its poor binding, cannot engage V394L GCase and cannot be hydrolyzed, whereas GC can. Vaccaro et al
) showed that the kcat
for GS was ~100-fold less than that for GC using purified WT GCase. If this also applies to the V394L enzyme and the binding constant is diminished, the accumulation of GS could be explained on the basis of a kinetic phenomenon, based on the decreased ability of V394L to cleave the less preferred substrate.
Finally, the studies here address the question of whether the toxic agent in neuronopathic Gaucher disease is GC or GS. The 4L;C* mouse accumulates GS to greater relative levels than GC; 4L;C* is nearly a pure GS storage disease that leads to central nervous dysfunction and eventual death. As a pure GC storage disease of CNS is not available, the question of this lipid's toxicity alone cannot be directly addressed. However, the in vitro studies showing toxicity of galactosylsphingosine and GS translate reasonably well to their CNS animal models, i.e. the twitcher mouse and the 4L;C* mouse, respectively. The in vivo toxicity of GS to neurons remains to be clarified. It will be important to address whether a diminution in GS can occur by the use of GC synthase inhibitors, and thereby diminish toxicity of GS. If it is true that GC synthase is responsible for all or most of the synthesis of GC and GS, compounds that inhibit this synthase could be therapeutic to the neuropathic forms of Gaucher disease.