Gaucher Disease Therapy Figure 8. Histopathology. (A) H&E stained brain stem and spinal cord sections showed axonal degeneration (arrow) in untreated 4L;C* and 4L;C* mice treated by 20 or 600 mg/kg/d IFG. The tissues were collected at terminal stage. (B) Electron microscopy of brain stem. Axonal inclusions (arrow) in brain stem were present in untreated 4L;C* (left panel) at 43 days, 20 mg/kg/d IFG treated 4L;C* mice at 55 days (middle panel) and 600 mg/kg/d IFG treated 4L;C* mice at 62 days (right panel). doi:10.1371/journal.pone.0019037.g008 accumulation of GS and GC in the brain [17]. Importantly, the saposin C-/- mice do not show proinflammation until after 6 months of age in the visceral or brain. In addition, the saposin C-/- mice do not develop any phenotype or pathology until after 10 months of age. In saposin C-/- mouse model, absence of saposin C reduced GCase protein by ,50% of WT levels and led to activity reductions, but GC and GS levels are not increased in the brain and visceral tissues [26]. The phenotype of 4L;C* mouse model mimics some neuronopathic variants of human Gaucher disease. Here, IFG treatment of 4L;C* mice enhanced the V394L GCase protein and activity levels up to 25% of WT levels in the livers and about 2-fold in the brain. This was not accompanied by significant reductions in the accumulated substrates, GC and GS. Indeed, substrate levels increased under high dose IFG (600 mg/kg/d) treatment. This inhibitory effect of IFG and the mutant GCase was clearly toxic/ lethal to developing pups as was evident when pregnant dames were administered this dose. Lower doses (20 mg/kg/d) did not have such toxic effect. To overcome such inhibitory effects, an on/off regimen had been suggested [16]. Here, no differences were found on the phenotype onset or life span between the on/ off and continuous treatments. Lacking significant reduction of substrate levels by IFG in this model could be attributed to inhibitory effect on GCase from locally excessive IFG, and/or absence of saposin C’s protection. Despite the lack of IFG effect on the GC and GS levels, the treatment extended 4L;C*’s lifespan by .25%. Importantly, the pathological microglial infiltration in the untreated mice was delayed in the treated mice until the terminal stage. Thus, IFG treatment slowed disease progression, but did not change the ultimate disease outcome. Intriguingly, in the IFG treated mice, proinflammation was suppressed in the middle stage of treatment (44 days). However, the effect was lost in the terminal state and this was not different from the end stage in untreated mice. Such effects on the anti-proinflammatory responses were dose depen- dent, suggesting specific effect of IFG. Proinflammatory responses have been associated with neurodegeneration of other lysosomal storages diseases [27], and anti-inflammatory treatments attenu- ated the phenotype and extending lifespan [28,29,30]. The molecular targets for IFG to have such anti-proinflammatory effects are unknown. TNFa and IL-6 are the proinflammation cytokines that are dependent on p38 signaling for their production [18], which has implications in neurodegenerative diseases [31,32]. IL-6 protein and mRNA levels remained elevated during IFG treatment. However, the reductions of brain TNFa and phosporylation of p38 with IFG treatment suggests that these pathways are involved in the IFG mediated proinflammatory attenuation. In cell lines, ceramide generated from GC through the salvage pathway by enhancing GCase activity can reduce p38-mediated decreases in IL-6 levels, which would be PLoS ONE | www.plosone.org 9 April 2011 | Volume 6 | Issue 4 | e19037