Gaucher Disease Therapy Figure 2. Life span and body weight. (A) Both treated and untreated 4L;C* mice had normal growth of their body weight till 35 days. The body weight of untreated 4L;C* mice started to decline at ,35 days. The mice on 20 mg/kg/d IFG showed decrease of body weight at ,35 days. The body weight of 4L;C* mice treated by 600 mg/kg/d IFG started decreasing at ,48 days. Their body weight grew slower than untreated 4L;C* mice. (B) The mice in both cohorts had significantly extended life span to average 54 days (p = 0.0002) by 20 mg/kg/d or 63 days (P,0.0001) by 600 mg/kg/d IFG treatment relative to the untreated mice (,48 days). The Kaplan-Meier survival curves were analyzed using the Mantel-Cox Test. doi:10.1371/journal.pone.0019037.g002 effect on the life span of 4L;C* mice (54 days) compared to starting the same dose prior to birth (63 days). In 5 litters in which IFG (600 mg/kg/d) was begun at 3–5 days prior birth, all such pups died on postnatal days 1 to 3. The pups in one litter survived when this treatment started 2 days prior to birth. The results indicate that earlier treatment started had better efficacy, but that non- toxic doses were needed before the greater benefit of higher doses could be observed; 600 mg/kg/d extended the life span compared 20 mg/kg/d, and the administration of 600 mg/kg/d prior to birth is toxic to the pups. IFG enhanced GCase activity and protein The mutant GCase activity and protein levels in the tissues were analyzed in 4L;C* mice (Fig. 3). In the liver, IFG administration enhanced the mutant GCase activity by 3-fold at 20 mg/kg/d and by 6-fold at 600 mg/kg/d compared to that in untreated 4L;C mice. The 6-fold increase of V394L GCase activity corresponded to ,25% of WT levels, and was similar to the maximal effect observed in cultured skin fibroblasts from V394L/V394L or 4L;C* mice (data not shown). On 600 mg/kg/ d, the mutant GCase activity was increased to a lesser extent in midbrain (1.4-fold) and spleen (1.9-fold). Lung mutant GCase activity was increased by 3.3-fold or 1.3-fold in the mice treated with IFG at 600 or 20 mg/kg/d, respectively. Immunoblot analyses using a specific anti-mouse GCase antibody showed that IFG increased GCase protein levels in the liver to WT levels (600 mg/kg/d) and to 21% of WT levels (20 mg/kg/d). In liver, these respective increases in GCase protein were about 28- or 5- fold compared to untreated 4L;C* (Fig. 3B). The results indicated a discrepancy between the activity and protein increases, and indicate that the IFG in vivo effect on activity was either inefficient or inhibitory. Also, tissue specific effects were observed with the liver enzyme having a greater response to IFG than that in the brain, spleen and lung. Whether this is due to enzyme differences in the various tissues or, more likely, IFG tissue distribution is unknown. Substrate levels in IFG treated mice GC and GS levels in 4L;C* tissues were determined by LC/MS. The tissues were collected from IFG treated (20 and 600 mg/kg/ d) 4L;C*, untreated 4L;C* and WT mice at 14, 28, 44 days, and at terminal stages. Compared to untreated 4L;C*, GC and GS levels in various tissues were either unchanged or increased (Fig. 4 and Fig. 5). The longer treatments with 600 mg/kg/d led to GC and/ or GS increases in visceral tissues (Fig. 4B and Fig. 5). The cerebral cortical levels were not significantly different in treated mice (Fig. 4A). At terminal stages, GC and GS levels in 600 mg/kg/d IFG treated brains were increased over baseline (Fig. 4A). Cerebral cortical lactosylceramide (LacCer) and lactosylsphin- gosine (LacSph) levels in 4L;C* mice were not reduced by IFG treatment (Fig. 6). In general, LacCer and LacSph levels were either increased or unchanged in 600 mg/kg/d IFG treated mice compared to either untreated or to 20 mg/kg/d treated mice. Ceramide levels in 4L;C cortex and liver were at WT levels and were not altered by IFG treatment (data not shown). These data indicated that GC and GS levels were not substantially reduced in IFG treated 4L;C* mice. High dose (600 mg/kg/d) IFG caused accumulation of GC or GS in selected visceral tissues. PLoS ONE | www.plosone.org 3 April 2011 | Volume 6 | Issue 4 | e19037