Gaucher Disease Therapy respectively. (B) GC and GS were accumulated in 4L;C* lung. IFG treatment did not reduce the substrate level in 4L;C* lung. Increased GC level was in 600 mg/kg/d IFG treated lung at terminal stage (Age was same as spleen in Fig. 5A). The data represent the mean6S.E. and analyzed by Student’s t-test. *, p,0.05 (n = 3 mice). The GC and GS levels were normalized by tissue weight. doi:10.1371/journal.pone.0019037.g005 reducing GFAP signal levels than the lower dose in age matched samples. p38 is involved in mediating inflammatory cytokine expression [18,19]. By immunoblotting with anti-Phosphorylated/active-p38 antibody, decreased phospho-p38 levels were detected in IFG treated 4L;C* midbrain, and the effect was dose dependent (Fig. 7D). The mRNA levels of p38a, b, c, and d were not significantly altered in the treated and untreated 4L;C* relative to WT, as determined by qRT-PCR analyses (data not shown). TNFa and IL6 are up-regulated during inflammation. By qRT- PCR analyses, increased TNFa and IL-6 mRNA levels were found in untreated 4L;C* midbrains (Fig. 7E) and brain stems (data not shown). TNFa levels were reduced by ,50% in IFG treated mice at 44 days and remained at decreased levels until the terminal stage (51 or 57 days). IL-6 mRNA levels remained elevated during IFG treatment with either dose at 44 days and at terminal stages. These results suggest that IFG attenuates the proinflammatory responses of microglial cells and astrocytes in 4L;C* brain. Histology The 4L;C* mice display axonal degeneration in the brainstem and spinal cord [17]. Axonal degeneration was found by H&E staining of brain stem and spinal cord sections from the 4L;C* mice untreated or treated with either 20 or 600 mg/kg/d IFG (Fig. 8A). Ultrastructural studies showed axonal inclusions in both treated and untreated 4L;C* mouse brain stems (Fig. 8B), midbrain and spinal cord (data not shown). These findings show that IFG treatment did not fundamentally change the brain pathology in 4L;C* mice. Discussion Pharmacological chaperones are a class of small molecules that enhance enzyme stability and can assist in trafficking of mutant enzymes from endoplasmic reticulum to normal destination such as the lysosome for GCase. IFG is a specific reversible GCase inhibitor (Ki ,20 nM) of WT GCase [20]. The binding of IFG to the active site of GCase locks the enzyme in its substrate-bound conformation and thermodynamically stabilizes the enzyme [21]. IFG binding affinity for GCase is greater (Ki ,,12 nM) at the neutral pH of the ER and lesser (Ki ,.50 nM) at the acidic lysosomal pH (Liou and Grabowski observations). Importantly, IFG enhances mutant activity in fibroblast having the N370S [15,20] and the L444P GCase in lymphoid lines and fibroblasts [16] (Liou and Grabowski unpublished). Using partially purified N370S GCase, IFG stabilizes the enzyme to thermal denaturation [21] (Liou and Grabowski unpublished). These findings suggest the potential for IFG as a therapeutic mutant enzyme enhancer that either improves enzyme stability or trafficking to the lysosome or both. The latter would facilitate dissociation of the GCase-IFG complex in the lysosome and allowing the GCase to bind its natural substrates. The finding that GCase in IFG treated cells have enhanced activity after dilution of the inhibitor implies that the free GCase retains an enhanced conformation for some time Figure 6. Analyses of lactosylceramide and lactosylsphingosine levels by LC/MS. Lactosylceramide (LacCer) level was not decreased in IFG treated 4L;C* cortex relative to the untreated mice. Increased lactosylsphingosine (LacSph) was detected in 600 mg/kg/d IFG treated cortex at 44 days. In the liver, LacSph level was not altered, but LacCer was increased in 600 mg/kg/d IFG treated mice at 44 days. The data represent the mean6S.E. and analyzed by Student’s t-test. **, p,0.01; *, p,0.05 (n = 3 mice). The GSL levels were normalized by protein level in the tissues lysate. doi:10.1371/journal.pone.0019037.g006 PLoS ONE | www.plosone.org 7 April 2011 | Volume 6 | Issue 4 | e19037