Gaucher Disease Therapy Figure 7. Proinflammation. (A) The brain stem and spinal cord sections were stained with anti-CD68 antibody, a macrophage-like marker. Positive CD68 signals (brown) on microglial cells demonstrated proinflammation. IFG (20 or 600 mg/kg/d) treated 4L;C* showed decreases of proinflammation at 44 days and increases of proinflammation at 59 or 64 days (terminal stage). (B) Enhanced GFAP staining (green) was in untreated 44 days old 4L;C* brain stem. IFG (20 mg/kg/d) treated 4L;C* showed decreases of GFAP signal at 44 days. GFAP signal were increased at terminal stage at 57 days. (C) GFAP protein in 44 days midbrain tissues was determined by immunoblot. Compared to untreated 4L;C*, GFAP levels were decreased in 20 mg/kg/d IFG and further reduced in 600 mg/kg/d IFG treated midbrain. (D) Phospho-p38 in 44 days midbrain tissues was analyzed by immunoblot. Increased Phospho-p38 was in untreated 4L;C* midbrain relative to WT. Phospho-p38 level was decreased in 20 mg/kg/d IFG treated midbrain and nearly to WT level in 600 mg/kg/d IFG treated mice. (E) TNFa and IL-6 mRNA in midbrain was determined by qRT-PCR. TNFa and IL-6 mRNA levels were increased by 35- and 13-fold in the untreated 4L;C* midbrain, respectively. In IFG (20 or 600 mg/kg/d) treated midbrain, TNFa mRNA were reduced at 44 or 45 days and at terminal stage (51 or 57 days). IL-6 mRNA level was not altered by IFG treatment. The data represent the mean6S.E. and analyzed by Student’s t-test (n = 3 mice, assayed in duplicate). doi:10.1371/journal.pone.0019037.g007 after dissociation. Importantly, orally administered IFG distributes to visceral organs and the brain [16], suggesting an improved delivery compared to enzyme therapy that has little or no delivery to lungs, bone marrow, lymphoid tissues, and brain [22]. However, with these promising in vitro assay results, in vivo demonstration of improved substrate clearance and therapeutic efficiency remained to be shown. In this study, a neuronopathic GCase-based mouse model having the V394L mutation was evaluated for IFG’s in vivo effect on enzyme activity and substrate levels in CNS and visceral organs. This model was generated by crossing V394L homozy- gous to saposin C mice. V394L homozygous mutant mice have residual enzyme activity of 4–10% of WT levels that is sufficient to maintain substrate flux and not present major GC or GS accumulation or cause a significant abnormal phenotype [23,24]. Since saposin C deficiency reduces GCase stability to proteolysis [25], the 4L;C* mice have decreased 4L GCase and develop a major neurological phenotype associated with PLoS ONE | www.plosone.org 8 April 2011 | Volume 6 | Issue 4 | e19037