anti-inflammatory [19]. In the brain, the total levels of GC and ceramide were not affected by IFG, and, therefore, the IL-6 pathway does not appear functional in this in vivo system. However, p38 phosphorylation was significantly reduced suggest- ing some connection. One intriguing possibility is that while the total levels of GC and ceramide remained the same, their subcellular distribution may have been different. Based on chaperone function, we could speculate that levels of GC and ceramide may have been partly corrected in the target organelle (e.g. lysosome) but not in other compartments. Alternatively, off-target effects of IFG might be evoked. IFG is an iminosugar that is a member of the class of inhibitors for glycosidases and glycosyltransferases. Iminosugars have therapeu- tic applications in the diseases associated with the metabolism of glycoconjugates to treat tumor metastasis, influenza virus infec- tion, and lysosomal storage diseases [33,34]. Single oral dose of 600 mg/kg to the rat achieves IFG concentration of 1.7 mM in the brain [16]. The continuous dosing of IFG (20 and 600 mg/kg/d) used here could have achieved much higher concentration in the brain than the Ki for GCase (20 nM). At such high concentrations, IFG could inhibit other glycosidase involved in inflammatory pathways, e.g. chitinase [35]. This study revealed the importance of in vivo analyses of the effects of pharmacological small molecules on mutant enzyme’s function. Clearly, the ultimate effects on substrate reductions are essential for GCase or other lysosomal enzymes. Thus, ex vivo results of increasing GCase activity and protein by chaperones are insufficient to conclude that in vivo substrate degradation will occur and need to be interpreted cautiously when evaluating additional new compounds. Materials and Methods Materials The following were from commercial sources: NuPAGE 4–12% Bis-Tris gel, NuPAGE MES SDS running buffer (Invitrogen, Carlsband, CA). 4-methyl-umbelliferyl-b-D-glucopyranoside (4MU-Glc; Biosynth AG, Switzerland). Sodium taurocholate (Calbiochem, La Jolla, CA). M-PER Mammalian Protein Extraction Reagent and BCA protein Assay reagent (Pierce, Rockford, IL). HybondTM-ECLTM nitrocellulose membrane and ECL detection reagent (Amersham Biosciences, Piscataway, NJ). Anti-fade/DAPI, Methyl green, ABC Vectastain and Alkaline phosphatase kit II (Black) (Vector Laboratory, Burlingame, CA). Rat anti-mouse CD68 monoclonal antibodies (Serotec, Oxford, UK). Mouse anti-GFAP monoclonal antibody (Sigma, St. Louis, MO). Rabbit anti-active p38 polyclonal antibody (Promega, Madison, WI). IFG tartrate was provided by Amicus Therapeutics (Cranbury, NJ). Animal care and drug treatment 4L;C* mice generation was as described [17]. Briefly, 4L;C* (V394L/V394L, saposin C-/-) mice were obtained from cross breeding saposin C+/-, V394L/V394L male and female mice. The strain background of 4L;C* was C57BL/6J/129SvEV. In utero IFG treatment was accomplished by providing the pregnant dames drinking water containing the requisite amount of IFG for 3–5 days prior to birth. In addition, mothers were kept on IFG during the weaning period so that the pups had continuous exposure to IFG via breast milk. Following weaning the mice were administrated IFG in drinking water under various specified regimens. Average daily water intake for each mouse is ,5 ml. At postnatal day 7, cohorts were treated with IFG either 600 or 20 mg/kg/d. Body weight, cage activity, and neurological Gaucher Disease Therapy phenotypes of the mice were monitored daily. The mice were maintained in microisolators in accordance with institutional guidelines under Institutional Animal Care and Use Committee (IACUC) approval at Cincinnati Children’s Hospital Research Foundation (CCHRF). IACUC at CCHRF approved the study described in this manuscript with Animal Use Protocol number 8D10085. Other dosing schemes were as indicated in the text. Histology and immunohistochemistry After being anesthetized, the mice were perfused with saline followed by 4% paraformaldehyde prior tissues dissection. The tissues fixed in 10% formalin were processed for paraffin sections and stained with hematoxylin and eosin (H&E). Paraffin sections of brain and spinal cords were stained with mouse anti-GFAP as an astrocyte marker. Biotinylated goat anti-mouse IgG and streptavidin conjugated fluorescent 480 (green) were applied to the sections. DAPI (blue) in antifade was used for cell nuclei staining. Tissues fixed in 4% paraformaldehyde were processed for frozen blocks. Frozen sections were stained with rat anti-CD68 monoclonal antibody [36]. Methyl green was used for cell nuclei staining. Karnovsky’s fixative was used for ultrastructural studies. Glycosphingolipid analyses Glycosphingolipids (GSLs) in the brain and liver lysates were extracted and analyzed by LC/MS at the Lipidomic Core at the Medical University of South Carolina [37]. GC and galactosylcer- amide in brain were separated for the LC/MS analyses. GC and GS levels in the tissue were normalized to protein content in the lysate. GSLs in spleen and lung tissue (5 mg) were extracted [36], and GC and GS analyses were carried out at CCHRF by ESI-LC- MS/MS using a Waters Quattro Micro API triple quadrupole mass spectrometer (Milford, MA) interfaced with Acquity UPLC system. Nitrogen was used as nebulizer and argon was used as collision gas. The source temperature was maintained at 120uC, and the desolvation temperature was kept at 450uC. The drying gas (N2) was maintained at ca. 800 L/h whereas the cone flow gas was off. The multiplier was set at an absolute value of 650. Online chromatographic separation was achieved using a Supelcosil-LC- 18-DB column (3363.0 mm i.d. 3.0 mm). Gradient elution with methanol and water charged with ammonium acetate and formic acid were employed. Optimized parameters for GC and GS were determined with individual standard compounds (Avanti Polar lipids, Inc.). For quantification of GCs, the ESI-MS/MS was operated in the multiple reaction monitoring (MRM) mode for monitoring transition pair of the individual protonated precursor ions and their common product ion m/z 264. GS was monitored by mass transition m/z 462.3.282.4. Calibration curves were built for C16, C18 and C24:1 GC using C12 GC as the internal standard. Quantification of GCs with various fatty acid chain lengths was realized by using the curve of each GC species with closest number of chain length. The quantification of GS was based on the curve using C8 GS as internal standard. The linear response for GCs and GS was in the range of 50 pg–25 ng. The extracted spleen and lung samples were suspended in methanol containing internal standard and injected into the LC/MS. The GC and GS levels in the spleen and lung were normalized to tissue weight. Analyses of GCase activity Tissues were collected from saline perfused mice, homogenized in 0.25% Na taurocholate and 0.25% Triton X-100 and their GCase activities were determined fluorometrically [24,38]. Assay mixtures were incubated in the presence of CBE for 60 min (37uC) PLoS ONE | www.plosone.org 10 April 2011 | Volume 6 | Issue 4 | e19037