to correct for the presence of non-lysosomal GCase and b- glucosidases that cleave 4MU-Glc. WT mice tissues were run in parallel as control. Data were analyzed by Student’s t-test. Immunoblots Mouse liver GCase protein detection by immunoblot was as described [36]. The amounts of GCase protein were quantitated with ImageQuant software and normalized to b-actin in the same samples. Midbrain tissues were homogenized in M-PER Mam- malian Protein Extraction Reagent and subjected to electropho- resis on NuPAGE 4-12% Bis-Tris gel. The protein was transferred to HybondTM-ECLTM nitrocellulose membrane. GFAP protein in the samples was reacted with anti-GFAP antibody [39]. Anti- active p38 polyclonal antibody was used to detect Phosphorylated p38 (Phospho-p38) in the samples resolved on 10% Bis-Tris gel [19]. The signals were detected with ECL detection reagent (GE Healthcare, Piscataway, NJ) according to manufacturer’s instructions. qRT-PCR RNAs in midbrain or brain stem tissues were extracted using TOTALLY RNA kit (Ambion Inc.) and purified with RNeasy Mini kit (Qiagen). Reverse transcription of total RNA (1 mg) for each sample was carried out using RT2 First Strand Kit (SA Biosciences). Quantitative RT-PCR (qRT-PCR) was performed References 1. Grabowski GA, Petsko GA, Kolodny EH (2010) Gaucher disease. In: Valle D, Beaudet A, Vogelstein B, Kinzler KW, Antonarakis SE et al, eds. The Online Metabolic and Molecular Bases of Inherited Diseases. New York: The McGraw- Hill Companies, Inc. 2. Gupta N, Oppenheim IM, Kauvar EF, Tayebi N, Sidransky E (2010) Type 2 Gaucher disease: Phenotypic variation and genotypic heterogeneity. Blood Cells Mol Dis. 3. Weinreb NJ, Charrow J, Andersson HC, Kaplan P, Kolodny EH, et al. (2002) Effectiveness of enzyme replacement therapy in 1028 patients with type 1 Gaucher disease after 2 to 5 years of treatment: a report from the Gaucher Registry. Am J Med 113: 112–119. 4. Wenstrup RJ, Kacena KA, Kaplan P, Pastores GM, Prakash-Cheng A, et al. (2007) Effect of enzyme replacement therapy with imiglucerase on BMD in type 1 Gaucher disease. J Bone Miner Res 22: 119–126. 5. Grabowski GA, Kacena K, Cole JA, Hollak CE, Zhang L, et al. (2009) Dose- response relationships for enzyme replacement therapy with imiglucerase/ alglucerase in patients with Gaucher disease type 1. Genet Med 11: 92–100. 6. Burrow TA, Cohen MB, Bokulic R, Deutsch G, Choudhary A, et al. (2007) Gaucher disease: progressive mesenteric and mediastinal lymphadenopathy despite enzyme therapy. J Pediatr 150: 202–206. 7. Radin NS (1994) Rationales for cancer chemotherapy with PDMP, a specific inhibitor of glucosylceramide synthase. Mol Chem Neuropathol 21: 111–127. 8. Weinreb NJ, Barranger JA, Charrow J, Grabowski GA, Mankin HJ, et al. (2005) Guidance on the use of miglustat for treating patients with type 1 Gaucher disease. Am J Hematol 80: 223–229. 9. McEachern KA, Fung J, Komarnitsky S, Siegel CS, Chuang WL, et al. (2007) A specific and potent inhibitor of glucosylceramide synthase for substrate inhibition therapy of Gaucher disease. Mol Genet Metab 91: 259–267. 10. Marshall J, McEachern KA, Chuang WL, Hutto E, Siegel CS, et al. (2010) Improved management of lysosomal glucosylceramide levels in a mouse model of type 1 Gaucher disease using enzyme and substrate reduction therapy. J Inherit Metab Dis 33: 281–289. 11. Lukina E, Watman N, Arreguin EA, Banikazemi M, Dragosky M, et al. (2010) A phase 2 study of eliglustat tartrate (Genz-112638), an oral substrate reduction therapy for Gaucher disease type 1. Blood 116: 893–899. 12. Fan JQ (2008) A counterintuitive approach to treat enzyme deficiencies: use of enzyme inhibitors for restoring mutant enzyme activity. Biol Chem 389: 1–11. 13. Sawkar AR, Cheng WC, Beutler E, Wong CH, Balch WE, et al. (2002) Chemical chaperones increase the cellular activity of N370S beta -glucosidase: a therapeutic strategy for Gaucher disease. Proc Natl Acad Sci U S A 99: 15428–15433. 14. Mu TW, Fowler DM, Kelly JW (2008) Partial restoration of mutant enzyme homeostasis in three distinct lysosomal storage disease cell lines by altering calcium homeostasis. PLoS Biol 6: e26. 15. Steet RA, Chung S, Wustman B, Powe A, Do H, et al. (2006) The iminosugar isofagomine increases the activity of N370S mutant acid beta-glucosidase in Gaucher Disease Therapy on ABI Prism 7000 sequence detection system. The primers for mouse TNFa, IL6, and GAPDH were from SA Biosciences. In PCR mixtures, cDNA aliquots were analyzed using 400 nM of each primer and RT2 Real-Time SYBR Green/ROX PCR master mix (SA Biosciences). PCR amplifications were run in duplicate as follows: 2 min at 50uC and 10 min at 95uC, followed by 40 cycles of 15 s at 95uC and 1 min at 60uC. Relative quantification of TNFa or IL6 referenced to internal control, GAPDH, was analyzed by DDCt Analysis. The fold changes of each gene were normalized by WT samples. Three mice from each group were used and the experiments were repeated twice. Acknowledgments The authors thank Venette Inskeep, Juying Xu, Spence Perry, Sean Gray and Didi Zhu for their technical assistance, Lisa McMillin, Meredith Farmer and Sabina Sylvest for skilled tissue preparation, Chris Woods for the photograph preparation and Michelle Cooley for her clerical expertise. Author Contributions Conceived and designed the experiments: YS GAG. Performed the experiments: HR BL BQ MZ WZ JB KK YS. Analyzed the data: YS WZ KK KDRS YAH GAG. Contributed reagents/materials/analysis tools: KDRS YAH. Wrote the paper: YS KDRS YAH GAG. Gaucher fibroblasts by several mechanisms. Proc Natl Acad Sci U S A 103: 13813–13818. 16. Khanna R, Benjamin ER, Pellegrino L, Schilling A, Rigat BA, et al. (2010) The pharmacological chaperone isofagomine increases the activity of the Gaucher disease L444P mutant form of beta-glucosidase. FEBS J. 17. Sun Y, Liou B, Ran H, Skelton MR, Williams MT, et al. (2010) Neuronopathic Gaucher disease in the mouse: viable combined selective saposin C deficiency and mutant glucocerebrosidase (V394L) mice with glucosylsphingosine and glucosylceramide accumulation and progressive neurological deficits. Hum Mol Genet 19: 1088–1097. 18. Zhang J, Shen B, Lin A (2007) Novel strategies for inhibition of the p38 MAPK pathway. Trends Pharmacol Sci 28: 286–295. 19. Kitatani K, Sheldon K, Anelli V, Jenkins RW, Sun Y, et al. (2009) Acid beta- glucosidase 1 counteracts p38delta-dependent induction of interleukin-6: possible role for ceramide as an anti-inflammatory lipid. J Biol Chem 284: 12979–12988. 20. Lieberman RL, Wustman BA, Huertas P, Powe AC, Jr., Pine CW, et al. (2007) Structure of acid beta-glucosidase with pharmacological chaperone provides insight into Gaucher disease. Nat Chem Biol 3: 101–107. 21. Lieberman RL, D’Aquino JA, Ringe D, Petsko GA (2009) Effects of pH and iminosugar pharmacological chaperones on lysosomal glycosidase structure and stability. Biochemistry 48: 4816–4827. 22. Xu YH, Sun Y, Barnes S, Grabowski GA (2010) Comparative therapeutic effects of velaglucerase alfa and imiglucerase in a Gaucher disease mouse model. PLoS One 5: e10750. 23. Xu YH, Reboulet R, Quinn B, Huelsken J, Witte D, et al. (2008) Dependence of reversibility and progression of mouse neuronopathic Gaucher disease on acid beta-glucosidase residual activity levels. Mol Genet Metab. 24. Xu YH, Quinn B, Witte D, Grabowski GA (2003) Viable mouse models of acid beta-glucosidase deficiency: the defect in Gaucher disease. Am J Pathol 163: 2093–2101. 25. Sun Y, Qi X, Grabowski GA (2003) Saposin C is required for normal resistance of acid beta-glucosidase to proteolytic degradation. J Biol Chem 278: 31918–31923. 26. Sun Y, Ran H, Zamzow M, Kitatani K, Skelton MR, et al. (2010) Specific saposin C deficiency: CNS impairment and acid beta-glucosidase effects in the mouse. Hum Mol Genet 19: 634–647. 27. Jeyakumar M, Thomas R, Elliot-Smith E, Smith DA, van der Spoel AC, et al. (2003) Central nervous system inflammation is a hallmark of pathogenesis in mouse models of GM1 and GM2 gangliosidosis. Brain 126: 974–987. 28. Jeyakumar M, Smith DA, Williams IM, Borja MC, Neville DC, et al. (2004) NSAIDs increase survival in the Sandhoff disease mouse: synergy with N- butyldeoxynojirimycin. Ann Neurol 56: 642–649. 29. Smith D, Wallom KL, Williams IM, Jeyakumar M, Platt FM (2009) Beneficial effects of anti-inflammatory therapy in a mouse model of Niemann-Pick disease type C1. Neurobiol Dis 36: 242–251. PLoS ONE | www.plosone.org 11 April 2011 | Volume 6 | Issue 4 | e19037