Isofagomine In Vivo Effects in a Neuronopathic Gaucher Disease Mouse Ying Sun1,3, Huimin Ran1,3, Benjamin Liou1,3, Brian Quinn1,3, Matt Zamzow1,3, Wujuan Zhang2,3, Jacek Bielawski4, Kazuyuki Kitatani4,5, Kenneth D. R. Setchell2,3, Yusuf A. Hannun 4 , Gregory A. Grabowski1,3* 1 The Division of Human Genetics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 2 The Division of Pathology and Laboratory Medicine, Cincinnati Children’s Hospital Medical Center, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 3 The Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States of America, 4 The Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, United States of America, 5 The Department of Clinical Laboratory, Tottori University Hospital, Tottori University, Yonago, Japan Abstract The pharmacological chaperone, isofagomine (IFG), enhances acid b-glucosidase (GCase) function by altering folding, trafficking, and activity in wild-type and Gaucher disease fibroblasts. The in vivo effects of IFG on GCase activity, its substrate levels, and phenotype were evaluated using a neuronopathic Gaucher disease mouse model, 4L;C* (V394L/V394L + saposin C-/-) that has CNS accumulation of glucosylceramide (GC) and glucosylsphingosine (GS) as well as progressive neurological deterioration. IFG administration to 4L;C* mice at 20 or 600 mg/kg/day resulted in life span extensions of 10 or 20 days, respectively, and increases in GCase activity and protein levels in the brain and visceral tissues. Cerebral cortical GC and GS levels showed no significant reductions with IFG treatment. Increases of GC or GS levels were detected in the visceral tissues of IFG treated (600 mg/kg/day) mice. The attenuations of brain proinflammatory responses in the treated mice were evidenced by reductions in astrogliosis and microglial cell activation, and decreased p38 phosphorylation and TNFa levels. Terminally, axonal degeneration was present in the brain and spinal cord from untreated and treated 4L;C* mice. These data demonstrate that IFG exerts in vivo effects by enhancing V394L GCase protein and activity levels, and in mediating suppression of proinflammation, which led to delayed onset of neurological disease and extension of the life span of 4L;C* mice. However, this was not correlated with a reduction in the accumulation of lipid substrates. Citation: Sun Y, Ran H, Liou B, Quinn B, Zamzow M, et al. (2011) Isofagomine In Vivo Effects in a Neuronopathic Gaucher Disease Mouse. PLoS ONE 6(4): e19037. doi:10.1371/journal.pone.0019037 Editor: Raphael Schiffmann, Baylor Research Institute, United States of America Received January 11, 2011; Accepted March 21, 2011; Published April 20, 2011 Copyright: 2011 Sun et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants to GAG (R01 NS/DK36681 and R21 HD059823) and YAH (CA087584), and to KK, the Japan Society for the Promotion of Science (Grant-in-Aid for Young Scientists-Start-up, 21890144). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: Greg.Grabowski@cchmc.org Introduction Gaucher disease is caused by mutations in GBA1 that encodes acid b-glucosidase (glucocerebrosidase, GCase, EC3.2.1.45). The resultant defective GCase leads to accumulation of the substrates, glucosylceramide (GC) and glucosylsphingosine (GS) [1]. The disease has three clinical variants: Type 1 is primarily a visceral and nonneuropathic disease whereas types 2 and 3 are a continuum of neuronopathic and visceral diseases ranging from acute (type 2, infantile) to subacute (type 3) progressive CNS degenerative diseases [1,2]. Enzyme therapy with regular infusions of mannose-terminated recombinant human enzymes (imiglucerase, Genzyme; velaglucer- ase alfa, Shire) has become the standard-of-care for significantly involved type 1 patients [3]. The effects on liver, spleen, hematologic parameters, and bone are dose dependent [4,5]. Children show excellent responses in growth and bone disease to enzyme therapy [6]. Alternatively, reductions in GC levels can be achieved by partial inhibition of GC synthase, an essential enzyme in the biosynthesis of complex glycosphingolipids [7], i.e. substrate reduction therapy (SRT). SRT with N-butyldeoxynojirimycin (miglustat) shows improvement in some Gaucher disease parameters [8]. Eliglustat Tartrate {(1R,2R)-Octanoic acid [2- (29,39-dihydro-benzo [1,4] dioxin-69-yl)-2-hydroxy-1-pyrrolidin-1- methyl-ethyl]-amide-L-tartaric acid salt}, an analog of 1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), is a novel potent inhibitor (IC50,24 nM) of GC synthase. This compound significantly reduces GC accumulation in visceral tissues and cells in the Gaucher disease mouse model [9,10]. In addition, phase 2 studies in humans show therapeutic effects that mimic those of higher dose enzyme therapy [11]. However, the compound does not reach significant levels in the brain since it is a pGP substrate. [9]. Directly modulating mutant enzyme activity by small molecules was proposed as an attractive alternative approach to treat Gaucher disease using pharmacological chaperones [12,13]. Competitive inhibitors specific for such enzymes could stabilize mutant enzyme proteins in endoplasm reticulum, assist the enzyme trafficking to lysosome, and restoring the activity [13,14]. IFG, a potent inhibitor of GCase, is an effective in vitro pharmacological chaperone that can enhance selected mutant GCases in fibroblasts [14,15]. IFG treatment of human patient fibroblasts facilitates mutant enzyme trafficking to lysosome and enhances protein and activity levels for the mutant GCases N370S, V394L, and L444P [14,15,16]. PLoS ONE | www.plosone.org 1 April 2011 | Volume 6 | Issue 4 | e19037