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Glucocerebrosidase 2 Gene Deletion Rescues Type 1 Gaucher Disease

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Glucocerebrosidase 2 Gene Deletion Rescues Type 1 Gaucher Disease Glucocerebrosidase 2 gene deletion rescues type 1 Gaucher disease Pramod K. Mistrya,1, Jun Liua,2, Li Sunb,c,2, Wei-Lien Chuangd, Tony Yuenb,c,e, Ruhua Yanga, Ping Lub,c, Kate Zhangd, Jianhua Lib,c, Joan Keutzerd, Agnes Stachnikb,c, Albert Mennonea, James L. Boyera, Dhanpat Jaina, Roscoe O. Bradyf, Maria I. Newb,e,1, and Mone Zaidib,c,1 aDepartment of Medicine, Yale School of Medicine, New Haven, CT 06520; bMount Sinai Bone Program, cDepartment of Medicine, and eDepartment of Pediatrics, Mount Sinai School of Medicine, New York, NY 10029; dGenzyme Sanofi, Framingham, MA 01701; and fNational Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20824 Contributed by Maria I. New, February 19, 2014 (sent for review December 11, 2013) The inherited deficiency of the lysosomal glucocerebrosidase basis of phenotypic diversity, unravel disease mechanism, and (GBA) due to mutations in the GBA gene results in Gaucher disease develop therapies have prompted the generation of mouse (GD). A vast majority of patients present with nonneuronopathic, models that recapitulate human GD1 (7–9). To this end, we have + type 1 GD (GD1). GBA deficiency causes the accumulation of two developed a GD1 mouse, Mx1–Cre :GD1, in which the Gba gene key sphingolipids, glucosylceramide (GL-1) and glucosylsphingo- is deleted in hematopoietic and mesenchymal lineage cells (4, 10, sine (LysoGL-1), classically noted within the lysosomes of mono- 11). This mouse displays hepatosplenomegaly, cytopenia, osteope- nuclear phagocytes. How metabolites of GL-1 or LysoGL-1 produced nia, Th1/Th2 hypercytokinemia, and an array of defects in early T- by extralysosomal glucocerebrosidase GBA2 contribute to the GD1 cell maturation, B-cell recruitment, and antigen presentation (4, 10). pathophysiology is not known. We recently recapitulated hepatos- Glucosylceramide synthase in Golgi complex converts ceramide plenomegaly, cytopenia, hypercytokinemia, and the bone-forma- to GL-1, which is used in the production of more complex tion defect of human GD1 through conditional deletion of Gba glycosphingolipids. As membranes turn over in the lysosome, + in Mx1–Cre :GD1 mice. Here we show that the deletion of Gba2 lysosomal glycosidases sequentially cleave off the sugar residues significantly rescues the GD1 clinical phenotype, despite enhanced from nonreducing end of glycosphingolipids, and GL-1 is even- elevations in GL-1 and LysoGL-1. Most notably, the reduced bone tually hydrolyzed to ceramide and glucose by lysosomal GBA. In MEDICAL SCIENCES volume and bone formation rate are normalized. These results sug- addition, acid ceramidase exhibits expanded enzymatic activity to gest that metabolism of GL-1 or LysoGL-1 into downstream bioac- deacylate GL-1 to LysoGL-1, which is then converted to sphin- tive lipids is a major contributor to the bone-formation defect. Direct gosine by GBA2 (Fig. 1A) (12–14). GBA deficiency in GD1 thus testing revealed a strong inhibition of osteoblast viability by nano- results in the accumulation of GL-1 and LysoGL-1 in lysosomes molar concentrations of sphingosine, but not of ceramide. These and subsequently in the cytosol (Fig. 1B) (15). Hence, cytosolic findings are consistent with toxicity of high circulating sphingosine GL-1 and LysoGL-1 potentially become substrates for the extra- levels in GD1 patients, which decline upon enzyme-replacement lysosomal neutral GBA, encoded by GBA2, which should result in therapy; serum ceramide levels remain unchanged. Together, com- the production of ceramide and sphingosine, respectively (16, 17). plementary results from mice and humans affected with GD1 not We speculate that if ceramide and/or sphingosine are the ac- only pinpoint sphingosine as being an osteoblast toxin, but also set tive disease-causing agents in GD1, then deleting Gba2 in Mx1– + forth Gba2 as a viable therapeutic target for the development of Cre :GD1 mice will rescue aspects of the GD1 phenotype, de- inhibitors to ameliorate certain disabling consequences of GD1. spite enhanced elevations of GL-1 and LysoGL-1 (Fig. 1C). We report visceral, hematologic, skeletal, and partial cytokine rescue macrophage | knockout mice | S1P of the GD1 phenotype in compound mutant mice. We also show aucher disease (GD) is the most common lysosomal storage Significance Gdisorder, with a frequency as high as 1 in 850 live births in Ashkenazi Jews. Inherited deficiency of lysosomal glucocere- Type 1 Gaucher disease (GD1) is a rare autosomal recessive brosidase (GBA) arising from biallelic mutations in the GBA – disorder caused by inherited mutations in the glucocerebrosidase gene underlies the clinical phenotype of GD (1 3). As a conse- (GBA1) gene. This disease results in a marked accumulation quence, the glucosphingolipids glucosylceramide (GL-1) and glu- of glycosphingolipid substrates, causing visceromegaly, cyto- cosylsphingosine (LysoGL-1) accumulate conspicuously within the penia, and osteopenia. Here, we have rescued this clinical – lysosomes of mononuclear phagocytes (1 4). These lipids spill phenotype in GD1 mice by genetically deleting Gba2, a gene over into circulation with modest elevation in serum GL-1 levels, encoding a downstream extralysosomal enzyme, GBA2. We but with dramatic increases in LysoGL-1 due to its high solubility also report that sphingosine production in GD1 patients may (5). It is not understood, however, how the metabolites of GL-1 contribute to the low-turnover bone loss. Our data suggest and LysoGL-1 produced by extralysosomal glucocerebrosidase that GBA2 and sphingosine are potential targets for pharma- (GBA2) contribute to GD pathophysiology. cological intervention in GD1 patients. Nonneuronopathic type 1 GD (GD1) manifests classically with striking hepatosplenomegaly, profound cytopenia, and a complex Author contributions: P.K.M., J.K., J.L.B., R.O.B., M.I.N., and M.Z. designed research; J. Liu, pattern of severe skeletal disease. However, some GD1 patients L.S., W.-L.C., R.Y., P.L., J. Li, A.S., and A.M. performed research; K.Z. contributed new reagents/analytic tools; P.K.M., J. Liu, L.S., W.-L.C., T.Y., K.Z., A.S., J.L.B., D.J., R.O.B., and can display broader phenotypes, including neurodegeneration, M.Z. analyzed data; and P.K.M., T.Y., M.I.N., and M.Z. wrote the paper. autoimmune diathesis, lymphoproliferative neoplasms, and Conflict of interest statement: P.K.M. and M.Z. have received honoraria and/or consulting osteopenia (1, 3). Furthermore, there is unexplained phenotypic fees from Genzyme. GBA variability among patients harboring identical mutations, 1To whom correspondence may be addressed. E-mail: [email protected], maria. between affected sibling pairs, and even between identical twins [email protected], or [email protected]. (6). Enzyme-replacement therapy (ERT) with macrophage-tar- 2J. Liu and L.S. contributed equally to this work. geted, recombinant glucocerebrosidase ameliorates certain, but This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. not all of, the manifestations (1). Thus, efforts to understand the 1073/pnas.1400768111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1400768111 PNAS Early Edition | 1of6 Downloaded by guest on October 4, 2021 Fig. 1. Aberration of sphingolipid pathways in Gba deficiency. (A) In normal mice, the lipid substrate glucosylceramide (GL-1) is converted by the lysosomal acid glucocerebrosidase Gba to ceramide and by ceramidase to glucosylsphingosine (LysoGL-1). (B) In Gba deficiency, the accumulating GL-1 and LysoGL-1 spill over into the cytoplasm, where the extralysosomal neutral glucocerebrosidase Gba2 converts them to ceramide and sphingosine, respectively. (C) We predict that if the defects in GD are due to products of GL-1 and LysoGL-1—namely, sphingosine or ceramide—the Gba−/− phenotype will be rescued in vivo by the deletion of Gba2. that sphingosine, but not ceramide, inhibits osteoblast survival extralysosomal Gba2 as a target to control at least some aspects of and that GD1 patients display elevated sphingosine levels that B-cell involvement in GD1. Group B cytokines, including IL-3, -13, are reduced upon imiglucerase therapy. The data identify monocyte chemotatic protein-1 (MCP-1), and TNF-α, were in- + sphingosine as one molecular mediator of GD1 pathophysiology creased in Mx1–Cre :GD1 mice, but were not sensitive to Gba2 and suggest that the extralysosomal GBA2 could potentially be deletion either alone or in combination with Gba deletion. targeted to ameliorate certain disabling manifestations of GD1. Group C cytokines that included IL-2, -6, granulocyte colony- stimulating factor (G-CSF), and keratinocyte-derived chemokine + Results (KC) were all elevated in Mx1–Cre :GD1 mice, but suppressed −/− We hypothesized that Gba2-encoded extralysosomal glucocere- in global Gba2 mice, compared with controls. Reduction of brosidase plays a pivotal role in GD pathophysiology. Having these Th1 cytokines in GD1 by targeting Gba2 may provide + generated a Mx1–Cre :GD1 mouse that recapitulates human additional therapeutic benefit in attenuating the accompanying GD1, we sought to delete Gba2 in this mouse to generate a systemic inflammation. Group D cytokines comprised IL-1β, + −/− compound Mx1–Cre :GD1:Gba2 mouse with a goal to rescue -9, macrophage inflammatory protein-1-beta (MIP-1β), and IFN- the GD1 clinical phenotype. To generate this mouse, we crossed γ, which were elevated in response to both Gba and Gba2 de- fl/fl fl/− Gba floxed mice—either wild-type Gba or Gba —with letion, but were not rescueable. The patterns of lack of rescue of +/− + Gba2 mice. The mice were finally bred with Mx1–Cre mice certain cytokines in the double mutant may be due to the con- + −/− to yield Mx1–Cre :GD1:Gba2 mice. We have previously tinued elevation of GL-1 and LysoGL-1 (Fig. 4A), which might + fl/fl fl/− shown that Mx1–Cre :Gba and Mx1–Cre+:Gba mice have itself drive this hypercytokinemia. This GL1/LysoGL1 elevation is an identical clinical phenotype (4). Hence, these mutants were consistent with the persistence of Gaucher cells in the double pooled for the rescue studies.
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