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Related Collections Phagocytes Transplantation Brief Reports Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  Blood, 1 January 2001, Vol. 97, No. 1, pp. 327-329 BRIEF REPORT Enhanced survival in Sandhoff disease mice receiving a combination of substrate deprivation therapy and bone marrow transplantation Mylvaganam Jeyakumar, Francine Norflus, Cynthia J. Tifft, Mario Cortina-Borja, Terry D. Butters, Richard L. Proia, V. Hugh Perry, Raymond A. Dwek, and Frances M. Platt From the Glycobiology Institute, Department of Biochemistry, University of Oxford, Oxford, and the School of Biological Sciences, University of Southampton, Southampton, United Kingdom; and the National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD.     Abstract Top Abstract Introduction Study design Results and discussion References Sandhoff disease is a lysosomal storage disorder characterized by GM2 ganglioside accumulation in the central nervous system (CNS) and periphery. It results from mutations in the HEXB gene, causing a deficiency in -hexosaminidase. Bone marrow transplantation (BMT), which augments enzyme levels, and substrate deprivation (using the glycosphingolipid biosynthesis inhibitor N-butyldeoxynojirimycin [NB-DNJ]) independently have been shown to extend life expectancy in a mouse model of Sandhoff disease. The efficacy of combining these 2 therapies was evaluated. Sandhoff disease mice treated with BMT and NB-DNJ survived significantly longer than those treated with BMT or NB-DNJ alone. When the mice were subdivided into 2 groups on the basis of their donor bone marrow-derived CNS enzyme levels, the high enzyme group exhibited a greater degree of synergy (25%) than the group as a whole (13%). Combination therapy may therefore be the strategy of choice for treating the infantile onset disease variants. (Blood. 2001;97:327-329) © 2001 by The American Society of Hematology.     Introduction Top Abstract Introduction Study design Results and discussion References The GM2 gangliosidoses are progressive, neurodegenerative lysosomal storage diseases.1 The hydrolysis of GM2 is catalyzed by -hexosaminidase. The HEXA and HEXB genes encode the -hexosaminidase  and  subunits, respectively.1 Mutations in the  and  subunit genes result in Tay-Sachs and Sandhoff diseases, respectively. Potential therapeutic approaches include enzyme augmentation (enzyme replacement, bone marrow transplantation [BMT], or gene therapy) and substrate deprivation.2,3 In BMT microglial cells of donor origin are thought to repopulate the brain, becoming perivascular macrophages and microglia, and to supply enzyme to neurones by secretion-recapture.4-6 Substrate deprivation utilizes an inhibitor of GSL biosynthesis, such as N-butyldeoxynojirimycin (NB-DNJ).7 By partially inhibiting GSL biosynthesis, the residual enzyme activity can hydrolyze the reduced influx of substrate into the lysosome, thus preventing storage. NB-DNJ inhibits the ceramide glucosyltransferase (glucosylceramide synthase, UDP-glucose-N-acylsphingosine D-glucosyltransferase, EC 2.4.1.80), which is the first enzyme in the GSL biosynthetic pathway.3,8 In a mouse model of Tay-Sachs disease, NB-DNJ treatment reduced GM2 accumulation in the brain.9 In the symptomatic mouse model of Sandhoff disease, NB-DNJ-treated mice10,11 survived 40% longer than untreated controls.10 BMT of Sandhoff mice extended life expectancy up to 8 months.12 Because the augmentation of enzyme through BMT and substrate deprivation may together show greater efficacy, we have treated Sandhoff mice with BMT and NB-DNJ and find that the 2 therapies act synergistically.     Study design Top Abstract Introduction Study design Results and discussion References Animals, treatment procedures, and behavioral tests Sandhoff mice were bone marrow transplanted at 10 to 16 days of age.12 Drug treatment was carried out as previously described.10,13 NB-DNJ was a gift from Searle/Monsanto (St Louis, MO) and Oxford GlycoSciences (Abingdon, United Kingdom). Mice were tested on the bar crossing or inverted screen as previously described.10 The Open Field Test (box 45 × 25 × 12 cm with a 150 cm2 floor grid) was conducted according to published methods.14 Biochemical analysis We used published methods to assay -hexosaminidase.10 BCA protein assay was used for protein determinations (Pierce, Chester, United Kingdom). GSL analysis was conducted as previously described.9 Statistical analysis Survival graphs were analyzed by the log-rank or the Mantel-Haenszel test.15 Log-likelihood test was used for -hexosaminidase enzyme level correlations. We used the Student t test to analyze -hexosaminidase, GSL levels, and locomotion scores. Bar-crossing and inverted-screen test data were analyzed using a nonparametric regression model with a logistic link to the data set. P values were estimated using likelihood ratio. The statistical software used was S-PLUS version 3.4 (MathSoft, Seattle, WA).     Results and discussion Top Abstract Introduction Study design Results and discussion References Survival of Sandhoff mice receiving combination therapy Bone marrow-transplanted Sandhoff (SH) mice were treated with NB-DNJ (600 mg/kg per day) from 10 to 11 weeks of age. Four groups were studied: untreated (SHUT), BMT monotherapy (SHBMT), NB-DNJ monotherapy (SHNB-DNJ), and combination therapy (BMT and NB-DNJ [SHBMT/NB-DNJ]). Mean survival time (Figure 1A) of the SHUT mice was 137 days. Survival was 166 days ± 4 (SHNB-DNJ), 196 days ± 8 (SHBMT), and 239 days ± 20 (SHBMT/NB-DNJ). All treated mice had significantly (P < .001) increased lifespans compared with SHUT mice (Figure 1A). SHBMT/NB-DNJ mice survived significantly (P < .001) longer than SHBMT or SHNB-DNJ mice. The effect of NB-DNJ on BMT was 13% more than additive (P < .089, Figure 1A). When the SHBMT mice were subdivided (50% of the mice into each category) on the basis of central nervous system -hexosaminidase levels (2.26% ± 0.11% and 3.31% ± 0.94%, low- and high-enzyme groups, respectively; P = .108), the high-enzyme group exhibited 25% synergy (P < .001). The low-enzyme group exhibited an additive effect (P = .71) of NB-DNJ and BMT. Between the SHBMT and SHBMT/NB-DNJ groups, a similar level of -hexosaminidase enzyme was measured in brain (P = .54) and spinal cord (P = .42) (Figure 1B) and in peripheral tissues (Figure 1C). In SHBMT, there was no correlation between survival and -hexosaminidase enzyme levels in brain, spinal cord, or peripheral tissues (P < .226). However, in the SHBMT/NB-DNJ group, there was a significant correlation between survival and -hexosaminidase levels in brain and spinal cord (P < .016, Pearson's correlation analysis) but not in liver, spleen, and kidney. View larger version (23K): [in this window] [in a new window]   Figure 1. Survival of treated and untreated Sandhoff mice. (A) Mice (n = 4-6 per group) were monitored daily, and a humane endpoint was applied when they became moribund and unable to right themselves when laid on their sides. In all the treated groups, the increase in survival relative to SHUT was statistically significant (P < .001). The synergistic effect was estimated using Bootstrap method. Synergy quotient (SQ) was defined as SQ = [(BMT  UT ) + (NB  DNJ  UT)]/(BMT/NB  DNJ  UT), where UT, BMT, NB  DNJ, and BMT/NB  DNJ are the mean survival of the Sandhoff mice untreated, treated with BMT, treated with NB-DNJ, and treated with BMT and NB-DNJ, respectively. If there is synergy, the SQ is less than one. (B) Analysis of -hexosaminidase activity in nerve tissues and (C) in visceral organs of the Sandhoff mice after BMT treatment. Each organ was assayed in triplicate. The means for the different animals were averaged (± SEM) and expressed as a percentage of wild-type levels. n = 4 to 6 animals per group. Neurologic function SHBMT, SHNB-DNJ, and SHBMT/NB-DNJ groups differed from the SHUT group in both the bar-crossing (P < .01) (Figure 2A) and inverted-screen tests (P < .01) (Figure 2B). The SHBMT group performed better in the bar-crossing test than the SHNB-DNJ group (P < .001), which performed better than the SHUT group (P < .05). The SHBMT/NB-DNJ group performed better than all other treatment groups (P < .023). The rate of decline in the SHBMT/NB-DNJ group was significantly (P < .01) slower than in all other groups. In the inverted-screen test, performance started to decline at approximately 127 days for SHUT but at approximately 140, 175, and 220 days for the SHNB-DNJ, SHBMT, and SHBMT/NB-DNJ groups, respectively. The rates of decline were similar across groups. All groups displayed similar locomotor activity at 12 weeks, comparable to wild-type mice (Figure 2C). However, at 18 and 24 weeks of age, there were significant differences in activity between SHUT and SHNB-DNJ in the BMT (P < .01) and the non-BMT (P < .001) groups (Figure 2C). View larger version (36K): [in this window] [in a new window]   Figure 2. Representative neurologic function of treated and untreated Sandhoff mice. Each animal was tested for its ability to cross a horizontal bar, measuring balance and motor co-ordination (A), and for the amount of time it spent upside down on an inverted screen, measuring muscle strength (B). Data are mean ± 0.4 of the SD. n = 4 to 6 animals per group. Locomotor activity of the NB-DNJ-treated and untreated mice was measured (C). Each animal at 12, 18, and 24 weeks of age was tested for locomotion in an "open-field." Data are mean ± SD (n = 4-6 animals per group). Between treated and untreated mice, data are statistically significant at 18 weeks (P < .05) and 24 weeks (P < .01) of age in control and BMT-treated Sandhoff mice. Glycosphingolipid analysis at end stage GM2 and GA2 were measured at terminal stage of the disease (Table 1). In the brains of SHNB-DNJ, SHBMT, and SHBMT/NB-DNJ mice, the GA2 levels were comparable to those in SHUT (P  .478), even though they lived longer than the controls. All treated groups had the same GA2 storage burden as the untreated mice, suggesting a slower rate of GA2 accumulation. The brain GM2 levels were similar in the SHNB-DNJ, SHBMT, and SHUT mice (P  .614), but the SHBMT/NB-DNJ mice had approximately 30% higher levels than the other groups (P  .017). In spinal cord the GM2 storage levels were comparable to those in brain. However, the spinal cord GA2 levels were significantly lower in the SHBMT (21%, P < .001) and in the SHBMT/NB-DNJ (12%, P < .01) groups than in the SHUT group. This may be due to higher levels of donor enzyme in the spinal cord (approximately 12% of normal; Figure 1B). In the SHNB-DNJ group, the liver GM2 level was reduced by approximately 17% (P < .01) whereas the GA2 level was comparable to that of the SHUT (P = .26) at end stage. However, in the SHBMT and SHBMT/NB-DNJ groups, the liver GSL composition approximated that of the wild type because of high donor-enzyme levels (Figure 1C).                                View this table: [in this window] [in a new window]   Table 1. Analysis of storage glycosphingolipids GM2 and GA2 at the terminal stage of disease in Sandhoff mice The onset of symptoms and the rates of disease progression were delayed significantly in SHBMT/NB-DNJ relative to the monotherapy groups. By increasing the level of enzyme, the therapeutic response to NB-DNJ was enhanced. In brain, -hexosaminidase activity from BMT was relatively small (approximately 2% to 5% of normal). The survival increase in the SHBMT/NB-DNJ group was 13% more than the sum of the 2 monotherapy effects, indicating synergy. The survival of the combination group revealed 2 cohorts, those that survived the longest and those that died at an earlier time point. Survival correlated with the enzyme level in the brain. Synergy was approximately 25% for the high enzyme group compared to 13% for the group as a whole. The additional brain enzyme reconstitution needed to achieve approximately 25% synergy was only approximately 1.05% of the wild-type level. Mechanisms for the clinical benefit of BMT may extend beyond augmentation of enzyme levels. This may involve an inflammatory component with activated microglia contributing to neuronal dysfunction or death, as has been proposed for Alzheimer disease. The replacement of diseased microglial cells with wild-type cells by BMT, in combination with direct substrate reduction in neurones by NB-DNJ, may have led to synergy. Glycosphingolipid analysis at terminal stage showed that for SHBMT/NB-DNJ there was an increased storage burden of GM2 and enhanced survival. This suggests that there is not a simple threshold level of brain GM2 storage, which leads to disease. It supports a more complex mechanism of pathogenesis, as suggested previously.12 Levels of GA2 are similar in all groups at their respective endpoints, suggesting that in the mouse there may be a threshold level of GA2 that elicits disease. It is unclear why there is no elevated storage in the BMT group, despite their enhanced survival. After approximately 4 months it is possible that the rate of synthesis is lowered because of neuronal dysfunction, death, or both or that BMT only impacts the storage levels later in the disease course. In conclusion, NB-DNJ therapy shows promise in treating GSL storage diseases, which involve neuropathology. These 2 therapeutic approaches act synergistically, provided that a high enough level of enzyme reconstitution can be achieved.     Acknowledgments We thank Searle/Monsanto and Oxford GlycoSciences for NB-DNJ, Julia McAvoy and David Smith for excellent technical assistance, and Rob Deacon for his advice on behavioral tests.     Footnotes Submitted March 27, 2000; accepted September 7, 2000. F.M.P. is a Lister Institute Research Fellow. M.J. is supported by a Biotechnology and Biological Science Research Council studentship. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Frances M. Platt, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, United Kingdom; e-mail: fran{at}glycob.ox.ac.uk.     References Top Abstract Introduction Study design Results and discussion References 1. Gravel RA, Clarke JTR, Kaback MM, Mahuran D, Sandhoff K, Suzuki K. The GM2 gangliosidoses. In: Scriver CR,Beadet AL,Sly WS,Valle D, eds. The Metabolic and Molecular Bases of Inherited Disease. Vol 2. New York: McGraw Hill; 1995:2839-2879. 2. Radin NS. Treatment of Gaucher disease with an enzyme inhibitor. Glycoconj J. 1996;13:153-157[CrossRef][Medline] [Order article via Infotrieve]. 3. Platt FM, Butters TD. New therapeutic prospects for the glycosphingolipid lysosomal storage diseases. Biochem Pharmacol. 1998;56:421-430[CrossRef][Medline] [Order article via Infotrieve]. 4. Hoogerbrugge PM, Brouwer OF, Bordigoni P, et al. Allogeneic bone marrow transplantation for lysosomal storage diseases: The European Group for Bone Marrow Transplantation [see comments]. Lancet. 1995;345:1398-1402[CrossRef][Medline] [Order article via Infotrieve]. 5. Walkley SU, Dobrenis K. Bone marrow transplantation for lysosomal diseases [comment]. Lancet. 1995;345:1382-1383[CrossRef][Medline] [Order article via Infotrieve]. 6. Krivit W, Sung JH, Shapiro EG, Lockman LA. Microglia: the effector cell for reconstitution of the central nervous system following bone marrow transplantation for lysosomal and peroxisomal storage diseases. Cell Transplant. 1995;4:385-392[CrossRef][Medline] [Order article via Infotrieve]. 7. Platt FM, Neises GR, Dwek RA, Butters TD. N-butyldeoxynojirimycin is a novel inhibitor of glycolipid biosynthesis. J Biol Chem. 1994;269:8362-8365[Abstract/Free Full Text]. 8. Platt FM, Neises GR, Karlsson GB, Dwek RA, Butters TD. N-butyldeoxygalactonojirimycin inhibits glycolipid biosynthesis but does not affect N-linked oligosaccharide processing. J Biol Chem. 1994;269:27108-27114[Abstract/Free Full Text]. 9. Platt FM, Neises GR, Reinkensmeier G, et al. Prevention of lysosomal storage in Tay-Sachs mice treated with N-butyldeoxynojirimycin. Science. 1997;276:428-431[Abstract/Free Full Text]. 10. Jeyakumar M, Butters TD, Cortina-Borja M, et al. Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin. Proc Natl Acad Sci U S A. 1999;96:6388-6393[Abstract/Free Full Text]. 11. Sango K, Yamanaka S, Hoffmann A, et al. Mouse models of Tay-Sachs and Sandhoff diseases differ in neurologic phenotype and ganglioside metabolism. Nat Genet. 1995;11:170-176[CrossRef][Medline] [Order article via Infotrieve]. 12. Norflus F, Tifft CJ, McDonald MP, et al. Bone marrow transplantation prolongs life span and ameliorates neurologic manifestations in Sandhoff disease mice. J Clin Invest. 1998;101:1881-1888[Medline] [Order article via Infotrieve]. 13. Platt FM, Reinkensmeier G, Dwek RA, Butters TD. Extensive glycosphingolipid depletion in the liver and lymphoid organs of mice treated with N-butyldeoxynojirimycin. J Biol Chem. 1997;272:19365-19372[Abstract/Free Full Text]. 14. Gerlai R, Friend W, Becker L, O'Hanlon D, Marks A, Roder J. Female transgenic mice carrying multiple copies of the human gene for S100 beta are hyperactive. Behav Brain Res. 1993;55:51-59[CrossRef][Medline] [Order article via Infotrieve]. 15. Harrington DP, Fleming TR. A class of rank test procedures for censored survival data. Biometrika. 1982;69:553-556[Abstract/Free Full Text]. © 2001 by The American Society of Hematology.   CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: G. Douillard-Guilloux, N. Raben, S. Takikita, L. Batista, C. Caillaud, and E. Richard Modulation of glycogen synthesis by RNA interference: towards a new therapeutic approach for glycogenosis type II Hum. Mol. Genet., December 15, 2008; 17(24): 3876 - 3886. [Abstract] [Full Text] [PDF] D. Elstein, A. Dweck, D. Attias, I. Hadas-Halpern, S. Zevin, G. Altarescu, J. F. M. G. Aerts, S. van Weely, and A. 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