Enhanced survival in Sandhoff disease mice receiving a combination of substrate deprivation therapy and bone marrow transplantation

Abstract

IntroductionThe G M2 gangliosidoses are progressive, neurodegenerative lysosomal storage diseases. 1 The hydrolysis of G M2 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-Nacylsphingosine 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 G M2 accumulation in the brain. 9 In the symptomatic mouse model of Sandhoff disease, NB-DNJtreated mice 10,11 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 designAnimals, 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 ϫ 25 ϫ 12 cm with a 150 cm 2 floor grid) was conducted according to published methods. 14 Biochemical analysisWe 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 analysisSurvival 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 Survival of Sandhoff mice receiving combination therapyBone marrow-transplanted Sandhoff (SH) mice were treated with NB-DNJ (6...

Early deficits in motor coordination and cognitive dysfunction in a mouse model of the neurodegenerative lysosomal storage disorder, Sandhoff disease

“…4C) indicative of increased "anxiety-like" behavior in the hexb−/−mice. We have employed a behavioral test battery to assess a mouse model of Sandhoff disease that we backbred onto the C57BL/6J background.We demonstrated deficits in motor activity, and coordination consistent with those previously reported [1,7,8,13,14,16,18,20,22,25,27,28] suggesting no significant effect of genetic background. However, we were also able to detect deficits as early as 66 days using the balance beam test.…”

“…Furthermore, the fact that all mice improve with successive trials in the rotarod and that the motor incoordination was worse in the harder balance beam task would tend to argue against solely peripheral causes, such as muscle wasting and hind limb rigidity. Finally, the deficits in motor coordination and/or learning are in accord with the neuronal loss and cellular pathology reported in the cerebellum, basal ganglia and striatum [16,18,22,25,28].The age of onset of motor deficits previously reported has widely varied, even when the same test was employed, such as the rotarod [18,20,22,25,27,28] or horizontal beam crossing test [1,4,[13][14][15], in which deficits were first detected as late as 104 days [13].Varied findings for the same test may be attributable to different genetic backgrounds (the original mice were not purebred and colony breeding schemes have differed between reports) and/or to different experimental parameters. Evidence for onset of motor incoordination as early as 60-70 days in LSDs, as we have detected using the balance beam test, is uncommon [15,27,28].…”

“…The age of onset of motor deficits previously reported has widely varied, even when the same test was employed, such as the rotarod [18,20,22,25,27,28] or horizontal beam crossing test [1,4,[13][14][15], in which deficits were first detected as late as 104 days [13].…”

“…The Sandhoff models have engendered much research directed at overcoming the challenges [1,2,4,11,14,15,18,20]. The knockout model [25] presented here displays ataxia, tremor, muscle wasting, and deficits in motor reflex, coordination and balance [1,4,7,8,13,14,16,18,20,22,25,27,28].…”

“…Both latency (time to cross the beam from end to end) and number of slips (each time a paw fell under the beam midline) were assessed as measures of motor coordination. In the rotarod test, latency to fall from a rotating rod (acceleration increased by 0.5 cm/s every 5 s) was scored automatically with infrared sensors in a Rotamex 5 rotarod (Columbus Inst, Ohio) with four trials per day over 2 days.Behavioral tests were primarily conducted on mice of 79(±2) days old, an age that in most previous assays showed no deficits, but were also performed on some at 66(± 2) and 97(± 2) days when had been typically reported as normal or displaying deficits, respectively [1,4,7,8,13,14,16,18,20,22,25,27,28]. All data are shown as mean ± S.E.M.…”