Delayed symptom onset and increased life expectancy in Sandhoff disease mice treated with N-butyldeoxynojirimycin

Abstract

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the beta-subunit of beta-hexosaminidase. GM2 ganglioside fails to be degraded and accumulates within lysosomes in cells of the periphery and the central nervous system (CNS). There are currently no therapies for the glycosphingolipid lysosomal storage diseases that involve CNS pathology, including the GM2 gangliosidoses. One strategy for treating this and related diseases is substrate deprivation. This would utilize an inhibitor of glycosphingolipid biosynthesis to balance synthesis with the impaired rate of catabolism, thus preventing storage. One such inhibitor is N-butyldeoxynojirimycin, which currently is in clinical trials for the potential treatment of type 1 Gaucher disease, a related disease that involves glycosphingolipid storage in peripheral tissues, but not in the CNS. In this study, we have evaluated whether this drug also could be applied to the treatment of diseases with CNS storage and pathology. We therefore have treated a mouse model of Sandhoff disease with the inhibitor N-butyldeoxynojirimycin. The treated mice have delayed symptom onset, reduced storage in the brain and peripheral tissues, and increased life expectancy. Substrate deprivation therefore offers a potentially general therapy for this family of lysosomal storage diseases, including those with CNS disease.

Figure 1

Representative behavioral analysis of Sandhoff mice treated from 6 weeks of age with 2,400 mg/kg per day of NB-DNJ. The data are from male siblings (n = 3 per group). The data are summarized as the means ± SD at each time point. (A) Performance of the mice in the bar-crossing test. (B) Performance of the mice in the inverted screen. (C) Activity levels on the inverted screen. ○, Untreated wild-type mice; ▵, untreated Sandhoff mice; and ▴, NB-DNJ-treated Sandhoff mice. Comparable data were obtained from female mice (data not shown).

Figure 2

(A) Graphical representation of HPTLC analysis of brain and liver G_M2 and G_A2 in Sandhoff mice in the presence or absence of NB-DNJ. Mice were treated from weaning at 3 weeks with 4,800 mg/kg per day of NB-DNJ, and GSL profiles were compared at 16 weeks of age relative to untreated age-matched controls. The HPTLC plates were analyzed by densitometry in relation to authentic GSL standards. (B) Graphical representation of HPTLC analysis of brain region G_M2 and G_A2 content at the end stage of untreated controls (125 days) and NB-DNJ-treated Sandhoff mice (170 days). CC, cerebral cortex; THP, thalamus/hypothalamus; MPM, midbrain, pons, and medulla; CBL, cerebellum; SPN, spinal cord. (C) Comparison of G_M2 and G_A2 levels in whole brain and liver at the end stage of diseases in untreated (125 days) and NB-DNJ-treated mice (170 days). The data are presented as the mean ± SD for three mice per group. Solid bars depict data from untreated mice, and open bars depict data from NB-DNJ-treated mice. Representative data are shown. The data set shown is from male mice, and comparable data were obtained from females (data not shown).

Figure 3

(A) PAS staining of Sandhoff brain sections in the hippocampus (dentate gyrus). (A and C) Untreated brain. (B and D) NB-DNJ-treated brain. The images shown are representative of at least three animals per group. (Bar = 50 μm.) The reduction in PAS staining in NB-DNJ-treated mouse brains also was observed in other storage regions of the brain (data not shown). The data are from female mice, and comparable data were obtained from males (data not shown).

Figure 4

Electron microscopy (low and high magnification) on brain and liver from untreated and NB-DNJ-treated Sandhoff mice (4,800 mg/kg per day for 90 days). Neurons from the polymorph layer of the dentate gyrus: untreated neuron (a and b), NB-DNJ-treated neuron (c and d), untreated hepatocyte (e and f), NB-DNJ-treated hepatocyte (g and h), untreated Kupffer cell (i and j), and NB-DNJ-treated Kupffer cell (k and l). The images are representative of data obtained from at least three mice. The images shown are from female mice, and comparable data were obtained from males (data not shown). (Bar = 0.5 μm.)

Figure 5

Detection of apoptotic cells in the thalamus of untreated Sandhoff mouse at 17 weeks of age (A) and an NB-DNJ-treated mouse at 17 weeks of age (B). The apoptotic cells appear brown (arrows). (Bar = 50 μm.) The sections were derived from male mice, and comparable data were obtained from females (data not shown).