Intracranial transplantation of neural stem cells (NSCs) delayed disease onset, preserved motor function, reduced pathology and prolonged survival in a mouse model of Sandhoff disease, a lethal gangliosidosis. Although donor-derived neurons were electrophysiologically active within chimeric regions, the small degree of neuronal replacement alone could not account for the improvement. NSCs also increased brain beta-hexosaminidase levels, reduced ganglioside storage and diminished activated microgliosis. Additionally, when oral glycosphingolipid biosynthesis inhibitors (beta-hexosaminidase substrate inhibitors) were combined with NSC transplantation, substantial synergy resulted. Efficacy extended to human NSCs, both to those isolated directly from the central nervous system (CNS) and to those derived secondarily from embryonic stem cells. Appreciating that NSCs exhibit a broad repertoire of potentially therapeutic actions, of which neuronal replacement is but one, may help in formulating rational multimodal strategies for the treatment of neurodegenerative diseases.

Mouse models of the GM2 gangliosidoses [Tay-Sachs, late onset Tay-Sachs (LOTS), Sandhoff] and GM1 gangliosidosis have been studied to determine whether there is a common neuro-inflammatory component to these disorders. During the disease course, we have: (i) examined the expression of a number of inflammatory markers in the CNS, including MHC class II, CD68, CD11b (CR3), 7/4, F4/80, nitrotyrosine, CD4 and CD8; (ii) profiled cytokine production [tumour necrosis factor alpha (TNF alpha), transforming growth factor (TGF beta 1) and interleukin 1 beta (IL1 beta)]; and (iii) studied blood-brain barrier (BBB) integrity. The kinetics of apoptosis and the expression of Fas and TNF-R1 were also assessed. In all symptomatic mouse models, a progressive increase in local microglial activation/expansion and infiltration of inflammatory cells was noted. Altered BBB permeability was evident in Sandhoff and GM1 mice, but absent in LOTS mice. Progressive CNS inflammation coincided with the onset of clinical signs in these mouse models. Substrate reduction therapy in the Sandhoff mouse model slowed the rate of accumulation of glycosphingolipids in the CNS, thus delaying the onset of the inflammatory process and disease pathogenesis. These data suggest that inflammation may play an important role in the pathogenesis of the gangliosidoses.

Sandhoff disease is a neurodegenerative disorder resulting from the autosomal recessive inheritance of mutations in the HEXB gene, which encodes the ␤-subunit of ␤-hexosaminidase. G M2 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 G M2 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.

Many neurodegenerative diseases are characterized by the accumulation of undegradable molecules in cells or at extracellular sites in the brain. One such family of diseases is the lysosomal storage disorders, which result from defects in various aspects of lysosomal function. Until recently, there was little prospect of treating storage diseases involving the CNS. However, recent progress has been made in understanding these conditions and in translating the findings into experimental therapies. We review the developments in this field and discuss the similarities in pathological features between these diseases and some more common neurodegenerative disorders.

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Gangliosides are found at high levels in neuronal tissues where they play a variety of important functions. In the gangliosidoses, gangliosides accumulate because of defective activity of the lysosomal proteins responsible for their degradation, usually resulting in a rapidly progressive neurodegenerative disease. However, the molecular mechanism(s) leading from ganglioside accumulation to neurodegeneration is not known. We now examine the effect of ganglioside GM2 accumulation in a mouse model of Sandhoff disease (one of the GM2 gangliosidoses), the Hexb؊/؊ mouse. Microsomes from Hexb؊/؊ mouse brain showed a significant reduction in the rate of Ca 2؉ -uptake via the sarco/endoplasmic reticulum Ca 2؉ -ATPase (SERCA), which was prevented by feeding Hexb؊/؊ mice with N-butyldeoxynojirimycin (NB-DNJ), an inhibitor of glycolipid synthesis that reduces GM2 storage. Changes in SERCA activity were not due to transcriptional regulation but rather because of a decrease in V max . Moreover, exogenously added GM2 had a similar effect on SERCA activity. The functional significance of these findings was established by the enhanced sensitivity of neurons cultured from embryonic Hexb؊/؊ mice to cell death induced by thapsigargin, a specific SERCA inhibitor, and by the enhanced sensitivity of Hexb؊/؊ microsomes to calcium-induced calcium release. This study suggests a mechanistic link among GM2 accumulation, reduced SERCA activity, and neuronal cell death, which may be of significance for delineating the neuropathophysiology of Sandhoff disease.

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...

Lysosomal storage disorders (LSDs) occur at a frequency of 1 in every 5,000 live births and are a common cause of pediatric neurodegenerative disease. The relatively small number of patients with LSDs and lack of validated biomarkers are substantial challenges for clinical trial design. Here, we evaluated the use of a commercially available fluorescent probe, Lysotracker, that can be used to measure the relative acidic compartment volume of circulating B cells as a potentially universal biomarker for LSDs. We validated this metric in a mouse model of the LSD Niemann-Pick type C1 disease (NPC1) and in a prospective 5-year international study of NPC patients. Pediatric NPC subjects had elevated acidic compartment volume that correlated with age-adjusted clinical severity and was reduced in response to therapy with miglustat, a European Medicines Agency-approved drug that has been shown to reduce NPC1-associated neuropathology. Measurement of relative acidic compartment volume was also useful for monitoring therapeutic responses of an NPC2 patient after bone marrow transplantation. Furthermore, this metric identified a potential adverse event in NPC1 patients receiving i.v. cyclodextrin therapy. Our data indicate that relative acidic compartment volume may be a useful biomarker to aid diagnosis, clinical monitoring, and evaluation of therapeutic responses in patients with lysosomal disorders.