Abstract
In Sandhoff disease (SD), the activity of the lysosomal hydrolytic enzyme, β-hexosaminidase (Hex), is lost due to a Hexb gene defect, which results in the abnormal accumulation of the substrate, GM2 ganglioside (GM2), in neuronal cells, causing neuronal loss, microglial activation, and astrogliosis. We established induced pluripotent stem cells from the cells of SD mice (SD-iPSCs). In the present study, we investigated the occurrence of abnormal differentiation and development of a neural lineage in the asymptomatic phase of SD in vitro using SD mouse fetus-derived neural stem cells (NSCs) and SD-iPSCs. It was assumed that the number of SD mouse fetal brain-derived NSCs was reduced and differentiation was promoted, resulting in the inhibition of differentiation into neurons and enhancement of differentiation into astrocytes. The number of SD-iPSC-derived NSCs was also reduced, suggesting that the differentiation of NSCs was promoted, resulting in the inhibition of differentiation into neurons and enhancement of that into astrocytes. This abnormal differentiation of SD-iPSCs toward a neural lineage was reduced by the glucosylceramide synthase inhibitor, miglustat. Furthermore, abnormal differentiation toward a neural lineage was reduced in SD-iPSCs with Hexb gene transfection. Therefore, differentiation ability along the time axis appears to be altered in SD mice in which the differentiation ability of NSCs is promoted and differentiation into neurons is completed earlier, while the timing of differentiation into astrocytes is accelerated. These results clarified that the abnormal differentiation of SD-iPSCs toward a neural lineage in vitro was shown to reflect the pathology of SD.
Highlights
In Sandhoff disease (SD), the Hexb gene encoding the β-subunit of the lysosomal hydrolytic enzyme, β-hexosaminidase consisting of Hex A and Hex B subunits, is mutated and the enzyme activities of both subunits are simultaneously lost, resulting in an inability to hydrolyze GM2 ganglioside and the accumulation of GM2 mainly in neurons
Neurons and astrocytes increased in SD mouse fetal brain-derived neurospheres
When miglustat (5 μM) was added during the preparation of cerebral cortex-like tissue with SD-iPSCs, the percentages of EdU/nestin double-positive cells was 30.4 ± 1.57%, showing no effect. These results suggest that differentiation from neural stem cells (NSCs) was promoted in cerebral cortex-like tissue prepared from SD-iPSCs, as observed in SD mouse fetal brain-derived neurospheres, decreasing NSCs and increasing neurons and astrocytes
Summary
In Sandhoff disease (SD), the Hexb gene encoding the β-subunit of the lysosomal hydrolytic enzyme, β-hexosaminidase consisting of Hex A and Hex B subunits, is mutated and the enzyme activities of both subunits are simultaneously lost, resulting in an inability to hydrolyze GM2 ganglioside and the accumulation of GM2 mainly in neurons. Changes that occur during the period from ontogeny to the asymptomatic phase have not yet been clarified, abnormal neurites of neurons prepared from the hippocampal region of SD mouse fetuses [4] and nerve cell death in the dorsal root ganglion prepared from 4-week-old SD mice in the asymptomatic phase [5] have been reported. These findings suggest that changes in neurons in the embryonic stage and asymptomatic phase are the partial cause of subsequent cranial nervous system dysfunction. In order to elucidate the intrinsic cause, the changes that occur before the development of symptoms need to be reproduced in vitro
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