Lactosylceramide synthases encoded by B4galt5 and 6 genes are pivotal for neuronal generation and myelin formation in mice

Abstract

It is uncertain which β4-galactosyltransferase (β4GalT; gene name, B4galt), β4GalT-5 and/or β4GalT-6, is responsible for the production of lactosylceramide (LacCer) synthase, which functions in the initial step of ganglioside biosynthesis. Here, we generated conditional B4galt5 knockout ( B4galt5 cKO) mice, using Nestin-Cre mice, and crossed these with B4galt6 KO mice to generate B4galt5 and 6 double KO (DKO) mice in the central nervous system (CNS). LacCer synthase activity and major brain gangliosides were completely absent in brain homogenates from the DKO mice, although LacCer synthase activity was about half its normal level in B4galt5 cKO mice and B4galt6 KO mice. The DKO mice were born normally but they showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Histological analyses showed that myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of the DKO mice. Neuronal cells, differentiated from neurospheres that were prepared from the DKO mice, showed impairments in neurite outgrowth and branch formation, which can be explained by the fact that neurospheres from DKO mice could weakly interact with laminin due to lack of gangliosides, such as GM1a. Furthermore, the neurons were immature and perineuronal nets (PNNs) were poorly formed in DKO cerebral cortices. Our results indicate that LacCer synthase is encoded by B4galt5 and 6 genes in the CNS, and that gangliosides are indispensable for neuronal maturation, PNN formation, and axonal and myelin formation.

Author summary

Gangliosides are membrane-bound glycosphingolipids that contain sialic acid residues and are abundant in the mammalian nervous system, suggesting that they play pivotal roles in neural functions. We generated conditional β 4-galactosyltransferase-5 ( B4galt5) knockout (KO) and double B4galt5/B4galt6 KO (DKO) mice to completely ablate lactosylceramide (LacCer) synthase in the central nervous system (CNS). LacCer functions in the initial step of ganglioside biosynthesis. DKO mice were born normally but showed growth retardation and motor deficits at 2 weeks and died by 4 weeks of age. Myelin-associated proteins were rarely found localized in axons in the cerebral cortex, and axonal and myelin formation were remarkably impaired in the spinal cords of DKO mice. Neurospheres prepared from DKO mice could weakly interact with laminin, probably due to the lack of gangliosides in these mice. This defect might have caused the impaired neurite outgrowth in neuronal cells from DKO mice and poor formation of perineuronal nets (PNNs) with immature neurons in the cerebral cortices of DKO mice. Our results indicate pivotal roles for gangliosides in the CNS, including neuronal maturation, PNN formation, and axonal and myelin formation.