Cerebellar neurons lacking complex gangliosides degenerate in the presence of depolarizing levels of potassium

Abstract

Mice engineered to lack GM2͞GD2 synthase (GalNAc-T), with resultant deficit of GM2, GD2, and all gangliotetraose gangliosides, were originally described as showing a relatively normal phenotype with only a slight reduction in nerve conduction. However, a subsequent study showed that similar animals suffer axonal degeneration, myelination defects, and impaired motor coordination. We have examined the behavior of cerebellar granule neurons from these neonatal knockouts in culture and have found evidence of impaired capacity for Ca 2؉ regulation. These cells showed relatively normal behavior when grown in the presence of physiological or moderately elevated K ؉ but gradually degenerated in the presence of high K ؉ . This degeneration in depolarizing medium was accompanied by progressive elevation of intracellular calcium and onset of apoptosis, phenomena not observed with normal cells. No differences were detected in cells from normal vs. heterozygous mice. These findings suggest that neurons from GalNAc-T knockout mice are lacking a calcium regulatory mechanism that is modulated by one or more of the deleted gangliosides, and they support the hypothesis that maintenance of calcium homeostasis is one function of complex gangliosides during, and perhaps subsequent to, neuronal development.GM1 ganglioside ͉ ganglioside-deficient neurons ͉ cerebellar granule neurons ͉ neuritogenesis ͉ intracellular calcium G angliosides are the major sialoglycoconjugates of the vertebrate central nervous system (1, 2) and have been proposed as important determinants in neuronal differentiation (3-5). Early support for this idea came from study of ganglioside storage disease, in which pyramidal neurons of the cerebral cortex were shown to sprout ectopic dendrites from the ''meganeurite'' ganglioside storage area with formation of aberrant synapses (6). The gangliotetraose family, consisting of GM1 and its oligosialo derivatives (GD1a, GD1b, GT1b, GQ1b, etc.), are the predominant forms in the neuron and are biosynthesized by a series of Golgi-localized enzymes that add sugars sequentially to the membrane-anchoring ceramide moiety (7,8). One approach to the study of their mechanistic roles has been development of genetically altered mice or cell lines that overexpress or lack one or more specific ganglioside. Neuro-2a cells overexpressing GD3 and complex gangliosides of the b-series underwent spontaneous neuritogenesis and cholinergic differentiation (9), whereas blockade of GM3 synthase with antisense vector reduced neuritogenesis in cerebellar granule neurons (CGN). † On the other hand, embryonic stem cells deficient in GD3 synthase could be induced to express a complex neurite network, suggesting that b-series gangliosides are not essential for neuronal differentiation of uncommitted precursor cells (10). NG-CR72 cells, mutants of the NG108-15 line deficient in GM1 synthase, were found to respond positively to dendritogenic agents with prolific neurite outgrowth but negatively to axonogenic stimuli with apoptosis (11). The latter effe...

Enhanced Susceptibility to Kainate-Induced Seizures, Neuronal Apoptosis, and Death in Mice Lacking Gangliotetraose Gangliosides: Protection with LIGA 20, a Membrane-Permeant Analog of GM1

“…In keeping with those findings, cultured CNS neurons from genetically altered mice lacking GM1 and other N-acetylgalactosamine(GalNac)-containing gangliosides because of disrupted GM2/GD2 synthase [UDP-N-acetyl-D-galactosaminyltransferase (GalNAcT); EC 2.4.1.92] (Fig. 1 B) suffered dysregulation of Ca 2ϩ homeostasis (Wu et al, 2001a). The apoptotic effect of this dysregulation in cultured CNS neurons, induced by both glutamate and high K ϩ , was partially attenuated by GM1 and more effectively by LIGA 20, a semisynthetic analog of GM1 ( Fig.…”

Neuromuscular synaptic transmission in aged ganglioside-deficient mice

“…GM2s-KO cerebellar neurons have been shown to degenerate when cultured under K + -induced depolarization, caused by an underlying impairment of cellular Ca 2+ regulation (Wu et al, 2001). We tested in acute experiments whether a similar effect occurs at aged GM2s-KO motor nerve terminals by measuring the increase in MEPP frequency evoked by increased extracellular K + concentrations.…”

“…Cultured GM2s-KO neurons have been shown to degenerate upon K + -induced depolarization, caused by an underlying impairment of cellular Ca 2+ regulation (Wu et al, 2001). In order to investigate possible effects at the GM2s-KO NMJ, we measured MEPP frequency in extracellular K + concentrations varying from 4.5-30 mM and compared it to WT NMJs (n=3-7 mice per condition per genotype).…”

“…Gangliosides have been suggested to play a role in the membrane flux of Ca 2+ and its cellular homeostasis (Ledeen and Wu, 2006;Rahmann et al, 1998;Wu et al, 2001). Therefore, we explored synaptic transmission at NMJs from muscle preparations incubated at low (0.2 mM) and high (5 mM) extracellular Ca 2+ , at 25 C bath temperature.…”

Gangliosides of the nuclear membrane: A crucial locus of cytoprotective modulation

“…Calcium is well known to have a critical role in apoptosis [Mattson and Chan, 2003], the nucleus being especially vulnerable. Cerebellar granule neurons (CGN) from mice engineered to lack GM2/GD2 synthase, with resultant deficit of GM2, GD2 and all gangliotetraose gangliosides were found deficient in calcium regulatory capability that resulted in apoptotic death when the cells were grown in the presence of high K þ [Wu et al, 2001]. That this was due to absence of GM1 was suggested in the observation that CGN from these knockout mice could be rescued from elevated K þ as well as excitotoxic levels of glutamate by GM1, and even more effectively by LIGA-20, a semisynthetic derivative of GM1 .…”