Mutant NG108-15 cells (NG-CR72) deficient in GM1 synthase respond aberrantly to axonogenic stimuli and are vulnerable to calcium-induced apoptosis: they are rescued with LIGA-20

The neuroblastoma x glioma NG108-15 hybrid cell line, a widely used model f or the study of neuronal differentiation, contains a variety of ganglioside s including GM1 and its sialosylated derivative, GD1a. To investigate the r ole of these a-series gangliotetraose gangliosides in neuritogenesis, we ha ve obtained a mutated subclone of NG108-15 that is deficient in that family of gangliosides. NG108-15 cells were grown in the presence of cholera toxi n, which killed the large majority of cells, and from the cholera-resistant survivors we isolated a clone, NG-CR72, that lacks GM1: and GD1a in the pl asma and nuclear membranes. GM2 concentration was significantly higher in t he plasma membrane. Enzyme assay indicated deficiency of UDP-Gal:GM2 galact osyltransferase (GM1 synthase), which was confirmed by incorporation studie s with [H-3]sphingosine. These cells resembled wildtype NG108-15 in extendi ng dendritic processes in response to dendritogenic agents (retinoic acid, dibutyryl cAMP) but responded aberrantly to axonogenic stimuli (KCl, ionomy cin) by extending unstable neurites that showed the cytoskeletal staining c haracteristic of dendrites. Moreover, mutant cells treated with the Ca2+-el evating axonogenic agents underwent apoptosis over time, attributed to dysf unction of Ca2+ regulatory mechanisms normally mediated by GM1. Such agents caused dramatic and sustained elevation of intracellular Ca2+ in mutant ce lls, in contrast to modest and temporary elevation in wild-type cells. Exog enous GM1, inserted into the plasma membrane, had no discernable protective effect on NG-CR72 cells whereas LIGA-20, a membrane-permeant derivative of GM1 that entered both plasma and nuclear membranes, blocked apoptosis, per mitted extension of stable neurites, and attenuated the abnormal elevation of intracellular Ca2+.