Elevation of intracellular glucosylceramide levels results in an increase in endoplasmic reticulum density and in functional calcium stores in cultured neurons

Gaucher disease is a glycosphingolipid storage disease caused by defects in the activity of the lysosomal hydrolase, glucocerebrosidase (GlcCerase), r esulting in accumulation of glucocerebroside (glucosylceramide, GlcCer) in lysosomes, The acute neuronopathic type of the disease is characterized by severe loss of neurons in the central nervous system, suggesting that a neu rotoxic agent might be responsible for cellular disruption and neuronal dea th. We now demonstrate that upon incubation with a chemical inhibitor of Gl cCerase, conduritol-B-epoxide (CBE), cultured hippocampal neurons accumulat e GlcCer, Surprisingly, increased levels of tubular endoplasmic reticulum e lements, an increase in [Ca2+](i) response to glutamate, and a large increa se in [Ca2+](i) release from the endoplasmic reticulum in response to caffe ine were detected in these cells. There was a direct relationship between t hese effects and GlcCer accumulation since co-incubation with CBE and an in hibitor of glycosphingolipid synthesis, fumonisin B-1, completely antagoniz ed the effects of CBE, Similar effects on endoplasmic reticulum morphology and [Ca2+](i) stores were observed upon incubation with a short-acyl chain, nonhydrolyzable analogue of GlcCer, C-8-glucosylthioceramide. Finally, neu rons with elevated GlcCer levels were much more sensitive to the neurotoxic effects of high concentrations of glutamate than control cells; moreover, this enhanced toxicity was blocked by pre-incubation with ryanodine, sugges ting that [Ca2+](i) release from ryanodine-sensitive intracellular stores c an induce neuronal cell death, at least in neurons with elevated GlcCer lev els. These results may provide a molecular mechanism to explain neuronal dy sfunction and cell death in neuronopathic forms of Gaucher disease.