Susceptibility of cerebellar granule neurons from GM2/GD2 synthase-null mice to apoptosis induced by glutamate excitotoxicity and elevated KCl: Rescue by GM1 and LIGA20

Our previous study showed an impaired regulation of Ca ²⁺ homeostasis in cultured cerebellar granule neurons (CGN) from neonatal mice lacking GM2, GD2 and all gangliotetraose gangliosides, due to disruption of the GM2/GD2 synthase (GalNAc-T) gene. In the presence of depolarizing concentration (55 mM) K ⁺, these cells showed persistent elevation of intracellular Ca ²⁺ ([Ca ²⁺] _i) leading to apoptosis and cell destruction. This was in contrast to CGN from normal littermates whose survival was enhanced by high K ⁺. In this study we demonstrate that glutamate has the same effect as K ⁺ on CGN from these ganglioside-deficient knockout (KO) mice and that apoptosis in both cases is averted by exogenous GM1. Even more effective rescue was obtained with LIGA20, a semi-synthetic derivative of GM1. LC ₅₀ of glutamate in the KO cells was 3.1 μM, compared to 46 μM in normal CGN. [Ca ²⁺] _i measurement with fura-2 revealed no difference in glutamate-stimulated Ca ²⁺ influx between the 2 cell types. However, reduction of [Ca ²⁺] _i following application of Mg ²⁺ was significantly impaired in the mutant CGN. The rescuing effects of exogenous GM1 and LIGA20 corresponded to their ability to restore Ca ²⁺ homeostasis. The greater potency of LIGA20 is attributed to its greater membrane permeability with resultant ability to insert into both plasma and nuclear membranes at low concentration (≤1μM); GM1 at the same concentration was incorporated only into the plasma membrane and required much higher concentration to influence Ca ²⁺ homeostasis and CGN viability.