Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary neurons: Role of P2 and NGFRp75 receptors

In this study, we monitored the direct expression of P2 receptors for extracellular ATP in cerebellar granule neurons undergoing metabolism impairment. Glucose deprivation for 30-60 min inhibited P2Y₁ receptor protein, only weakly modulated P2X₁, P2X₂ and P2X₃, and up-regulated by about two-fold P2X₄, P2X₇ and P2Y₄. The P2X/Y antagonist basilen blue, protecting cerebellar neurons from hypoglycemic cell death, maintained within basal levels only the expression of P2X₇ and P2Y₄ proteins, but not P2X₄ or P2Y₁. Glucose starvation transiently increased (up to three-fold) the expression of NGFRp75 receptor protein and strongly stimulated the extracellular release of nerve growth factor (NGF; about 10-fold). Exogenously added NGF then augmented hypoglycemic neuronal death by about 60%, increasing the percentage of Höechst-positive nuclei (from approximately 62 to 95%), reducing lactate dehydrogenase (LDH) release (from about 50 to 14%) and significantly overstimulating the hypoglycemia-induced expression of P2X₇ and P2Y₄. Conversely, extracellular ATP augmented hypoglycemic neuronal death by about 80%, reducing the number of Höechst-positive nuclei (from approximately 62% to 14%), augmenting LDH outflow (by about 30%) and further increasing the hypoglycemia-induced expression of NGFRp75. Our results indicate that P2 and NGFRp75 receptors are modulated during glucose starvation and that extracellular ATP and NGF drive features of, respectively, necrotic and apoptotic hypoglycemic cell death, aggravating the consequences of metabolism impairment in cerebellar primary neurons.