Spermine-induced toxicity in cerebellar granule neurons is independent of its actions at NMDA receptors.
The neurotoxic actions of polyamines such as spermine have been linked to their modulation of NMDA receptors, resulting in an excitotoxic cell death. Here, we demonstrate that chronic exposure to the polyamine spermine and acute exposure to the combination of spermine and glutamate result in significant toxicity to primary cultures of cerebellar granule neurons (CGNs). However, in both cases this cell death (a) lacks the characteristic cell swelling associated with the necrotic cell death induced by glutamate and (b) is characterized by the widespread formation of apoptotic nuclei. Whereas dizocilpine (MK-801) blocks the synergistic cell death resulting from acute exposure to spermine plus glutamate, neither MK-801 nor the calcium chelator EGTA appreciably attenuates CGN death resulting from chronic exposure to spermine. Consistent with previous reports, glutamate, both acute and chronic, causes CGN death that is characterized by cell swelling, sensitivity to MK-801 and EGTA, and only small numbers of apoptotic nuclei. Spermine-induced toxicity is not blocked by either the protein synthesis inhibitor cycloheximide or the pancaspase inhibitor tert-butoxycarbonyl-Asp-(O-methyl) fluoromethyl ketone. However, the antioxidant butylated hydroxyanisole is an effective blocker of spermine-induced CGN death, suggesting a free-radical component to this cell death. The intact spermine molecule, rather than a catabolic by-product, is required for cell death because the amine oxidase inhibitors N1,N2-bis(2,3-butadienyl)-1,4-butanediamine and aminoguanidine fail to block this toxicity. Thus, in CGNs, spermine-induced toxicity does not occur by its modulation of NMDA receptors, although, under some circumstances, NMDA receptor activation can modulate spermine-induced toxicity.