NMDA-evoked excitotoxicity increases tissue transglutaminase in cerebellar granule cells.

In neuronal cells, excessive activation of glutamate receptors causes excitotoxic damage culminating in apoptotic and necrotic cell death. The molecular mechanism of excitotoxicity has been associated with excessive Ca(2+) influx and overload, triggering biochemical events that lead to cell death and tissue degeneration. Following mild insults via NMDA-receptor activation, central neurons undergo several biochemical modifications recognizable as early events in apoptotic machinery.Tissue transglutaminase, the most ubiquitous among cell transglutaminases, catalyzes the Ca(2+)-dependent protein cross-linking probably associated with morphological changes in several neurodegenerative disorders. The possible involvement of this enzyme in excitotoxicity-mediated events was investigated in primary cultures of cerebellar granule cells exposed for 30 min to NMDA (100 microM) in Locke's buffer. Under these conditions time-dependent increases in transglutaminase activity were observed. Tissue transglutaminase expression reached the highest levels within 3-4 h of NMDA exposure. Similarly, high levels of incorporation of fluorescent substrates were observed in living cells. Confocal laser microscopy analysis showed that fluorescein-labelled structures were distributed within the cytoplasm and close to the membranes of NMDA-exposed cells. These effects were dependent on the Ca(2+) influx triggered by the excitotoxic stimulus. Morphological changes in NMDA-treated cells gave evidence of significant cell damage which appeared within 5-6 h of NMDA exposure. These results suggest that increases in tissue transglutaminase may be associated to the effects of NMDA-induced excitotoxicity. Therefore, it is reasonable to hypothesize that if tissue transglutaminase levels and activity are up-regulated under such conditions, the protein cross-linking could be likely involved in excitotoxic response.