Functional Role of RNA Polymerase II and P70 S6 Kinase in KCl Withdrawal-induced Cerebellar Granule Neuron Apoptosis

KCl withdrawal-induced apoptosis in cerebellar granule neurons is associated with aberrant cell cycle activation, and treatment with cyclin-dependent kinase (Cdk) inhibitors protects cells from undergoing apoptosis. Because the Cdk inhibitor flavopiridol is known to inhibit RNA polymerase II (Pol II)-dependent transcription elongation by inhibiting the positive transcription elongation factor b (P-TEFb, a complex of CDK9 and cyclin T), we examined whether inhibition of RNA Pol II protects neurons from apoptosis. Treatment of neurons with 5, 6-dichloro-1-β-d-ribobenzimidazole (DRB), an RNA Pol II-dependent transcription elongation inhibitor, and flavopiridol inhibited phosphorylation and activation of Pol II and protected neurons from undergoing apoptosis. In addition to Pol II, neurons subjected to KCl withdrawal showed increased phosphorylation and activation of p70 S6 kinase, which was inhibited by both DRB and flavopiridol. Immunostaining analysis of the neurons deprived of KCl showed increased nuclear levels of phospho-p70 S6 kinase, and neurons protected with DRB and flavopiridol showed accumulation of the kinase into large spliceosome assembly factor-positive speckle domains within the nuclei. The formation of these foci corresponded with cell survival, and removal of the inhibitors resulted in dispersal of the speckles into smaller foci with subsequent apoptosis induction. Because p70 S6 kinase is known to induce translation of mRNAs containing a 5′-terminal oligopyrimidine tract, our data suggest that transcription and translation of this subset of mRNAs may contribute to KCl withdrawal-induced apoptosis in neurons. Neurons undergoing apoptosis show increased expression of cell cycle regulatory proteins. The expression of CDK7 and CDK9 and phosphorylation and activation of RNA polymerase II and P70S6K are increased in apoptotic neurons, and their inhibition prevents apoptosis. The P70S6K-dependent translation of specific mRNAs may contribute to neuronal vulnerability. Inhibitors of P70S6K could be beneficial in preventing neurodegeneration and neuronal apoptosis.