Increasing evidence suggests that c-Jun N-terminal kinase (JNK) is an important kinase mediating neuronal apoptosis in Parkinson's disease (PD) model induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In order to study roles of JNK activity in neuronal apoptosis in this model, we blocked JNK activity in vivo using a specific inhibitor of JNK, SP600125. Our data showed that MPTP-induced phospho-c-Jun of substantial nigral neurons, caused apoptosis of dopaminergic neurons, and decreased the dopamine level in striatal area. We found that inhibiting JNK with SP600125 reduced the levels of c-Jun phosphorylation, protected dopaminergic neurons from apoptosis, and partly restored the level of dopamine in MPTP-induced PD in C57BL/6N mice. These results indicate that JNK pathway is the major mediator of the neurotoxic effects of MPTP in vivo and inhibiting JNK activity may represent a new and effective strategy to treat PD.

Bcl-2-interacting mediator of cell death (Bim), a proapoptotic BH3-only protein, plays a critical role in neuronal apoptosis. Cerebellar granule neurons (CGNs) depend on activity for their survival and undergo apoptosis when deprived of depolarizing concentration of KCl. While it has been proposed that the activation of c-Jun NH2-terminal protein kinase (JNK)/c-Jun pathway contributes to the upregulation of bim gene in neurons subjected to survival signaling withdrawal, here we show that neither inhibition of JNK activity nor expression of dominant-negative c-Jun suppresses the expression of bim gene induced by activity deprivation in CGNs. We conclude that induction of bim gene is independent of the activation of JNK/c-Jun signaling pathway by activity deprivation during apoptosis of CGNs.

Agents that elevate intracellular cyclic AMP (cAMP) levels promote neuronal survival in a manner independent of neurotrophic factors. Inhibitors of phosphatidylinositol 3 kinase and dominant-inactive mutants of the protein kinase Akt do not block the survival effects of cAMP, suggesting that another signaling pathway is involved. In this report, we demonstrate that elevation of intracellular cAMP levels in rat cerebellar granule neurons leads to phosphorylation and inhibition of glycogen synthase kinase 3b (GSK-3b). The increased phosphorylation of GSK-3b by protein kinase A (PKA) occurs at serine 9, the same site phosphorylated by Akt. Purified PKA is able to phosphorylate recombinant GSK-3b in vitro. Inhibitors of GSK-3 block apoptosis in these neurons, and transfection of neurons with a GSK-3b mutant that cannot be phosphorylated interferes with the prosurvival effects of cAMP. These data suggest that activated PKA directly phosphorylates GSK-3b and inhibits its apoptotic activity in neurons.

Cerebellar granule neuron (CGN) survival depends on activity of the myocyte enhancer factor-2 (MEF2) transcription factors. Neuronal MEF2 activity is regulated by depolarization via a mechanism that is presently unclear. Here, we show that depolarization-mediated MEF2 activity and CGN survival are compromised by overexpression of the MEF2 repressor histone deacetylase-5 (HDAC5). Furthermore, removal of depolarization induced rapid cytoplasm-to-nuclear translocation of endogenous HDAC5. This effect was mimicked by addition of the calcium/calmodulin-dependent kinase (CaMK) inhibitor KN93 to depolarizing medium. Removal of depolarization or KN93 addition resulted in dephosphorylation of HDAC5 and its co-precipitation with MEF2D. HDAC5 nuclear translocation triggered by KN93 induced a marked loss of MEF2 activity and subsequent apoptosis. To selectively decrease CaMKII, CGNs were incubated with an antisense oligonucleotide to CaMKIIa. This antisense decreased CaMKIIa expression and induced nuclear shuttling of HDAC5 in CGNs maintained in depolarizing medium. Selectivity of the CaMKIIa antisense was demonstrated by its lack of effect on CaMKIV-mediated CREB phosphorylation. Finally, antisense to CaMKIIa induced caspase-3 activation and apoptosis, whereas a missense control oligonucleotide had no effect on CGN survival. These results indicate that depolarization-mediated calcium influx acts through CaMKII to inhibit HDAC5, thereby sustaining high MEF2 activity in CGNs maintained under depolarizing conditions.

The transcription factor myocyte-enhancer factor 2 (MEF2) has been shown to be required for the survival of different types of neurons. However, the death- or survival-inducing second messenger pathways that regulate MEF2 activity remain to be fully elucidated. Membrane depolarization by KCl induces neuronal survival that is dependent upon MEF2-mediated gene transactivation. Here we report that membrane depolarizationinduced activation of MEF2 requires the cAMP-protein kinase A (PKA) pathway. Inhibition of the activity of cAMP-PKA pathway attenuates membrane depolarization-induced activation of MEF2 activity and neuronal survival, whereas enhancing the activity of this pathway prevents KCl withdrawal-induced inhibition of MEF2 and neuronal apoptosis. Moreover, PKA directly phosphorylates MEF2 at Thr-20 in vitro to increase MEF2 DNA binding activity. A mutation of Thr-20 to Ala renders MEF2 resistant to PKA phosphorylation in vitro and reduces its DNA binding activity. Transfection of this T20A mutant blocks survival and induces apoptosis in cultured cortical and cerebellar granule neurons. This study identifies the transcription factor MEF2 as a target of cAMP-PKA pathway and demonstrates that PKA phosphorylation of MEF2 is a key step in modulating its DNA binding activity and ability to promote neuronal survival.