Minocycline has been shown to have remarkably neuroprotective qualities, but underlying mechanisms remain elusive. We reported here the robust neuroprotection by minocycline against glutamate-induced apoptosis through regulations of p38 and Akt pathways. Pre-treatment of cerebellar granule neurons (CGNs) with minocycline (10–100 lM) elicited a dose-dependent reduction of glutamate excitotoxicity and blocked glutamate-induced nuclear condensation and DNA fragmentations. Using patch-clamping and fluorescence Ca2+ imaging techniques, it was found that minocycline neither blocked NMDA receptors, nor reduced glutamatecaused rises in intracellular Ca2+. Instead, confirmed by immunoblots, minocycline in vivo and in vitro was shown to directly inhibit the activation of p38 caused by glutamate. A p38-specific inhibitor, SB203580, also attenuated glutamate excitotoxicity. Furthermore, the neuroprotective effects of minocycline were blocked by phosphatidylinositol 3-kinase (PI3-K) inhibitors LY294002 and wortmannin, while pharmacologic inhibition of glycogen synthase kinase 3b (GSK3b) attenuated glutamate-induced apoptosis. In addition, immunoblots revealed that minocycline reversed the suppression of phosphorylated Akt and GSK3b caused by glutamate, as were abolished by PI3-K inhibitors. These results demonstrate that minocycline prevents glutamate-induced apoptosis in CGNs by directly inhibiting p38 activity and maintaining the activation of PI3-K/Akt pathway, which offers a novel modality as to how the drug exerts protective effects.

Parkinson's disease is characterized by the pathologicalloss of dopaminergic neurons in the substantia nigra. The current therapy for Parkinson's disease is aimed to replace the lost transmitter. But the ultimate objective in the neurodegenerative therapy is the functional restoration and/or cessation of progression of neuronal loss. Given the critical role that the c-Jun N-terminal kinase (JNK) pathway plays in regulating the cellular processes that are involved in Parkinson's disease, the importance of JNK in this disease's pathogenesis is being increasingly recognized. Much evidence suggests that JNK plays an important role in mediating 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)/1-methyl-4-phenylpyridnium ion (MPP+)-induced neurotoxicity. Therefore, direct blockade of JNK may prevent or effectively slow the progression of Parkinson's disease. Studies including our own showed that the inhibition of JNK with SP-600125, a specific inhibitor of JNK, protects dopaminergic neurous both from MPP+-induced neuronal apoptosis in vitroand in MPTP Parkinson's disease model. These results support JNK inhibition as a potential strategy in treating Parkinson's disease.

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.

Myocyte enhancer factor 2 (MEF2) proteins are important regulators of gene expression during the development of skeletal, cardiac, and smooth muscle. MEF2 proteins are also present in brain and recently have been implicated in neuronal survival and differentiation. In this study we examined the cellular mechanisms regulating the activity of MEF2s during apoptosis of cultured cerebellar granule neurons, an established in vitro model for studying depolarization-dependent neuronal survival. All four MEF2 isoforms (A, B, C, and D) were detected by immunoblot analysis in cerebellar granule neurons. Endogenous MEF2A and MEF2D, but not MEF2B or MEF2C, were phosphorylated with the induction of apoptosis. The putative sites that were phosphorylated during apoptosis are functionally distinct from those previously reported to enhance MEF2 transcription. The increased phosphorylation of MEF2A and MEF2D was followed by decreased DNA binding, reduced transcriptional activity, and caspase-dependent cleavage to fragments containing N-terminal DNA binding domains and C-terminal transactivation domains. Expression of the highly homologous N terminus of MEF2A (1–131 amino acids) antagonized the transcriptional activity and prosurvival effects of a constitutively active mutant of MEF2D (MEF2D-VP16). We conclude that MEF2A and MEF2D are prosurvival factors with high transcriptional activity in postmitotic cerebellar granule neurons. When these neurons are induced to undergo apoptosis by lowering extracellular potassium, MEF2A and MEF2D are phosphorylated, followed by decreased DNA binding and cleavage by a caspase-sensitive pathway to N-terminal fragments lacking the transactivation domains. The degradation of MEF2D and MEF2A and the generation of MEF2 fragments that have the potential to act as dominant-inactive transcription factors lead to apoptotic cell death.

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.