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.

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.

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.

The neuroprotective properties of bis (7)-tacrine, a novel dimeric acetylcholinesterase (AChE) inhibitor, on glutamate-induced excitotoxicity were investigated in primary cultured cerebellar granule neurons (CGNs). Exposure of CGNs to 75

We explored the feasibility of studying nuclear matrix protein (NMP) expressions of the hepatocytes in normal and cirrhotic rat livers with liver regeneration after partial hepatectomy. Sixteen Wistar healthy rats were studied with experimental liver regeneration and/or liver cirrhosis. Two-dimensional (2-D) gel electrophoresis was used to generate these NMP compositions from these rat liver samples. Several antibodies against cytokeratin, vimentin, actin, B23, HNF4alpha, and heat shock protein 70 were used for identification by Western blot. Totally, 41 strongly stained protein spots were characterized on the 2-D gels. Thirty-four protein spots were detected in all of these rat livers, of which, cytokeratin, vimentin, actin, HNF4alpha, and heat shock protein 70 were identified. B23 was detected in the regenerated livers. Three protein spots (s33, s34, and s35) were detectable only inNMPpreparation extracted from the regenerating rat livers after hepatectomy. Another three protein spots (s36, s37, and s38) were detectable only in NMP preparation extracted from thioacetamide-induced cirrhotic rat livers. Under these conditions including experimental liver regeneration and/or liver cirrhosis, Over thirty higher abundance NMPs of hepatocytes were consistently expressed and considered as common and basic NMPs. Some of the NMPs are specific for liver regeneration and may play a critical role in cell proliferation and cell cycle, and some are specific for liver cirrhosis.

AIM: A broad-range proteomic approach was applied to investigate the complexity of the mechanisms involved in pancreatic regeneration for identification of new treatment targets and potential markers of pancreatic stem cells. METHODS: A regeneration pancreatic model was induced by partial (90%) pancreatectomy in rats. Changes in protein expression in rat regeneration pancreas at 3 d after partial pancreatectomy, as compared to sham surgery, were analyzed using two-dimensional gel electrophoresis (2-DE), mass spectrometry (MS), and mass fingerprinting. RESULT: Two-DE displayed 91 spots with at least 1.5-fold of differentially expression at the time point of 3 days after pancreatectomy and 53 differentially expressed proteins were identified by peptide mass fingerprinting(PMF). These included embryogenic and cell proliferation-related, lipid and energy metabolism-related, protein and amino acid metabolism-related proteins, signal transduction proteins. CONCLUSION: The proteome profiling technique provided a broad-based and effective approach for the rapid assimilation and identification of adaptive protein changes while pancreas regeneration induced by pancreatectomy. Our data highlight the globe proteome during the pancreatic proliferation and differentiation processes, which is very important and will lead to better understand regulation mechanism of the pancreatic regeneration, and to ultimately reach the target of discovering protein biomarkers of pancreatic stem cells.

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.

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.