Cdk5, a cyclin-dependent kinase, is critical for neuronal development, neuronal migration, cortical lamination, and survival. Its survival role is based, in part, on "cross-talk" interactions with apoptotic and survival signaling pathways. Previously, we showed that Cdk5 phosphorylation of mitogen-activated protein kinase kinase (MEK)1 inhibits transient activation induced by nerve growth factor (NGF) in PC12 cells. To further explore the nature of this inhibition, we studied the kinetics of NGF activation of extracellular signal-regulated kinase (Erk)1/2 in cortical neurons with or without roscovitine, an inhibitor of Cdk5. NGF alone induced an Erk1/2-transient activation that peaked in 15 min and declined rapidly to baseline. Roscovitine, alone or with NGF, reached peak Erk1/2 activation in 30 min that was sustained for 48 h. Moreover, the sustained Erk1/2 activation induced apoptosis in cortical neurons. Significantly, pharmacological application of the MEK1 inhibitor PD98095 to roscovitine-treated cortical neurons prevented apoptosis. These results were also confirmed by knocking down Cdk5 activity in cortical neurons with Cdk5 small interference RNA. Apoptosis was correlated with a significant shift of phosphorylated tau and neurofilaments from axons to neuronal cell bodies. These results suggest that survival of cortical neurons is also dependent on tight Cdk5 modulation of the mitogen-activated protein kinase signaling pathway.

Cdk5 Modulation of Mitogen-activated Protein Kinase Signaling Regulates Neuronal Survival

Apoptosis mediated by p53 in rat neural AF5 cells following treatment with hydrogen peroxide and staurosporine.

L-3, 4-Dihydroxyphenylalanine (L-DOPA)-induced dyskinesia (LID) is a major clinical complication in the treatment of Parkinson’s disease (PD). This debilitating side effect likely reflects aberrant compensatory responses for a combination of dopaminergic neuron denervation and repeated L-DOPA administration. Abnormal endogenous opioid signal transduction pathways in basal ganglia have been well documented in LID. Opioid receptors have been targeted to alleviate the dyskinesia. However, the exact role of this altered opioid activity is remains under active investigation. In the present review, we discuss the current understanding of opioid signal transduction in the basal ganglia and how the malfunction of opioid signaling contributes to the pathophysiology of LID. Further study of the opioid system in LID may lead to new therapeutic targets and improved treatment of PD patients.

/pdf/opioid-system-in-l-dopa-induced-dyskinesia-3tsygrzok7.pdf

Opioid system in L-DOPA-induced dyskinesia.

Most small molecule drugs act on living systems by physically interacting with specific proteins and modulating target function. Identification of drug binding targets, within the complex milieu of the human proteome, remains a challenging task of paramount importance in drug discovery. Existing approaches for target identification employ complex workflows with limited throughput. Here, we present the isothermal shift assay (iTSA), a mass spectrometry method for proteome-wide identification of drug targets within lysates or living cells. Compared with prevailing methods, iTSA uses a simplified experimental design with increased statistical power to detect thermal stability shifts that are induced by small molecule binding. Using a pan-kinase inhibitor, staurosporine, we demonstrate improved performance over commonly used thermal proteome profiling methods, identifying known targets in cell lysates and living cells. We also demonstrate the identification of both known targets and additional candidate targets for the kinase inhibitor harmine in cell and tissue lysates.

/pdf/an-isothermal-shift-assay-for-proteome-scale-drug-target-1ttyaxrlah.pdf

An isothermal shift assay for proteome scale drug-target identification.

Hippocampal sclerosis (HS) is the most common cause of chronic medically refractory epilepsy in adults Histologically, HS is characterized by segmental neuronal loss and gliosis Although neuronal loss is important to the pathophysiology of HS, the molecular mechanisms underlying the neuronal loss remain uncertain Recently, it has been appreciated that proteins important in neurodevelopment may also have a role in neurodegeneration Cyclin-dependent kinase 5 (cdk5), known to be crucial in development of the normal cerebral cortex, has now been shown as pivotal in several cell death paradigms, including apoptosis and necrosis Deregulation of cdk5 by p25 causes hyperphosphorylation of tau and may contribute to pathology in several neurodegenerative conditions Furthermore, it has been shown that after transient forebrain ischemia, cdk5 causes specific death of CA1 neurons in the rat hippocampus by direct phosphorylation of the NR2A subunit of the NMDA receptor and subsequent excitotoxicity Because apoptosis, necrosis, and excitotoxicity are all thought to contribute to neuronal loss in HS, we hypothesized that abnormalities of the cdk5 pathway would accompany this disorder Surgically resected cases of HS with adjacent histologically normal lateral temporal cortex were examined for cdk5 and its activator p35/p25 We consistently found increased immunoreactivity for p35/p25 in surviving neurons within areas of neuronal loss compared with areas where neurons were preserved Western blots showed the ratio of p25 to p35 to be greater in diseased hippocampi than in the adjacent histologically normal temporal lobe Histone-based kinase assays demonstrated increased activity of the p25-cdk5 complex in HS compared with the temporal lobe despite neuronal loss in the hippocampal samples Our results suggest that p25 is pathologically increased in HS and that deregulation of cdk5 by p25 might contribute to neuronal death in this condition

Deregulation of cdk5 in Hippocampal Sclerosis

AIM:
To investigate whether cyclin-dependent kinase 5 and its regulatory protein p35 was involved in staurosporine-induced apoptosis of cortical neuronal cultures.
METHODS:
Primary cerebral cortical neurons were exposed to 300 nmol/L staurosporine. After incubation for different time, morphological alterations were observed with phase-contrast microscopy, fluorescence microscopy, and transmission electron microscopy. DNA fragmentation was detected by agarose gel electrophoresis. The protein levels of Cdk4, p53, Cdk5, and its regulatory protein p35 following staurosporine treatment were measured by Western blotting. The Cdk5 activity was assayed for histone H1 kinase activity by autoradiography.
RESULTS:
The typical morphological changes of apoptosis were observed and the nuclear DNA fragmentation showed the characteristic "ladder" pattern after the cells were treated by staurosporine. The Cdk5 protein level increased markedly at 3 h and continued to 24 h. The p35 level increased at 3 h after being exposed to staurosporine, and decreased at 12 h. The cleavage of p35 to p25 was also detected at 12 h and increased at 24 h. There was no increase in Cdk5 kinase activity despite the increased cleavage of p35. The protein level of Cdk4 protein increased at 3 h and then decreased gradually from 6 h, but it was still higher than that in the vehicle cultures at 12 h. The p53 level decreased obviously at 3 h after staurosporine treatment and then seemed to increase at 12 h, but remained lower than that of vehicle cultures.
CONCLUSION:
Staurosporine-induced increase in Cdk5 protein levels and the cleavage of p35 to p25 may contribute to neuronal apoptosis.

Wen Zhang

Papers

Involvement of cyclin dependent kinase 5 and its activator p35 in staurosporine-induced apoptosis of cortical neurons.

Parathyroid hormone-related peptide (1-34) reduces alveolar bone loss in type 1 diabetic rats.