Commitment of 1-methyl-4-phenylpyrinidinium ion-induced neuronal cell death by proteasome-mediated degradation of p35 cyclin-dependent kinase 5 activator.
ABSTRACT: The dysfunction of proteasomes and mitochondria has been implicated in the pathogenesis of Parkinson disease. However, the mechanism by which this dysfunction causes neuronal cell death is unknown. We studied the role of cyclin-dependent kinase 5 (Cdk5)-p35 in the neuronal cell death induced by 1-methyl-4-phenylpyrinidinium ion (MPP+), which has been used as an in vitro model of Parkinson disease. When cultured neurons were treated with 100 microM MPP+, p35 was degraded by proteasomes at 3 h, much earlier than the neurons underwent cell death at 12-24 h. The degradation of p35 was accompanied by the down-regulation of Cdk5 activity. We looked for the primary target of MPP+ that triggered the proteasome-mediated degradation of p35. MPP+ treatment for 3 h induced the fragmentation of the mitochondria, reduced complex I activity of the respiratory chain without affecting ATP levels, and impaired the mitochondrial import system. The dysfunction of the mitochondrial import system is suggested to up-regulate proteasome activity, leading to the ubiquitin-independent degradation of p35. The overexpression of p35 attenuated MPP+-induced neuronal cell death. In contrast, depletion of p35 with short hairpin RNA not only induced cell death but also sensitized to MPP+ treatment. These results indicate that a brief MPP+ treatment triggers the delayed neuronal cell death by the down-regulation of Cdk5 activity via mitochondrial dysfunction-induced up-regulation of proteasome activity. We propose a role for Cdk5-p35 as a survival factor in countering MPP+-induced neuronal cell death.
Project description:Cyclin-dependent kinase 5(CDK5) in complex with its activator, p35 (protein of 35 kDa), is essential for early neurodevelopment in mammals. However, endogenous cleavage of p35 to p25 is associated with neuron death and neurodegenerative disease. Here we show that a peptide (p10') encoding the N-terminal domain of p35 protects against CDK5/p25-induced toxicity in neurons. p10' also prevented the death of neurons treated with the neurotoxin, 1-methyl-4-phenylpyridinium (MPP(+)), which induces conversion of endogenous p35 to p25, and Parkinson disease (PD)-like symptoms in animals. MPP(+) induces CDK5/p25-dependent phosphorylation of peroxiredoxin 2 (Prx2), resulting in inhibition of its peroxireductase activity and accumulation of reactive oxygen species (ROS). We found that p10' expression inhibited both Prx2 phosphorylation and ROS accumulation in neurons. In addition, p10' inhibited the p25-induced appearance of antigen of the Ki67 antibody (Ki67) and phosphohistone H2AX (?H2AX), classic markers of cell cycle activity and DNA double-strand breakage, respectively, associated with neuron death. Our results suggest that p10 (protein of 10 kDa) is a unique prosurvival domain in p35, essential for normal CDK5/p35 function in neurons. Loss of the p10 domain results in CDK5/p25 toxicity and neurodegeneration in vivo.
Project description:When cell cycle re-activation occurs in post-mitotic neurons it places them at increased risk for death. The cell cycle/cell death association has been reported in many neurodegenerative diseases including Alzheimer disease (AD), yet the mechanisms by which a normal neuron suppresses the cycle remain largely unknown. Recently, our laboratory has shown that Cdk5 (cyclin-dependent kinase 5) is a key player in this protective function. When a neuron is under stress, Cdk5 is transported to the cytoplasm; this eliminates its cell cycle suppression activity and the neuron re-enters S-phase. In the current study we show that a similar principle applies during a normal cell cycle. When a neuronal cell enters S phase, Cdk5 is transported to the cytoplasm where it is ubiquitinated by the E3 ligase APC-Cdh1. Ubiquitinated Cdk5 is then rapidly degraded by the proteasome. The ubiquitination site of Cdk5 appears to be in the p35 binding area; in the presence of high levels of p35, the ubiquitination of Cdk5 was blocked, and the degradation in S phase was attenuated. The data suggest an unsuspected role for Cdk5 during the progression of a normal cell cycle and offer new pharmaceutical targets for regulating neuronal cell cycling and cell death.
Project description:CDK5 activation promotes ischemic neuronal death in stroke, with the recognized activation mechanism being calpain-dependent p35 cleavage to p25. Here we reported that CDK5-Tyr15 phosphorylation by zinc induced CDK5 activation in brain ischemic injury. CDK5 activation and CDK5-Tyr15 phosphorylation were observed in the hippocampus of the rats that had been subjected to middle cerebral artery occlusion, both of which were reversed by pretreatment with zinc chelator; while p35 cleavage and calpain activation in ischemia were not reversed. Zinc incubation resulted in CDK5-Tyr15 phosphorylation and CDK5 activation, without increasing p35 cleavage in cultured cells. Site mutation experiment confirmed that zinc-induced CDK5 activation was dependent on Tyr15 phosphorylation. Further exploration showed that Src kinase contributed to zinc-induced Tyr15 phosphorylation and CDK5 activation. Src kinase inhibition or expression of an unphosphorylable mutant Y15F-CDK5 abolished Tyr15 phosphorylation, prevented CDK5 activation and protected hippocampal neurons from ischemic insult in rats. We conclude that zinc-induced CDK5-Tyr15 phosphorylation underlies CDK5 activation and promotes ischemic neuronal death in stroke.
Project description:Recent studies have shown the involvement of cyclin-dependent kinase 5 (Cdk5) in cell cycle regulation in postmitotic neurons. In this study, we demonstrate that Cdk5 and its co-activator p35 were detected in the nuclear fraction in neurons and Cdk5/p35 phosphorylated retinoblastoma (Rb) protein, a key protein controlling cell cycle re-entry. Cdk5/p35 phosphorylates Rb at the sites similar to those phosphorylated by Cdk4 and Cdk2. Furthermore, increased Cdk5 activity elevates activity of E2F transcription factor, which can trigger cell cycle re-entry, leading to neuronal cell death. A normal Cdk5 activity in neurons did not induce E2F activation, suggesting that Cdk5 does not induce cell cycle re-entry under normal conditions. Taken together, these results indicate that Cdk5 can regulate cell cycle by its ability to phosphorylate Rb. Most importantly, increased Cdk5 activity induces cell cycle re-entry, which is especially detrimental for survival of postmitotic neurons.
Project description:Huntington's disease (HD) is a progressive neurodegenerative disorder caused by an expanded CAG trinucleotide repeat sequence in the huntingtin gene. The resulting poly-glutamine expansion in the huntingtin protein imparts a novel toxic gain of function causing selective loss of medium spiny neurons (MSNs) in the striatum. Although the exact mechanism of cell death is unclear, recent evidence suggests involvement of NMDA-receptor mediated excitotoxicity and aberrant cyclin dependent kinase 5 (cdk5) activity in striatal cells undergoing neurodegeneration. In this study we directly tested the effect of reduced levels of p25 and p35, two proteins required for cdk5 activation, on striatal neurodegeneration using mice with targeted deletion of p35. Quinolinic acid (QA) injected into the striatum of mice causes NMDA-receptor mediated cell death, and these QA-induced striatal lesions were examined in p35 hemizygous null (p35+/-) and wildtype (WT) mice. Striatal QA lesion volumes were 30% smaller in p35+/- mice than in WT mice. Furthermore, primary neuronal cultures of MSNs from P0 p35+/- pups displayed 33% less apoptotic neurons following NMDA treatment than those from WT pups. Examination of YAC128 mouse model of HD showed elevated p25 levels in striatum following intrastriatal QA injection. Our findings provide direct evidence for p25 and p35 involvement in excitotoxic neurodegeneration of MSNs and suggest a role for the cdk5 pathway in HD striatal neurodegeneration.
Project description:Our previous data suggested that the human basic helix-loop-helix transcription factor achaete-scute homologue-1 (hASH1) may stimulate both proliferation and migration in the lung. In the CNS, cyclin-dependent kinase 5 (Cdk5) and its activator p35 are important for neuronal migration that is regulated by basic helix-loop-helix transcription factors. Cdk5/p35 may also play a role in carcinogenesis. In this study, we found that the neuronal activator p35 was commonly expressed in primary human lung cancers. Cdk5 and p35 were also expressed by several human lung cancer cell lines and coupled with migration and invasion. When the kinase activity was inhibited by the Cdk5 inhibitor roscovitine or dominant-negative (dn) Cdk5, the migration of lung cancer cells was reduced. In neuroendocrine cells expressing hASH1, such as a pulmonary carcinoid cell line, knocking down the gene expression by short hairpin RNA reduced the levels of Cdk5/p35, nuclear p35 protein, and migration. Furthermore, expression of hASH1 in lung adenocarcinoma cells normally lacking hASH1 increased p35/Cdk5 activity and enhanced cellular migration. We were also able to show that p35 was a direct target for hASH1. In conclusion, induction of Cdk5 activity is a novel mechanism through which hASH1 may regulate migration in lung carcinogenesis.
Project description:In response to neurotoxic signals, postmitotic neurons make attempts to reenter the cell cycle, which results in their death. Although several cell cycle proteins have been implicated in cell cycle-related neuronal apoptosis (CRNA), the molecular mechanisms that underlie this important event are poorly understood. Here, we demonstrate that neurotoxic agents such as ?-amyloid peptide cause aberrant activation of mitogen-activated kinase kinase (MEK)-extracellular signal-regulated kinase (ERK) signaling, which promotes the entry of neurons into the cell cycle, resulting in their apoptosis. The MEK-ERK pathway regulates CRNA by elevating the levels of cyclin D1. The increase in cyclin D1 attenuates the activation of cyclin-dependent kinase 5 (cdk5) by its neuronal activator p35. The inhibition of p35-cdk5 activity results in enhanced MEK-ERK signaling, leading to CRNA. These studies highlight how neurotoxic signals reprogram and alter the neuronal signaling machinery to promote their entry into the cell cycle, which eventually leads to neuronal cell death.
Project description:Cyclin-dependent kinase 5 (Cdk5) plays a critical role during neurodevelopment, synaptic plasticity, and neurodegeneration. Cdk5 activity depends on association with neuronal proteins p35 and p25, a proteolytic product of p35. Cdk5 regulates the actin cytoskeletal dynamics that are essential for neuronal migration, neuritic growth, and synaptogenesis. However, little is known about the interaction of actin and Cdk5 and its effect on neuronal Cdk5 activity. In a previous study, we observed that Cdk5/p35 activity is negatively correlated with co-immunoprecipitated F-actin (filamentous actin) amounts in the mouse brain, and suggested that F-actin inhibits the formation of the Cdk5/p35 complex [Journal of Neuroscience (2008) vol. 28, p. 14511]. The experiments reported here were undertaken to elucidate the relationship between actin and the formation of the Cdk5/p35 complex and its activity. Instead of an F-actin-mediated inhibition, we propose that G-actin (globular actin) in the F-actin preparations is responsible for inhibiting Cdk5/p35 and Cdk5/p25 kinase activity. We found that F-actin binds to p35 but not p25 or Cdk5. We have shown that G-actin binds directly to Cdk5 without disrupting the formation of the Cdk5/p35 or Cdk5/p25 complexes. G-actin potently suppressed Cdk5/p35 and Cdk5/p25 activity when either histone H1 or purified human tau protein were used as substrates, indicating a substrate-independent inhibitory effect of G-actin on Cdk5 activity. Finally, G-actin suppressed the activity of Cdk5 immunoprecipitated from wild type and p35-deficient mouse brain, suggesting that G-actin suppresses endogenous Cdk5 activity in a p35-independent manner. Together, these results suggest a novel mechanism of actin cytoskeletal regulation of Cdk5/p35 activity.
Project description:The mechanisms underlying persistent fibroblast activation and myofibroblast phenoconversion in underlying multi-organ fibrosis in systemic sclerosis (SSc) remain incompletely understood, hindering effective therapies to slow or reverse the process. Cyclin-dependent kinase 5 (CDK5) is a pleiotropic member of the CDK family originally identified in neuronal cells. In contrast to other CDKs, CDK5 activity depends on its CDK5R1 subunit p35. Here we demonstrate that expression of p35 and CDK5 activity are induced by TGF-ß in fibroblasts and adipocytic cell types. Levels of p35 are markedly elevated in both SSc skin biopsies and explanted SSc fibroblasts, as well as in fibrotic skin in mice. Ectopic p35 and CDK5 suppressed adipogenic markers while stimulating collagen production and myofibroblast markers, whereas RNAi-mediated CDK5 knockdown abrogated TGF-? fibrotic responses in a Smad-independent manner. Pharmacological inhibitors of CDK5 likewise prevented and reversed TGF-? responses in fibroblast monolayers and in ex vivo human skin organ cultures, ameliorated collagen overproduction in SSc fibroblasts, and prevented and reversed skin fibrosis in two distinct mouse models of SSc. Together, these results reveal a previously unrecognized key function for p35/CDK5 as a mediator of mesenchymal cell fibrotic responses. The results suggest a potential pathogenic role for elevated p35 expression and CDK5 activity in SSc, and raise the possibility that their selective pharmacological targeting might represent a novel treatment approach in fibrosis.
Project description:Cyclin-dependent kinase 5 (Cdk5), a cdc2-related kinase expressed in postmitotic neurons, is activated by association with a brain-specific activator, p35. It has been suggested that the conversion of p35 to p25 by the protease calpain is involved in neuronal cell death. However, p35 protein is turned over rapidly via proteasomal degradation in living neurons. In this study we show that the phosphorylation of p35 by Cdk5 suppresses the cleavage to p25 by calpain, whereas phosphorylation facilitates the proteasomal degradation of p35. The phosphorylation site in p35 that might be involved in preventing calpain cleavage was distinct from the phosphorylation site involved in facilitating proteasomal degradation. A phosphorylated form of p35 that was resistant to cleavage by calpain was more prevalent in the fetal brain, whereas the unphosphorylated form of p35 occurred in the adult brain. These results suggest that the phosphorylation of p35 serves as a protective mechanism that suppresses the generation of p25 in developing brains.