Cdk5 Overactivation Research Articles

Cdk5 Promotes Mitochondrial Fission via Drp1 Phosphorylation at S616 in Chronic Ethanol Exposure-Induced Cognitive Impairment.

Excessive alcohol consumption can lead to alterations in brain structure and function, even causing irreversible learning and memory disorders. The hippocampus is one of the most sensitive areas to alcohol neurotoxicity in the brain. Accumulating evidence indicates that mitochondrial dysfunction contributes to alcohol neurotoxicity. However, little is known about the underlying molecular mechanisms. In this study, we found that chronic exposure to ethanol caused abnormal mitochondrial fission/fusion and morphology by activating the mitochondrial fission protein dynamin-related protein 1 (Drp1) and upregulating Drp1 receptors, such as fission protein 1 (Fis1), mitochondrial dynamics protein of 49kDa (Mid49), and mitochondrial fission factor (Mff), combined with decreasing optic atrophy 1 (Opa1) and mitochondrial fusion protein mitofusin 1 (Mfn1) levels. In addition, mitochondrial division inhibitor 1 (mdivi-1) abrogated ethanol-induced mitochondrial dysfunction and improved hippocampal synapses and cognitive function in ethanol-exposed mice. Chronic ethanol exposure also resulted in cyclin-dependent kinase 5 (Cdk5) overactivation, as shown by the increase in the levels of Cdk5 and its activator P25 in the hippocampus. Furthermore, a Cdk5/P25 inhibitor (roscovitine) or Cdk5 knockdown using small interfering RNA (LVi-Cdk5) exerted neuroprotection by inhibiting abnormal mitochondrial fission through Drp1 phosphorylation at Ser616 and mitochondrial translocation after chronic ethanol exposure. Taken together, the present study demonstrated that inhibition of aberrant Cdk5 activation attenuates hippocampal neuron injury and cognitive deficits induced by chronic exposure to ethanol through Drp1-mediated mitochondrial fission and mitochondrial dysfunction. Interfering with this pathway might serve as a potential therapeutic approach to prevent ethanol-induced neurotoxicity in the brain.

Targeting CDK5 in Astrocytes Promotes Calcium Homeostasis Under Excitotoxic Conditions.

Glutamate excitotoxicity triggers overactivation of CDK5 and increases calcium influx in neural cells, which promotes dendritic retraction, spine loss, increased mitochondrial calcium from the endoplasmic reticulum, and neuronal death. Our previous studies showed that CDK5 knockdown (KD) in astrocytes improves neurovascular integrity and cognitive functions and exerts neuroprotective effects. However, how CDK5-targeted astrocytes affect calcium regulation and whether this phenomenon is associated with changes in neuronal plasticity have not yet been analyzed. In this study, CDK5 KD astrocytes transplanted in CA3 remained at the injection site without proliferation, regulated calcium in the CA1 hippocampal region after excitotoxicity by glutamate in ex vivo hippocampal slices, improving synapsin and PSD95 clustering. These CDK5 KD astrocytes induced astrocyte stellation and neuroprotection after excitotoxicity induced by glutamate in vitro. Also, these effects were supported by CDK5 inhibition (CDK5i) in vitro through intracellular stabilization of calcium levels in astrocytes. Additionally, these cells in cocultures restored calcium homeostasis in neurons, redistributing calcium from somas to dendrites, accompanied by dendrite branching, higher dendritic spines and synapsin-PSD95 clustering. In summary, induction of calcium homeostasis at the CA1 hippocampal area by CDK5 KD astrocytes transplanted in the CA3 area highlights the role of astrocytes as a cell therapy target due to CDK5-KD astrocyte-mediated synaptic clustering, calcium spreading regulation between both areas, and recovery of the intracellular astrocyte-neuron calcium imbalance and plasticity impairment generated by glutamate excitotoxicity.

Inhibition of the CDK5/caspase-3 Pathway by p5-TAT Protects Hippocampal Neurogenesis and Alleviates Radiation-induced Cognitive Dysfunction

Radiation-induced cognitive dysfunction is a common complication associated with cranial radiation therapy. Inhibition of hippocampal neurogenesis and proliferation plays a critical role in this complication. Relieving hippocampal apoptosis may significantly protect hippocampal neurogenesis and proliferation. Previous studies have demonstrated that hyperactivity of cyclin-dependent kinase 5 (Cdk5) is closely related to apoptosis. The exact molecular changes and function of Cdk5 in radiation-induced cognitive dysfunction are still not clear. Whether inhibition of Cdk5 and the relevant caspase-3 could improve hippocampal neurogenesis and ameliorate radiation-induced cognitive dysfunction needs further exploration. We hypothesized that inhibition of the Cdk5/caspase-3 pathway by p5-TAT could protect hippocampal neurogenesis and alleviate radiation-induced cognitive dysfunction. In our study, we reported that radiation induced hyperactivity of Cdk5 accompanied by elevation of the levels of cleaved caspase-3, a marker of neuronal apoptosis. Inhibition of hippocampal neurogenesis and proliferation as well as cognitive dysfunction was also observed. p5-TAT, a specific inhibitor of Cdk5, decreased the overactivation of Cdk5 without affecting the levels of Cdk5 activators. Additionally, this treatment suppressed the expression of cleaved caspase-3. We further demonstrated that p5-TAT treatment reduced hippocampal dysfunction and improved behavioral performance. Therefore, Cdk5 inhibition by the small peptide p5-TAT is a promising therapeutic strategy for radiation-induced cognitive dysfunction.

Suppressing Cdk5 Activity by Luteolin Inhibits MPP+-Induced Apoptotic of Neuroblastoma through Erk/Drp1 and Fak/Akt/GSK3β Pathways.

Parkinson’s disease (PD) is characterized by the progressive degeneration of dopaminergic neurons. The cause of PD is still unclear. Oxidative stress and mitochondrial dysfunction have been linked to the development of PD. Luteolin, a non-toxic flavonoid, has become interested in an alternative medicine, according to its effects on anti-oxidative stress and anti-apoptosis, although the underlying mechanism of luteolin on PD has not been fully elucidated. This study aims to investigate whether luteolin prevents neurotoxicity induction by 1-methyl-4-phenylpyridinium iodide (MPP+), a neurotoxin in neuroblastoma SH-SY5Y cells. The results reveal that luteolin significantly improved cell viability and reduced apoptosis in MPP+-treated cells. Increasing lipid peroxidation and superoxide anion (O2−), including mitochondrial membrane potential (Δψm) disruption, is ameliorated by luteolin treatment. In addition, luteolin attenuated MPP+-induced neurite damage via GAP43 and synapsin-1. Furthermore, Cdk5 is found to be overactivated and correlated with elevation of cleaved caspase-3 activity in MPP+-exposed cells, while phosphorylation of Erk1/2, Drp1, Fak, Akt and GSK3β are inhibited. In contrast, luteolin attenuated Cdk5 overactivation and supported phosphorylated level of Erk1/2, Drp1, Fak, Akt and GSK3β with reducing in cleaved caspase-3 activity. Results indicate that luteolin exerts neuroprotective effects via Cdk5-mediated Erk1/2/Drp1 and Fak/Akt/GSK3β pathways, possibly representing a potential preventive agent for neuronal disorder.

Inhibition of cyclin-dependent kinase 5 activity alleviates diabetes-related cognitive deficits.

Cognitive deficit is a prevalent and underestimated complication of diabetes, and the underlying cellular and molecular mechanisms are not well understood. Aberrant activity of cyclin-dependent kinase (Cdk)5 is implicated in a number of neurodegenerative diseases. The present study examined the role of Cdk5 in the progression of diabetes-related cognitive deficits. We showed that the Cdk5 protein expression and kinase activity were significantly increased in diabetic mice at 16 wk. In primary cultured hippocampal neurons exposed to 30 mM glucose, Cdk5 protein and kinase activity were also elevated in a time-dependent manner. Moreover, the high glucose exposure led to an aberrant Cdk5 activation due to its activator p25 that was cleaved from p35 by calpain. Both in diabetic mice and in cultured hippocampal neurons exposed to high glucose, inhibition of Cdk5 activity with roscovitine (Ros) or short hairpin RNA (shRNA) decreased the protein levels of cleaved caspase-3 and the ratio of Bax and Bcl-2. The apoptotic rate detected by TUNEL in vivo or Annexin V and propidium iodide staining for flow cytometry in vitro also had obvious reduction. In addition, high glucose exposure resulted in the increase of phosphorylated (phospho)-MAPK kinase (MKK)6, phospho-p38, and c-Jun, which were rescued by Ros or Cdk5 shRNA. It is more important that the cognitive deficits of diabetic mice were also effectively alleviated by Ros. These results indicate that aberrant Cdk5 activity triggered hippocampal neuron apoptosis by activating MKK6/p38 MAPK cascade in hyperglycemia. Inhibition of Cdk5 overactivation attenuates neuronal apoptosis and cognitive deficits and contributes to the relief of diabetic neurotoxicity in the brain.-Liu, W., Zhou, Y., Liang, R., Zhang, Y. Inhibition of cyclin-dependent kinase 5 activity alleviates diabetes-related cognitive deficits.

P5-TAT: A Novel Medicine Targeting CDK5 Over-Activation for Radiation-Induced Brain Injury

P5-TAT: A Novel Medicine Targeting CDK5 Over-Activation for Radiation-Induced Brain Injury

Golgi fragmentation induced by overactivated cyclin-dependent kinase 5 is associated with isoflurane-induced neurotoxicity.

Overactivated cyclin-dependent kinase 5 (Cdk5) induces Golgi fragmentation, which interrupts the processing and trafficking of secretory cargo and subsequently synaptic plasticity and synaptogenesis, and even leads to neuronal cell death. Cdk5 overactivation and subsequent Golgi fragmentation are involved in many neurodegenerative diseases. However, whether isoflurane-induced neurotoxicity is relevant to aberrant Cdk5 activation and subsequent Golgi fragmentation remains unknown. In the present study, we explored the underlying molecular mechanisms of isoflurane-induced neurotoxicity in primary cultured hippocampal neurons. After treatment with 2% isoflurane for 6 h, immunofluorescence staining and transmission electron microscopy were used to examine the Golgi structure. Neuronal viability was evaluated using the 3-(4,5-dimethyithiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay and TUNEL staining. Cdk5 activity was assessed using histone H1 as a substrate. Our results showed that Cdk5 activity and the number of fragmented Golgi increased significantly after isoflurane exposure. This was accompanied by an increase in neuronal death. Meanwhile, pharmacological inhibition of Cdk5 activity by 8 µM roscovitine alleviated isoflurane-induced Golgi fragmentation and neurotoxicity. Cumulatively, this study shows that aberrant Cdk5 activation-induced Golgi fragmentation is relevant to isoflurane neurotoxicity and indicates that a Cdk5 inhibitor may be a potential therapeutic candidate for the prevention of isoflurane-induced neurotoxicity. Video abstract: http://links.lww.com/WNR/A445.

Inhibition of cyclin-dependent kinase 5 affects early neuroinflammatory signalling in murine model of amyloid beta toxicity

BackgroundCyclin-dependent kinase 5 (Cdk5) belongs to the family of proline-directed serine/threonine kinases and plays a critical role in neuronal differentiation, migration, synaptogenesis, plasticity, neurotransmission and apoptosis. The deregulation of Cdk5 activity was observed in post mortem analysis of brain tissue of Alzheimer’s disease (AD) patients, suggesting the involvement of Cdk5 in the pathomechanism of this neurodegenerative disease. However, our recent study demonstrated the important function of Cdk5 in regulating inflammatory reaction.MethodsSince the role of Cdk5 in regulation of inflammatory signalling in AD is unknown, we investigated the involvement of Cdk5 in neuroinflammation induced by single intracerebroventricular (icv) injection of amyloid beta protein (Aβ) oligomers in mouse. The brain tissue was analysed up to 35 days post injection. Roscovitine (intraperitoneal administration) was used as a potent Cdk5 inhibitor. The experiments were also performed on human neuroblastoma SH-SY5Y as well as mouse BV2 cell lines treated with exogenous oligomeric Aβ.ResultsOur results demonstrated that single injection of Aβ oligomers induces long-lasting activation of microglia and astrocytes in the hippocampus. We observed also profound, early inflammatory response in the mice hippocampus, leading to the significant elevation of pro-inflammatory cytokines expression (e.g. TNF-α, IL-1β, IL-6). Moreover, Aβ oligomers elevated the formation of truncated protein p25 in mouse hippocampus and induced overactivation of Cdk5 in neuronal cells. Importantly, administration of roscovitine reduced the inflammatory processes evoked by Aβ in the hippocampus, leading to the significant decrease of cytokines level.ConclusionsThese studies clearly show the involvement of Cdk5 in modulation of brain inflammatory response induced by Aβ and may indicate this kinase as a novel target for pharmacological intervention in AD.

Cdk5 at crossroads of protein oligomerization in neurodegenerative diseases: facts and hypotheses.

Cyclin-dependent kinase 5 (Cdk5) is involved in proper neurodevelopment and brain function and serves as a switch between neuronal survival and death. Overactivation of Cdk5 is associated with many neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. It is believed that in those diseases Cdk5 may be an important link between disease-initiating factors and cell death effectors. A common hallmark of neurodegenerative disorders is incorrect folding of specific proteins, thus leading to their intra- and extracellular accumulation in the nervous system. Abnormal Cdk5 signaling contributes to dysfunction of individual proteins and has a substantial role in either direct or indirect interactions of proteins common to, and critical in, different neurodegenerative diseases. While the roles of Cdk5 in α-synuclein (ASN) - tau or β-amyloid peptide (Aβ) - tau interactions are well documented, its contribution to many other pertinent interactions, such as that of ASN with Aβ, or interactions of the Aβ - ASN - tau triad with prion proteins, did not get beyond plausible hypotheses and remains to be proven. Understanding of the exact position of Cdk5 in the deleterious feed-forward loop critical for development and progression of neurodegenerative diseases may help designing successful therapeutic strategies of several fatal neurodegenerative diseases. Cyclin-dependent kinase 5 (Cdk5) is associated with many neurodegenerative disorders such as Alzheimer's or Parkinson's diseases. It is believed that in those diseases Cdk5 may be an important factor involved in protein misfolding, toxicity and interaction. We suggest that Cdk5 may contribute to the vicious circle of neurotoxic events involved in the pathogenesis of different neurodegenerative diseases.

The inhibition of Cdk5 activity after hypoxia/ischemia injury reduces infarct size and promotes functional recovery in neonatal rats

Recent studies indicate that over-activation of Cdk5 is a crucial pro-death signal and Cdk5 activity inhibition provides neuroprotection in animal stroke models. However, Cdk5 inhibitors are reported to affect physiological functions of Cdk5 and lead to serious side effects. Therefore, targeting Cdk5 or its activators without affecting physiological functions of Cdk5 is a therapeutic strategy for ischemic brain injury. In this study, we examined Cdk5 activity in a rat hypoxia/ischemia (HI) injury model. Cdk5 expression was not changed after HI injury, but Cdk5 activity significantly increased, which was demonstrated by the increased phorsphorylation—phosphorylation of Tau and glucocorticoid receptor (GR), two downstream signals of Cdk5. We further showed that the levels of Cdk5 activators p35 and p39 decreased after HI injury, while p25, which is converted from p35 and has a higher activator activity on Cdk5, increased markedly after HI injury. P5, a 24-residue mimetic peptide of p35, was reported to specifically inhibit the p25/Cdk5 signal pathway in an Alzheimer’s disease model. P5-TAT, which can cross the blood–brain barrier and cell membrane facilitated by TAT protein, was used in our study. We found that p5-TAT treatment did not change the levels of p35, p39, and p25, but reduced the phorsphorylation of Tau and GR, suggesting the inhibition of the p25/Cdk5 by the peptide p5-TAT. This was supported by the fact that p5 interacted with Cdk5, but not with Cdk5 activators. In addition, p5-TAT reduced cleaved caspase-3 level, a marker of neuronal apoptosis. We further demonstrated that p5-TAT pre-treatment reduced cerebral infarct volume; even when p5-TAT was delayed to be administered at 24h after HI injury, p5-TAT still promoted long-term functional recovery. Therefore, Cdk5 inhibition by the small peptide p5-TAT or its derivatives is a promising therapeutic strategy for the treatment of ischemic brain injury including hypoxic-ischemic encephalopathy and stroke.

Generation of the Cdk5 activator p25 is a memory mechanism that is affected in early Alzheimer's disease.

About 15 years ago it was proposed that generation of the truncated protein p25 contributes to toxicity in Alzheimer’s disease (AD). p25 is a calcium-dependent degradation product of p35, the principal activator of cyclin-dependent kinase 5 (Cdk5). The biochemical properties of p25 suggested that its generation would cause Cdk5 overactivation and tau hyperphosphorylation, a prerequisite for neurofibrillary tangle (NFT) formation. Whilst this model was appealing as it explained NFT formation, many laboratories could not confirm the finding of increased p25 generation in brain from AD patients. On the contrary, it emerged that p25 levels are reduced in AD. This reduction occurs primarily in the early stages of the disease. Further, p25 generation in the mouse hippocampus is associated with normal memory formation and p25 overexpression enhances synaptogenesis. Therefore, it transpires that p25 generation is a molecular memory mechanism that is impaired in early AD. I discuss the prospect that investigation of p25-regulated proteins will shed light into mechanisms underlying synaptic degeneration associated with memory decline in AD.

Valproic acid downregulates Cdk5 activity via the transcription of the p35 mRNA

The cyclin-dependent kinase 5 (Cdk5) is a neuron-specific Ser/Thr kinase that is activated by the regulatory subunit p35. Overactivation of Cdk5, which is induced by the cleavage of p35 by calpain, is implicated in neuronal death in various neurodegenerative diseases. In contrast, depletion of the Cdk5 activity renders neurons vulnerable to stresses. Recent reports suggest the involvement of Cdk5 in mental disorders. We hypothesized that perturbation of Cdk5 activity is related to mental conditions. To verify this hypothesis, we investigated the effect of valproic acid (VPA), which is a drug of choice for psychiatric disorders, on Cdk5 activity. VPA decreased the expression of p35 at both the protein and mRNA levels in cultured neurons, resulting in a decrease of Cdk5 activity. VPA decreased the p35 mRNA via histone deacetylase inhibition. The chronic administration of VPA also downregulated p35 in mouse brains. These results indicate that VPA regulates Cdk5 activity in neurons via p35 transcription mediated by HDAC inhibition.

Inhibition of aberrant cyclin-dependent kinase 5 activity attenuates isoflurane neurotoxicity in the developing brain

Aberrant CDK5 activity is implicated in a number of neurodegenerative disorders. Isoflurane exposure leads to neuronal apoptosis, and subsequent learning and memory defects in the developing brain. The present study was designed to examine whether and how CDK5 activity plays a role in developmental isoflurane neurotoxicity. Rat pups and hippocampal neuronal cultures were exposed to 1.5% isoflurane for 4 h. The protein and mRNA levels of CDK5, p35 and p25 were detected by western blot and QReal-Time PCR. CDK5 activity was evaluated in vitro using Histone H1 as a substrate. Roscovitine (an inhibitor of CDK5) was applied before isoflurane treatment, cleaved Caspase-3, Bcl-2, Bax, MEF2 and phospho-MEF2A-Ser-408 expressions were determined. Dominant-Negative CDK5 was transfected before isoflurane treatment. Neuronal apoptosis was evaluated by Flow cytometry (FCM) and TUNEL-staining. Cognitive functions were assessed by Morris water maze. We found that isoflurane treatment led to an aberrant CDK5 activation due to its activator p25 that was cleaved from p35 by calpain. Inhibition of CDK5 activity with Roscovitine enhanced Bcl-2, and decreased cleaved Caspase-3 and Bax expressions. In addition, isoflurane exposure resulted in a decrease of MEF2 and increase of phospho-MEF2A-Ser-408, which were rescued by Roscovitine or Dominant-Negative CDK5 transfection. Dominant-Negative CDK5 transfection also decreased the percentage of TUNEL-positive cells in isoflurane neurotoxicity. Moreover, Roscovitine remarkably alleviated the learning and memory deficits induced by postnatal isoflurane exposure. These results indicated that aberrant CDK5 activity-dependent MEF2 phosphorylation mediates developmental isoflurane neurotoxicity. Inhibition of CDK5 overactivation contributes to the relief of isoflurane neurotoxicity in the developing brain.

Retinoic Acid Induces Apoptosis of Prostate Cancer DU145 Cells through Cdk5 Overactivation

Retinoic acid (RA) has been believed to be an anticancer drug for a long history. However, the molecular mechanisms of RA actions on cancer cells remain diverse. In this study, the dose-dependent inhibition of RA on DU145 cell proliferation was identified. Interestingly, RA treatment triggered p35 cleavage (p25 formation) and Cdk5 overactivation, and all could be blocked by Calpain inhibitor, Calpeptin (CP). Subsequently, RA-triggered DU145 apoptosis detected by sub-G1 phase accumulation and Annexin V staining could also be blocked by CP treatment. Furthermore, RA-triggered caspase 3 activation and following Cdk5 over-activation were destroyed by treatments of both CP and Cdk5 knockdown. In conclusion, we report a new mechanism in which RA could cause apoptosis of androgen-independent prostate cancer cells through p35 cleavage and Cdk5 over-activation. This finding may contribute to constructing a clearer image of RA function and bring RA as a valuable chemoprevention agent for prostate cancer patients.

Quantitative analysis of in vivo phosphorylation of Cyclin-dependent kinase activator p35 by Phos-tag SDS-PAGE/immunoblotting

Phosphorylation is a major post-translational modification widely used in regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes downregulation of Cdk5 whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser8 and Thr138 as major phosphorylation sites using metabolic labeling of cultured cells followed by 2D-phosphopeptide mapping and phospho-specific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the in vivo phosphorylation states of p35. Using Phos-tag acrylamide, electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states, and quantified Ser8, Ser91 and Thr138 phosphorylation. This is the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.

Regulation of Androgen Receptor and Prostate Cancer Growth by Cyclin-dependent Kinase 5

Prostate cancer is the most frequently diagnosed male malignancy. The normal prostate development and prostate cancer progression are mediated by androgen receptor (AR). Recently, the roles of cyclin-dependent kinase 5 (Cdk5) and its activator, p35, in cancer biology are explored one after another. We have previously demonstrated that Cdk5 may regulate proliferation of thyroid cancer cells. In addition, we also identify that Cdk5 overactivation can be triggered by drug treatments and leads to apoptosis of prostate cancer cells. The aim of this study is to investigate how Cdk5 regulates AR activation and growth of prostate cancer cells. At first, the data show that Cdk5 enables phosphorylation of AR at Ser-81 site through direct biochemical interaction and, therefore, results in the stabilization of AR proteins. The Cdk5-dependent AR stabilization causes accumulation of AR proteins and subsequent activation. Besides, the positive regulations of Cdk5-AR on cell growth are also determined in vitro and in vivo. S81A mutant of AR diminishes its interaction with Cdk5, reduces its nuclear localization, fails to stabilize its protein level, and therefore, decreases prostate cancer cell proliferation. Prostate carcinoma specimens collected from 177 AR-positive patients indicate the significant correlations between the protein levels of AR and Cdk5 or p35. These findings demonstrate that Cdk5 is an important modulator of AR and contributes to prostate cancer growth. Therefore, Cdk5-p35 may be suggested as diagnostic and therapeutic targets for prostate cancer in the near future.

Abstract B45: The short-term effects of arsenic on Cdk5-dependent apoptosis of prostate cancer cells

Abstract Arsenic compounds have been used widely in medicine to treat a variety of diseases. However, it has also been believed to involve the carcinogenesis of prostate cancer and the hormone disruption might be one of the causes. Our previous results indicate that digoxin treatment leads to apoptosis of prostate cancer cells through Cdk5 overactivation, in which the regular activator of Cdk5, p35, is cleaved into more potent one, p25. Here, our latest data showed that arsenic also caused p35 cleavage and subsequently Cdk5 overactivation in prostate cancer cells. Similarly, this arsenic- induced stimulatory effect on Cdk5 resulted in apoptosis of prostate cancer cells accompanying with the degradation of androgen receptor in LNCaP cells. This result implies that androgen is no longer a trophic factor for prostate cancer cells after arsenic treatment. Although STAT3 was identified by us as a Cdk5 substrate responding to tumor growth, arsenic-triggered Cdk5 overactivation did not increase STAT3 phosphorylation. In conclusion, although long-term exposure of arsenic possibly causes prostate cancer, our present results provide evidence indicating that in vitro and short-term treatments of arsenic might lead to apoptosis of prostate cancer cell lines through Cdk5 overactivation pathway. This study may arouse the new indication for the old chemical and also shed light on the application on cancer therapy. Citation Information: Clin Cancer Res 2010;16(14 Suppl):B45.

Quantitative Measurement of in Vivo Phosphorylation States of Cdk5 Activator p35 by Phos-tag SDS-PAGE

Phosphorylation is a major post-translational modification widely used in the regulation of many cellular processes. Cyclin-dependent kinase 5 (Cdk5) is a proline-directed serine/threonine kinase activated by activation subunit p35. Cdk5-p35 regulates various neuronal activities such as neuronal migration, spine formation, synaptic activity, and cell death. The kinase activity of Cdk5 is regulated by proteolysis of p35: proteasomal degradation causes down-regulation of Cdk5, whereas cleavage of p35 by calpain causes overactivation of Cdk5. Phosphorylation of p35 determines the proteolytic pathway. We have previously identified Ser(8) and Thr(138) as major phosphorylation sites using metabolic labeling of cultured cells followed by two-dimensional phosphopeptide mapping and phosphospecific antibodies. However, these approaches cannot determine the extent of p35 phosphorylation in vivo. Here we report the use of Phos-tag SDS-PAGE to reveal the phosphorylation states of p35 in neuronal culture and brain. Using Phos-tag acrylamide, the electrophoretic mobility of phosphorylated p35 was delayed because it is trapped at Phos-tag sites. We found a novel phosphorylation site at Ser(91), which was phosphorylated by Ca(2+)-calmodulin-dependent protein kinase II in vitro. We constructed phosphorylation-dependent banding profiles of p35 and Ala substitution mutants at phosphorylation sites co-expressed with Cdk5 in COS-7 cells. Using the standard banding profiles, we assigned respective bands of endogenous p35 with combinations of phosphorylation states and quantified Ser(8), Ser(91), and Thr(138) phosphorylation. The highest level of p35 phosphorylation was observed in embryonic brain; Ser(8) was phosphorylated in all p35 molecules, whereas Ser(91) was phosphorylated in 60% and Thr(138) was phosphorylated in approximately 12% of p35 molecules. These are the first quantitative and site-specific measurements of phosphorylation of p35, demonstrating the usefulness of Phos-tag SDS-PAGE for analysis of phosphorylation states of in vivo proteins.

Cell-Penetrating Fragments of the Cdk5 Regulatory Subunit Are Protective in Models of Neurodegeneration.

Cdk5 is essential for neuronal differentiation processes in the brain. Activation of Cdk5 requires the association with the mostly neuron-specific p35 or p39. Overactivation of CDK5 by cleavage of p35 into p25 is thought to be involved in neurodegenerative processes. Here, we have tested an approach to inhibit pathological Cdk5 activation with a Tat-linked dominant-negative fragment of p25. It reduced cell death induced by staurosporine and showed a tendency to alleviate manganese-induced cell death, while it did not protect against 6-OHDA toxicity. Our results suggest that the Tat technique is a suitable tool to inhibit dysregulated CDK5.

Neurodegeneration in an Aβ‐induced model of Alzheimer’s disease: the role of Cdk5

Cdk5 dysregulation is a major event in the neurodegenerative process of Alzheimer's disease (AD). In vitro studies using differentiated neurons exposed to Abeta exhibit Cdk5-mediated tau hyperphosphorylation, cell cycle re-entry and neuronal loss. In this study we aimed to determine the role of Cdk5 in neuronal injury occurring in an AD mouse model obtained through the intracerebroventricular (icv) injection of the Abeta(1-40) synthetic peptide. In mice icv-injected with Abeta, Cdk5 activator p35 is cleaved by calpains, leading to p25 formation and Cdk5 overactivation. Subsequently, there was an increase in tau hyperphosphorylation, as well as decreased levels of synaptic markers. Cell cycle reactivation and a significant neuronal loss were also observed. These neurotoxic events in Abeta-injected mice were prevented by blocking calpain activation with MDL28170, which was administered intraperitoneally (ip). As MDL prevents p35 cleavage and subsequent Cdk5 overactivation, it is likely that this kinase is involved in tau hyperphosphorylation, cell cycle re-entry, synaptic loss and neuronal death triggered by Abeta. Altogether, these data demonstrate that Cdk5 plays a pivotal role in tau phosphorylation, cell cycle induction, synaptotoxicity, and apoptotic death in postmitotic neurons exposed to Abeta peptides in vivo, acting as a link between diverse neurotoxic pathways of AD.