lncRNA CDK5RAP3 regulates the proliferation and invasion of gastric cancer cells via miR-223-3p / 国际外科学杂志

Objective:To explore the expression of long non-coding RNA (lncRNA) CDK5RAP3 in gastric cancer tissue and its regulatory effect on gastric cancer cell proliferation and invasion.Methods:The expression differences of CDK5RAP3 in gastric cancer tissues and adjacent tissues were analyzed by TCGA database. By transfecting the pcDNA3.1-CDK5RAP3 plasmid into Hs-746T cells, a gastric cancer cell line overexpressing CDK5RAP3 (CDK5RAP3 group) was constructed, and the pcDNA3.1 plasmid was transfected into Hs-746T cells as a control group. The changes of CDK5RAP3 expression in the two groups of cells were detected by real-time quantitative PCR (qRT-PCR). The effects of overexpression of CDK5RAP3 on the proliferation and invasion of Hs-746T cells were detected by CCK-8 assay and Transwell assay, respectively. The binding sites of CDK5RAP3 and miR-223-3p were predicted by the starBase v2.0 database. The direct binding of CDK5RAP3 and miR-223-3p was verified by dual-luciferase reporter gene experiment. The expression levels of miR-223-3p in Hs-746T cells in each group were detected by qRT-PCR. Western blot was used to detect the expression levels of proliferation proteins and invasion proteins in Hs-746T cells in each group. The experimental data were analyzed by SPSS 17.0 software, and the measurement data conforming to the normal distribution were expressed as Mean±SD. The t-test was used to compare between two groups, and the one-way analysis of variance was used to compare the means of multiple groups. Results:Compared with adjacent tissues, the expression level of CDK5RAP3 in gastric cancer tissues was significantly lower ( P<0.01). The expressions of CDK5RAP3 in Hs-746T cells in the control group and CDK5RAP3 group were (1.08±0.77) and (10.63±2.14), respectively, and the difference was statistically significant ( P<0.01). Up-regulation of CDK5RAP3 significantly decreased the proliferation activity of Hs-746T cells ( P<0.05). The number of invasive cells in the control group and CDK5RAP3 group were (137.80±28.72) and (57.76±24.95), respectively, and the difference was statistically significant ( P<0.01). CDK5RAP3 could directly bind miR-223-3p ( P<0.01). The expression of miR-223-3p in Hs-746T cells in control group and CDK5RAP3 group were (6.22±1.20) and (1.01±0.98), respectively, and the difference was statistically significant ( P<0.01). Compared with the control group, up-regulation of CDK5RAP3 significantly reduced the expression levels of proliferation and invasive proteins. Conclusion:The expression of CDK5RAP3 is low in gastric cancer tissue, and CDK5RAP3 inhibits the proliferation and invasion of gastric cancer Hs-746T cells by targeting miR-223-3p.

miR-142-5p inhibits the invasion and migration of lung adenocarcinoma H1650 cells by affecting epithelial mesenchymal transformation / 中国肿瘤生物治疗杂志

@# Objective: To study the expression of miR-142-5p in lung adenocarcinoma tissues, and to explore its effect on proliferation, invasion, migration and epithelieal-mesenchymal transition (EMT) of H1650 cells and the potential mechanisms. Methods:Atotal of 107 pairs of lung adenocarcinoma tissues and corresponding para-cancerous tissues from patients, who underwent tumor resection and were pathologically confirmed at the Department of Thoracic Surgery, the Fourth Hospital of Hebei Medical University between Jan. 2014 and Jan. 2015, were collected for this study; in addition, human lung adenocarcinoma cell lines (H1650, HCC827, A549, H1975, PC9) and human bronchial epithelial BEAS-2B cells were also used in this study. qPCR was used to detect the expression of miR-142-5p in lung adenocarcinoma tissues and cell lines. The correlation between expression of miR-142-5p and clinical features was analyzed.After transfection with miR-142-5p mimics or miR-negative control (miR-NC) plasmid, the proliferation, invasion and migration of H1650 cells were detected with CCK-8, Transwell invasion assay and Wound healing assay, respectively. The bioinforamtics tool was used to predict the target genes of miR-142-5p, and Luciferase reporter gene assay was performed to validate the regulation of miR-142-5p on target gene. Western blotting (WB) was used to detect the expressions of cyclin-dependent kinase 5 (CDK5) and EMTrelated protein. Results: Compared to Para-cancerous tissues and BEAS-2B cells, the expression of miR-142-5p was lower in lung adenocarcinoma tissues and cell lines (all P<0.01). Of the 107 cases of lung adenocarcinoma tissues, 61 cases (57.01%) showed decreased miR-142-5 expression, which was correlated with the TNM stage and lymph node metastasis (both P<0.01). Transfection of miR-142-5p mimics significantly up-regulated the expression of miR-142-5p and decreased the proliferation, invasion and migration of H1650 cells (all P<0.05 or P<0.01). Bioinformatics showed that CDK5 was a target gene of miR-142-5p. Luciferase reporter gene assay and WB validated that miR-142-5p could significantly down-regulate CDK5 expression in H1650 cells, up-regulate the expression of E-cadherin and down-regulate the expressions of N-cadherin, Twist and Snail in H1650 cells (all P<0.01). Conclusion: miR-142-5p is low expressed in lung adenocarcinoma tissues and cell lines; it suppresses the EMT process to inhibit, invasion and migration of H1650 cells via down-regulating the expression of CDK5.

knocking out mediated by CRISPR-Cas9 genome editing for PD-L1 attenuation and enhanced antitumor immunity

Blocking the programmed death-ligand 1 (PD-L1) on tumor cells with monoclonal antibody therapy has emerged as powerful weapon in cancer immunotherapy. However, only a minority of patients presented immune responses in clinical trials. To develop an alternative treatment method based on immune checkpoint blockade, we designed a novel and efficient CRISPR-Cas9 genome editing system delivered by cationic copolymer aPBAE to downregulate PD-L1 expression on tumor cells specifically knocking out Cyclin-dependent kinase 5 () gene . The expression of PD-L1 on tumor cells was significantly attenuated by knocking out , leading to effective tumor growth inhibition in murine melanoma and lung metastasis suppression in triple-negative breast cancer. Importantly, we demonstrated that aPBAE/Cas9-Cdk5 treatment elicited strong T cell-mediated immune responses in tumor microenvironment that the population of CD8 T cells was significantly increased while regulatory T cells (Tregs) was decreased. It may be the first case to exhibit direct PD-L1 downregulation CRISPR-Cas9 genome editing technology for cancer therapy. It will provide promising strategy for preclinical antitumor treatment through the combination of nanotechnology and genome engineering.

Hypothermia treatment ameliorated cyclin-dependent kinase 5-mediated inflammation in ischemic stroke and improved outcomes in ischemic stroke patients

OBJECTIVES: The inflammatory response is a key mechanism of neuronal damage and loss during acute ischemic stroke. Hypothermia has shown promise as a treatment for ischemic stroke. In this study, we investigated the molecular signaling pathways in ischemic stroke after hypothermia treatment. METHODS: Cyclin-dependent kinase 5 (CDK5) was overexpressed or silenced in cultured cells. Nuclear transcription factor-κB (NF-κB) activity was assessed by measurement of the luciferase reporter gene. An ischemic stroke model was established in Sprague-Dawley (SD) rats using the suture-occluded method. Animals were assigned to three groups: sham operation control, ischemic stroke, and ischemic stroke + hypothermia treatment groups. Interleukin 1β (IL-1β) levels in the culture supernatant and blood samples were assessed by ELISA. Protein expression was measured by Western blotting. RESULTS: In HEK293 cells and primary cortical neuronal cultures exposed to hypothermia, CDK5 overexpression was associated with increased IL-1β, caspase 1, and NF-κB levels. In both a murine model of stroke and in patients, increased IL-1β levels were observed after stroke, and hypothermia treatment was associated with lower IL-1β levels. Furthermore, hypothermia-treated patients showed significant improvement in neurophysiological functional outcome. CONCLUSIONS: Overall, hypothermia offers clinical benefit, most likely through its effects on the inflammatory response.

The role of oxidative stress in inhibiting FOXO1 through activating Cdk5 in neuronal damage / 西安交通大学学报(医学版)

Objective: To investigate the role of oxidative stress in inhibiting forkhead box O1 (FOXO1) through activating cyclin-dependent kinase 5 (Cdk5) in neuronal damage. Methods: Cdk5 was coexpressed with p39 in HEK cells together with wild-type FOXO1 or a nonphosphorylatable FOXO1-S249A mutant; FOXO1 phosphorylation was immunoblotted through pS249-FOXO1 antibody which specific recognized FOXO1 Ser249 phosphorylation. Cdk5 or p39 was coexpressed in HEK cells together with FOXO1 and their interaction was explored through Co-IP. In primary cortical culture neurons, various Cdk5 activators (p25, p35, and p39) were overexpressed together with Cdk5. FOXO1 transcriptional activity was tested by luciferase assay. We detected the effect of Cdk5 inhibitor roscovitine on FOXO1 transcriptional activity in primary neurons. We compared FOXO1 subcellular localization of wild type with Cdk5 heterozygous mice cortical brain tissues through cytoplasmic and nuclear extracts. In addition, we administered oxidative stress (H2O2 and glutamate treatment) in primary neurons and examined the effect on the cdk5 activity by Western blot, FOXO1 transcriptional activity by luciferase assay, and neuronal damage through stained caspase3. Results: ① Identification of Cdk5/p39 was capable of phosphorylating the transcription factor FOXO1 at Ser249, and associated with it. ② Activated Cdk5 dramatically inhibited FOXO1 transcriptional activity. In contrast, roscovitine, Cdk5 inhibitor, significantly increased FOXO1 transcriptional activity and led to FOXO1 translocation into the nucleus. ③ FOXO1 protein was found predominately in the nucleus in the Cdk5 heterozygous mice. H2O2 or glutamate treatment promoted p35 to be cleaved to p25, and inhibited FOXO1 transcriptional activity. Importantly, this effect of oxidative stress was abolished when Cdk5 was silenced by shRNA. ⑤ Oxidative stress dramatically increased caspase3 immunostaining in primary neurons. Conclusion: Cdk5/p39 inhibits FOXO1 activity through phosphorylation and association. Moreover, oxidative stress inhibits FOXO1 activity through activating Cdk5, and leads to neuronal death.

Expressions of CDK5 and p35/p25 in the blood of patients with vascular cognitive impairment / 西安交通大学学报(医学版)

Objective: To investigate the expressions and possible roles of cyclin dependent kinase 5 (CDK5), p35 and P25 in the blood of patients with vascular cognitive impairment (VCI). Methods: Totally 91 cases of VCI were recruited as the study group, including 49 cases of non-dementia vascular cognitive impairment (VCIND) and 42 cases of vascular dementia (VAD), and 30 cases of cerebral apoplexy cognitive function (NC) and healthy control (N) group, respectively. RT-qPCR was used to detect the relative expression of CDK5 mRNA in the blood. Western blot method was used to detect the protein expressions of CDK5, p35 and p25 in each group. Results: The relative expression of CDK5 mRNA and the expression levels of CDK5, p35 and p25 protein in the four groups: VAD>VCIND>NC>N respectively, with significant differences among the groups (P<0.05). Conclusion: CDK5, p35 and p25 may be involved in the occurrence of VCI, and their expression levels are positively correlated with VCI.

Effect of Wuzang Wenyang Huayu Decoction on Neurofibrillary Tangles by Inhibiting Cyclin-dependent Kinase-5 Over-expression in SAMP8 Mice / 中国实验方剂学杂志

Objective: To explore the mechanism of Wuzang Wenyang Huayu decoction in improving the cognitive competence and the pharmacological mechanism for neurofibrillary tangles related to cyclin-dependent kinase-5(CDK-5).Method: The 10 SAMR1 mice were used as normal group,40 SAMP8 mice were randomly divided into model group,donepezil group (0.4 mg·kg-1·d-1),high and low dose Wuzang Wenyang Huayu decoction groups (5,1.25 g·kg-1·d-1).Drugs were administered by gastric lavage for 4 continuous weeks.Directional navigation and space exploration ability were evaluated with Morris amaze.Real-time PCR was used to measure the mRNA expression of CDK-5 in brain nerve tissues.Western blot was used to detect the protein expression of CDK-5 and phosphorylation of Tau protein.Meanwhile,neurofibrillary tangles in brain tissue were detected with silver staining method.Result: As compared with normal group,both CDK-5 expression and Tau protein phosphorylation in brain nerve tissues were remarkably increased in model group (PPPPPPConclusion: Wuzang Wenyang Huayu decoction can markedly improve the cognitive competence of SAMP8 mice,and the mechanism may be related to its inhibition on CDK-5 over-expression,and down-regulation of Tau protein phosphorylation and neurofibrillary tangles in brain tissue.

Effects of Stilbene Glycoside on Okadaic Acid-induced Tau Protein Phosphorylation in NG 108-15 Cells / 中国药房

OBJECTIVE： To observe the effects of stilbene glycosidec （TSG） on okadaic acid （OA）-induced Tau protein phosphorylation in NG108-15 cells， and to investigate the potential anti-Alzheimer’s disease （AD） mechanism of this compound. METHODS： AD model of NG108-15 cells was induced by OA. The survival rate of NG108-15 cells was observed by MTT assay after pretreated with low-dose， medium-dose and high-dose of TSG （50， 100， 200 μmol/L）. The apoptosis of NG108-15 cells was detected by AO/EB double fluorescence staining. The protein and mRNA expression of CDK5 and GSK3β， and the protein expression of Tau and p-Tau were detected by Western blotting assay and RT-PCR. The distribution of CDK5， GSK3β and Tau protein were detected by immunofluorescence. RESULTS： The normal morphology of NG108-15 cells was observed in normal control group， but CDK5， GSK3β and Tau protein were not found or few was found. Contracted or globular early apoptotic cells were observed in model gorup； the distribution of CDK5， GSK3β and Tau protein was increased， while survival rate of the cells was decreased； protein and mRNA expression of CDK5 and GSK3β as well as ratio of the relative expression of p-Tau to that of Tau （p-Tau/Tau） were all increased significantly （P＜0.05 or P＜0.01）. After pretreatment of TSG， the distribution of early apoptotic cells as well as CDK5， GSK3β and Tau protein were all decreased to some extent in administration groups， while survival rates of the cells were increased significantly. Protein expression of CDK5 and p-Tau/Tau in medium-dose group and high-dose group as well as mRNA expression of CDK5， protein and mRNA expression of GSK3β in administration group were decreased significantly （P＜0.05）. CONCLUSIONS： TSG can protect against AD model cells， the effects of which may be associated with improving survival rate of the cells， down-regulating the protein expression and gene transcription level of phosphokinase CDK5 and GSK3β， inhibiting Tau protein phosphorylation.

Effects of thin recipe of Buyang Huanwu decoction on CDK5 and apoptotic factors in an oxidative stress cell model / 中国药理学通报

Aim To explore the neuroprotective mechanism of thin recipe of Buyang Huanwu decoction on CDK5 and apoptotic factors in an oxidative stress cell model. Methods A PC 12 cell model of oxidative stress damage was established using hydrogen peroxide (H202) at the concentration of 0. 3 mmol • L"1 co-cultured for 2 h, PC 12 cells were divided into four groups, namely normal control, model control, Buyang Huanwu decoction and its thin recipe groups. We evaluated the content of extracellular L-LDH and intracellular GSH by ELISA, and the extracellular ROS levels by FCM. In addition, the expressions of CDK5 and Tau were further analyzed to facilitate the understanding of neuroprotective mechanism by IF, and the mR-NA levels of apoptotic factors such as caspase-3 and the ratio of Bax/Bcl-2 mRNA were determined by qPCR. Results The thin recipe of Buyang Huanwu decoction increased GSH activity, decreased L-LDH and ROS levels compared with model cells at the concentration of 1.0 g • L"1 (P < 0. 05). Furthermore, we observed lower expression of CDK5 and Tau, as well as less mRNA of caspase-3 and the ratio of Bax/Bcl-2 in the thin recipe of Buyang Huanwu decoction groups than that of model groups (P < 0. 05). Conclusions The thin recipe of Buyang Huanwu decoction could play the neuroprotective effects by CDK5 and Tau signal sites to inhibit neuronal apoptosis after oxi-dative stress damage of PC12, which illuminate the partial mechanism and the reasonability of this new recipe simplified from Buyang Huanwu decoction in molecular level.

Identificación in silico de moléculas potencialmente inhibidoras de CDK5, proteína relacionada con la enfermedad de Alzheimer / Identification in silico of potentially inhibitive molecules of CDK5, protein related with the Alzheimer´s disease

Introducción: La enfermedad de Alzheimer exhibe un compromiso neurodegenerativo e irreversible. Hoy, numerosas investigaciones promueven la inhibición de algunas quinasas para su tratamiento, de especial mención la CDK5. Objetivo: Identificación de nuevas moléculas con posibilidad de interactuar con la proteína quinasa dependiente de ciclina 5, CDK5, inhibiendo su función. Material y Métodos: Se realizó un estudio in silico, para lo cual se extrajeron 911 moléculas de pubchem, y mediante AutoDock Vina se hicieron acoplamientos moleculares con la proteína CDK5 extraída de Protein Data Bank y con un inhibidor conocido para la proteína. Además se realizó un acoplamiento inverso para la identificación de otros posibles blancos moleculares con los mejores ligandos seleccionados. Resultados: Con los resultados obtenidos fueron identificadas cinco moléculas con valores de afinidad entre -11,6 hasta -17,7 Kcal/mol que se unen en el sitio activo de la proteína, de igual forma que lo hace el inhibidor conocido de la misma, e interactúan con los residuos cisteína 83 y glutamina 81. Conclusiones: Las moléculas identificadas pueden interactuar con la CDK5 a nivel de su sitio activo, por lo que podrían actuar como inhibidores de esta quinasa. Esto abre una futura ventana terapéutica en el tratamiento de la enfermedad de Alzheimer)AU)

Introduction: The illness of Alzheimer exhibits a neurodegenerative and irreversible commitment. Today, numerous investigations promote the inhibition of some kinases to the treatment, of special mention the CDK5. Objective: Identification of new molecules witch are able to interact with the cicline dependent kinase protein 5, CDK5, inhibiting their function. Material and Methods: it was carried out a study in silico, for that 911 pubchem molecules were extracted, and by means of AutoDock Vina molecular joining were made with the protein CDK5 extracted from the Protein Data Bank and with a well-known inhibitor for the protein. It was also carried out an inverse joining for the identification of other possible molecular targets with the best selected ligands. Results: With the obtained results five molecules were identified with values of likeness among -11,6 until -17,7 Kcal/mol that joins in the active site of the protein, in the same form that makes it the well-known inhibitor of the CDK5, and interact with the residuals cysteine 83 and glutamine 81. Conclusions: The identified molecules can interact with the CDK5 at level of their active place, for what you/they could act as inhibitors of this quinasa. This opens a future therapeutic window in the treatment of the illness of Alzheimer(AU)

Changes of Cdk5 expression after focal brain contusion in rats / 中国法医学杂志

Objective This study aims to evaluate the changes of Cdk5 expression at the time of 3 hours to 10 days after moderate brain injury by blunt force impact in a rat model,and to demonstrate its forensic significance.Methods To establish a rat model of blunt focal brain contusion,and to observe the changes of Cdk5 expression in brain tissue at different timepoints after brain injury by immunohistochemistry and Western blot.Results A low expression level of Cdk5 was observed in the brain tissue of both normal and sham control groups.The expression of Cdk5 increased after 3 and 6 hours,remarkably increased at 12 hours,and reached the maximal level at 24 hours after focal brain injury.The Cdk5 level gradually decreased 3 days,5 days,7 days,and 10 days and reached the normal level 7 and 10 days after the injury,with no statistical difference (P＞0.05) compared with the normal and sham control groups.Conclusion The expression of Cdk5 increased in the peripheral area of contusion tissue after blunt brain injury in rats,showing single peak change,and dropped to normal level with the time extension.The change of Cdk5 expression may provide a new reference index for the prediction of early brain contusion.

Regulation of thin recipe of Buyang Huanwudecoction on Cdk5 expression of rats after cerebral ischemia / 中国药理学通报

Aim To evaluate the regulation of thin recipe of Buyang Huanwu decoction on cyclin-dependent kinase 5(Cdk5)expressions in hippocampus tissue of rats after cerebral ischemia.Methods Male SD rats were divided into sham-operation group,MCAO group,Buyang Huanwu decoction group(ig.3.15 g·kg-1)and its thin recipe composition group(ig.2.41 g·kg-1).Each group was then divided into five subgroups based on the time after administration for 1,3,7,14,28 d respectively.Cdk5 protein and mRNA levels in each group were examined by using immunohistochemistry,Western blot and real-time PCR respectively.Results The up-regulation of Cdk5 was observed in model rat hippocampus after cerebral ischemia 1 day,and kept increasing with the aggravation of ischemia injury,the peaked expression was observed after 7～14 d,while the downtrend was observed after 28 days compared with the corresponding sham-operation groups(P0.05).Conclusion The thin recipe of Buyang Huanwu decoction could exert the protective effect by regulating Cdk5 after cerebral ischemia.

Aberrant Activation of Cdc2/cyclin B1 Is Involved in Initiation of Cytoskeletal Pathology in Murine Niemann-Pick Disease Type C / 华中科技大学学报（医学）（英德文版）

Niemann-Pick disease type C (NPC) is a fatal,neurovisceral lipid storage disease,neuropathologically characterized by cytoplasmic sequestration of glycolipids in neurons,progressive neuronal loss,neurofibrillary tangles (NFTs) formation,and axonal spheroids (AS).Cytoskeletal pathology including accumulation of hyperphosphorylated cytoskeletal proteins is a neuropathological hallmark of the mouse model of NPC (npc mice).With a goal of elucidating the mechanisms underlying the lesion formation,we investigated the temporal and spatial characteristics of cytoskeletal lesions and the roles of cdc2,cdk4,and cdk5 in lesion formation in young npc mice.Cytoskeletal lesions were detectable in npc mice at three weeks of age.Importantly,concomitant activation of cdc2/cyclin B 1 kinase and accumulation of a subsequently generated cohort of phospho-epitopes were detected.The activation of cdk4/cyclin D1 and cdk5/p25 kinases was observed during the fourth week of life in npc mice,and this activation contributed to the lesion formation.We concluded that the progression of cytoskeletal pathology in npc mice older than four weeks is accelerated by the cumulative effect of cdc2,cdk4,and cdk5 activation.Furthermore,cdc2/cyclin B1 may act as a key initial player one week earlier.Targeting cell cycle activation may be beneficial to slow down the NPC pathogenesis.

The protective action and its mechanism of 5-hydroxy-1 H-indazole in SH-SY5 Y cells / 中国药理学通报

Aim To study the protection and possible mechanism of 5-hydroxy-1 H-indazole against 1-methyl-4-phenylpyridinium iodide ( MPP+)-induced SH-SY5 Y cell apoptosis. Methods An apoptotic model was es-tablished in human neuroblastoma SH-SY5 Y by MPP+in vitro. MTT analysis was used to evaluate the protec-tive effect of 5-hydroxy-1H-indazole. Immunochemistry and Hoechst33258 nuclear staining were used to ob-serve the neuroprotection and anti-apoptosis of 5-hy-droxy-1H-indazole. Western blot was used to detect the levels of P-tau ( Ser396 ) closely related to neuronal apoptosis and its upstream kinases:P-GSK-3β and CDK5 . Results MPP+ induced activation of GSK-3β, increase of activity of CDK5 , tau hyperphosphory-lation and neuronal cell apoptosis. However,5-hydrox-y-1 H-indazole reduced the activities of GSK-3β and CDK5,then decreased the level of tau hyperphosphory-lation and inhibited MPP+-induced SH-SY5 Y cells ap-optosis. Conclusions 5-hydroxy-1H-indazole could attenuate MPP+-induced SH-SY5 Y neuronal cell apop-tosis. Possible mechanism is that 5-hydroxy-1H-in-dazole inhibits GSK-3βand CDK5 two signal transduc-tion pathways to lower the level of tau phosphorylation, then plays a role of neuroprotection.

The role of roscovitine in tunicamycin induced podocyte injury / 天津医药

Objective To observe the protective effects of roscovitine on the podocyte injury induced by endoplasmic reticulum stress (ERS) caused by tunicamycin. Methods The differentiated podocytes cultured at 37℃were randomly di-vided into:(1) Control group, DMSO group and tunicamycin group (TM, 1.0μmol/L). The treatment was given for 3, 6 and 12 hours in three groups. (2) For control group, tunicamycin group, tunicamycin+roscovitine group (20, 40μmol/L, TM+ROS), the treatment was given for 12 hours. The podocyte apoptosis was detected by flow cytometry and TUNEL method. The ex-pressions of Cdk5, GRP78, Caspase-12 and CHOP were detected by Western blot assay. Results (1) Compared with con-trol group and DMSO group, the podocyte apoptosis was increased significantly in a time dependent manner after tunicamy-cin treatment in TM group;the protein expressions of Cdk5, GRP78, Caspase-12 and CHOP were also up-regulated signifi-cantly in TM group (P<0.05). (2) Flow cytometry and TUNEL analysis showed that tunicamycin induced apoptosis in podo-cytes, which was significantly inhibited by roscovitine in a concentration dependent manner in TM+ROS group as compared to that of TM group (P<0.05). The protein expressions of GRP78, Caspase-12 and CHOP were also significantly decreased in a concentration dependent manner in TM+ROS group compared to those of TM group (P<0.05). Conclusion Roscovi-tine, the inhibitor of Cdk5, can reduce the podocyte apoptosis induced by tunicamycin. The protective effects of roscovitine on podocytes can be a novel approach of treating diabetic nephropathy.

Progress in the study of new cancer target Cdk5 and its inhibitors / 药学学报

Cyclin-dependent kinase-5 (Cdk5) is a kind of Ser/Thr kinases in the signaling pathway, which regulates the neural development. The recent studies have confirmed that hyperactivation of Cdk5 is closely associated with the evolution, progression and apoptosis of tumors. The Cdk5 inhibitors have been extensively studied in the drug discovery against cancer. The structure features of these inhibitors and molecular mechanisms of their activities have provided clues for the drug development. In the second generation Cdk5 inhibitors, the ATP-binding pocket, a highly conserved site, has been targeted in the drug design in most cases. In addition, a growing number of peptides has been generated by targeting the protein/protein interfaces of Cdk5.

Role of Fuzhisan in reducing Tau protein hyperphosphorylation in cortical neurons through a cyclin-dependent kinase 5 pathway / 中国药理学通报

Aim Fuzhisan ( FZS ) , a Chinese herbal complex prescription that has been used for the treat-ment of AD for over 15 years, is known to enhance the cognitive ability in AD patients. In this study, to in-vestigate whether FZS reduces Aβ25-35-induced Tau protein hyperphosphorylation in neonatal rat cortical neurons by suppressing the cyclin-dependent kinase 5 ( CDK5 ) pathway. Methods Neonatal Wistar rats born within 24 h were selected to separate and purify their cortical neurons for culture in vitro. After 7-day culture of cortical neurons in vitro, 20 μmol · L-1 Aβ25-35 was used to act on them for 24 h. Medication groups were pretreated with FZS ( 20 mg · L-1 ) , CDK5 inhibitor roscovitine ( 15 μmol · L-1 ) and cal-pain preparation calpeptin (20 μmol·L-1 ) for 24 h, followed by reaction with 20 μmol·L-1 Aβ25-35 for 24 h. Tau protein phosphorylation levels at Ser396, Ser202 and Thr231 and the protein level of CDK5 acti-vator proteins p25/p35 were assayed by Western blot. Fluorescence intensity was measured with a fluores-cence microplate reader to reflect calpain activity. CDK5 kinase activity was assayed by immunoprecipita-tion. Results After 20 μmol·L-1 Aβ25-35 acting on cortical neurons for 24h, there were increments in the following: Tau protein phosphorylation levels at Ser396, Ser202 and Thr231, CDK5 kinase activity, CDK5 activator protein p25 level, and calpain activity. In the 20 mg·L-1 FZS treatment group, Aβ25-35 was suppressed markedly, resulting in increments in Tau protein phosphorylation levels at Ser396 , Ser202 and Thr231 , suppression of CDK5 kinase activity and p25 protein level, and elevation in calpain activity. Both CDK5 inhibitor roscovitine and calpain preparation cal-peptin, as positive control drugs, also played a role in suppressing Tau protein hyperphosphorylation. Con-clusion FZS can suppress Aβ25-35-induced Tau pro-tein hyperphosphorylation in cortical neurons through the calpain/CDK5 pathway.

Inhibition of roscovitine on the activity of Cdk5/p25 and phosphorylation of tau in retina of RCS rats after intravitreal injection / 中华实验眼科杂志

Background Study determined that retinitis pigmentosa has a similar pathogenesis mechanism to Alzheimer disease,and activity of cyclin-dependent kinase 5 (Cdk5) and its activators participates in the degeneration of central nervous system.Roscovitine,an inhibitor of Cdk5,can suppress activity of Cdk5/p25 pathway and therefore inhibit cell apoptosis.However,the influence of roscovitine on retinitis pigmentosa(RP) is unclear.Objective This study was to investigate the expressions of p35,p25 and tau in the retinas of RCS rats.Methods Roscovitine of 4 μl was intravitreally injected in the right eyes of 12 SPF 17-day-old RCS rats,and the fellow eyes were not intervened as the control eyes.The rats were sacrificed on eighth day (postnatal 25 days) and eighteenth day (postnatal 35 days),and whole retinas were isolated to evaluate the relative expressions of Cdk5,p35,p25 and tau phosphorylation by Western blot,and the activity of Cdk5/p25 was analyzed by quantitative colorimetric assay.The results were compared between the right eyes and fellow eyes by paired t test.The use and care of the rats complied with Ethic Statement of Experimental Animal of Ningxia Medical University.Results In the eighth and eighteenth day after injection,the relative expression values (A values) of p35 in rat retinas were 1.186±0.019 and 1.069± 0.019 in the injected eyes,showing significant decreases in comparison with 1.364±0.016 and 1.214±0.008 of the fellow eyes (t =-6.294,-6.477,both at P＜0.05);the relative expression values (A values) of p25 in rat retinas were 0.312±0.009 and 0.269±0.018 in the injected eyes,which was significantly lower than 0.595±0.013 and 0.473±0.011 of the fellow eyes (t=-36.508,-11.879,both at P＜0.05).No significant difference was found in the relative expression of Cdk5 protein between the injected eyes and the fellow eyes in various time points after injection (both at P＞0.05).The activities of Cdk5/p25 were (0.003 83 ±0.000 14) mol/(s · mg) and (0.002 01 ± 0.000 11) mol/(s · mg) in the injected eyes,with significant decreases in comparison with the (0.005 47±0.000 27)mol/(s · mg)and (0.003 35±0.000 15) mol/(s · mg) of the fellow eyes (t=-9.152,P=0.000;t=-9.248,P=0.000),and the tau phosphorylation levels followed the same pattern in the eighth and eighteenth day after injection (t =-9.854,-6.744,both at P＜0.05).Conclusions Intravitreal injection of roscovitine can inhibit the activity of Cdk5/p25 and tau phosphorylation level in retinas of RCS rats to certain extend.

The impact of celastrol on cognitive function and the expression of Cdk5, p25 and p35 in hippocampus in APPswe/PS1dE9 double transgenic mouse after partial hepatectomy / 临床麻醉学杂志

Objective To investigate the impact of celastrol on cognitive function and the ex-pression of Cdk5/p25 in hippocampus in APPswe/PS1dE9 double transgenic mouse after partial hepa-tectomy.Methods Three-month-old APPswe/PS1dE9 double transgenic mice (n=96)were randomly divided into surgery group (partial hepatectomy,group S),celastrol group (celastrol and partial hepa-tectomy,group C)and DMSO group (DMSO and partial hepatectomy,group O).Eight mice were se-lected randomly in each group and were Morris-water maze trained for 5 continuous days.Their learn-ing and memory abilities were evaluated at 1,3,7 and 14 d after the surgery,respectively.For the remaining mice in each group,the hippocampus were collected and the changes of Cdk5,p35 and p25 in hippocampus were measured by western blot at the time 1,3,7,14 d after partial hepatectomy. Six mice were killed at each time for data collection.Results The average escape latency of group C was significantly shorter than those of groups S and O at 3,7 and 14 d after partial hepatectomy (P <0.05).The percentages of time in target quadrant of groups S and O decreased significantly than that in group C (P <0.05).Western blot showed the expression of Cdk5 in group C was significantly low-er than that in groups S and O at 3,7 and 14 d after partial hepatectomy (P <0.05),and the same re-sult was also found in the expression of p25 at 1,3,7 and 14 d after partial hepatectomy (P <0.05). There was no significant difference in the expression of p35 between each group.Conclusion Celas-trol can improve the learning and memory ability in APPswe/PS1dE9 the double transgenic mouse, with the mechanism may be related to the decrease of Cdk5 and p25 in hippocampus.

CDK5-dependent inhibitory phosphorylation of Drp1 during neuronal maturation

Mitochondrial functions are essential for the survival and function of neurons. Recently, it has been demonstrated that mitochondrial functions are highly associated with mitochondrial morphology, which is dynamically changed by the balance between fusion and fission. Mitochondrial morphology is primarily controlled by the activation of dynamin-related proteins including dynamin-related protein 1 (Drp1), which promotes mitochondrial fission. Drp1 activity is regulated by several post-translational modifications, thereby modifying mitochondrial morphology. Here, we found that phosphorylation of Drp1 at serine 616 (S616) is mediated by cyclin-dependent kinase 5 (CDK5) in post-mitotic rat neurons. Perturbation of CDK5 activity modified the level of Drp1S616 phosphorylation and mitochondrial morphology in neurons. In addition, phosphorylated Drp1S616 preferentially localized as a cytosolic monomer compared with total Drp1. Furthermore, roscovitine, a chemical inhibitor of CDKs, increased oligomerization and mitochondrial translocation of Drp1, suggesting that CDK5-dependent phosphorylation of Drp1 serves to reduce Drp1's fission-promoting activity. Taken together, we propose that CDK5 has a significant role in the regulation of mitochondrial morphology via inhibitory phosphorylation of Drp1S616 in post-mitotic neurons.