Cell Line ; Cyclin-Dependent Kinase 5 ; metabolism ; Humans ; Lipopolysaccharides ; Microglia ; cytology ; drug effects ; metabolism ; Stilbenes ; pharmacology

Mitochondria continuously fuse and divide to maintain homeostasis. An impairment in the balance between the fusion and fission processes can trigger mitochondrial dysfunction. Accumulating evidence suggests that mitochondrial dysfunction is related to neurodegenerative diseases such as Parkinson's disease (PD), with excessive mitochondrial fission in dopaminergic neurons being one of the pathological mechanisms of PD. Here, we investigated the balance between mitochondrial fusion and fission in the substantia nigra of a non-human primate model of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. We found that MPTP induced shorter and abnormally distributed mitochondria. This phenomenon was accompanied by the activation of dynamin-related protein 1 (Drp1), a mitochondrial fission protein, through increased phosphorylation at S616. Thereafter, we assessed for activation of the components of the cyclin-dependent kinase 5 (CDK5) and extracellular signal-regulated kinase (ERK) signaling cascades, which are known regulators of Drp1(S616) phosphorylation. MPTP induced an increase in p25 and p35, which are required for CDK5 activation. Together, these findings suggest that the phosphorylation of Drp1(S616) by CDK5 is involved in mitochondrial fission in the substantia nigra of a non-human primate model of MPTP-induced PD.
1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine ; Cyclin-Dependent Kinase 5 ; Cyclin-Dependent Kinases ; Dopaminergic Neurons ; Homeostasis ; Mitochondria ; Mitochondrial Dynamics ; Neurodegenerative Diseases ; Parkinson Disease ; Phosphorylation ; Phosphotransferases ; Primates ; Substantia Nigra

BACKGROUND/AIMS: The primary pathophysiologic abnormality in achalasia is known to be a loss of inhibitory myenteric ganglion cells, which may result from an immune-mediated response or neuronal degeneration. The aim of this study was to identify proteins suggestive of an immune-mediated response or neuronal degeneration in the serum of achalasia patients using a proteomic analysis. METHODS: Blood samples were collected from five symptomatic achalasia patients and five sex- and age-matched healthy controls. Serum proteomic analysis was conducted, and the protein spots were identified using matrix-assisted laser desorption ionization/time-of-flight and a proteomics analyzer. The serum level of C3 was measured by enzyme-linked immunosorbent assay in nine patients with achalasia and 18 sex- and age-matched healthy controls. RESULTS: Of the 658 matched protein spots, 28 spots were up-regulated over 2-fold in the serum from achalasia patients compared with that from controls. The up-regulated proteins included complement C4B5, complement C3, cyclin-dependent kinase 5, transthyretin, and alpha 2 macroglobulin. The serum levels of C3 in achalasia patients were significantly higher than those of controls. CONCLUSIONS: The serum proteomic analysis of achalasia patients suggests an immune-mediated response or neuronal degeneration. Further validation studies in larger samples and the esophageal tissue of achalasia patients are required.
alpha-Macroglobulins ; Complement C3 ; Complement System Proteins ; Cyclin-Dependent Kinase 5 ; Enzyme-Linked Immunosorbent Assay ; Esophageal Achalasia ; Ganglion Cysts ; Humans ; Neurons ; Prealbumin ; Proteins ; Proteomics

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.
Animals ; Cells, Cultured ; Cyclin-Dependent Kinase 5/*metabolism ; Dynamins/analysis/*metabolism ; HeLa Cells ; Humans ; Mitochondria/metabolism ; Mitosis ; Neurons/*cytology/*metabolism ; Phosphorylation ; Rats

OBJECTIVETo explore the role of cyclin dependent kinase 5 (CDK5) in 2, 5-hexanedione (HD)-induced neuropathy.

METHODSThirty male Wistar rats weighted 200 approximately 240 g were divided randomly into three groups, i.e. control group, 200 mg/kg HD group and 400 mg/kg HD group (n = 10 for each group). HD was administered to rats by intraperitoneal injection at dosage of 200 or 400 mg/kg for 8 weeks (five times per week) to establish the intoxicated rats model. The relative contents of CDK5, p35 and p25 were determined in cerebrum, spinal cord and sciatic nerve of rats by Western Blotting.

RESULTSCompared with that of the control group rats, p35 contents were significantly decreased (P < 0.01) in the cytosolic fractions of cerebrum and spinal cord in both the 200 and 400 mg/kg HD intoxicated rats, while in the membrane fractions of spinal cord and sciatic nerve, p35 contents were increased significantly (P < 0.01). The changes of p25 showed the same pattern with p35. P25 contents were significantly reduced (P < 0.05) in the cytosolic (cerebrum and spinal cord) and membrane (cerebrum) fractions of both HD-treated rats and were elevated (P < 0.01) in the membrane fraction of spinal cord and cytosolic fraction of sciatic nerve. The relative amounts of CDK5 were significantly decreased (P < 0.01) in the cytosolic and membrane fractions of cerebrum in both the 200 and 400 mg/kg HD intoxicated rats. Except for membrane fraction of sciatic nerve, the significant increased (P < 0.01) of CDK5 were observed in the spinal cord and sciatic nerve of both the 200 and 400 mg/kg HD treated rats.

CONCLUSIONHD can induce significant changes of CDK5 and its activators p35, p25 in nerve tissues, which may be related to the neuropathy induced by HD.

Animals ; Cyclin-Dependent Kinase 5 ; metabolism ; Disease Models, Animal ; Hexanones ; poisoning ; Male ; Nerve Tissue ; metabolism ; Nervous System Diseases ; chemically induced ; Phosphotransferases ; metabolism ; Rats ; Rats, Wistar

OBJECTIVETo observe the effects of polydatin on learning and memory and cyclin-dependent kinase 5 (Cdk5) kinase activity in the hippocampus of rats with chronic alcoholism.

METHODSForty rats were randomly divided into 4 groups: control group, chronic alcoholism group, low and high polydatin group. The rat chronic alcoholism model was established by ethanol 3.0 g/(kg · d) (intragastric administration). The abstinence scoring was used to evaluate the rats withdrawal symptoms; cognitive function was measured by Morris water maze experiment; Cdk5 protein expression in the hippocampus was detected by immunofluorescence; Cdk5 kinase activity in the hippocampus was detected by liquid scintillation counting method.

RESULTSThe abstinence score, escape latency, Cdk5 kinase activity in chronic alcoholism group rats were significantly higher than those of control group (P < 0.05). The abstinence score, escape latency in high polydatin group rats were significantly lower than those of chronic alcoholism group (P < 0.05); Cdk5 kinase activity in high and low polydatin group rats was significantly lower than that of chronic alcoholism group( P < 0.05); immunofluorescence showed that the Cdk5 positive cells of chronic alcoholism group were significantly increased compared with control group (P < 0.05), and the Cdk5 positive cells of polydatin groups were significantly decreased compared with chronic alcoholism group ( P < 0.05).

CONCLUSIONPolydatin-reduced the chronic alcoholism damage may interrelate with regulation of Cdk5 kinase activity.

Alcoholism ; physiopathology ; Animals ; Cyclin-Dependent Kinase 5 ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Glucosides ; pharmacology ; Hippocampus ; drug effects ; enzymology ; Learning ; drug effects ; Memory ; drug effects ; Rats ; Stilbenes ; pharmacology

OBJECTIVETo study the effect of arsenic on neuronal cell apoptosis and the mRNA and protein expression of calpain 1, calpain 2, and cyclin-dependent kinases 5 (cdk5)/p25 and to provide a scientific basis for the research on neurotoxic mechanism of arsenic trioxide (As2O3).

METHODSPrimary cultured rat neurons were divided into untreated control group, dimethyl sulfoxide (DMSO) solvent control group, and 1, 5, and 10 µmol/L As2O3 treated groups. Eight hours after being treated with As2O3, cell apoptosis rate was determined by flow cytometry, the mRNA expression of calpain 1, calpain 2, cdk5, and p35 was measured by real-time fluorescence quantitative PCR, and the protein expression of calpain 1, calpain 2, cdk5, p35, and p25 was measured by Western blot.

RESULTSCompared with those in the untreated control group and DMSO solvent control group, the cell apoptosis rates in the 5 and 10 µmol/L As2O3 treated groups were significantly increased (P < 0.05). The mRNA expression levels of calpain 1 were 6.36±3.26, 7.11±5.13, and 7.47±2.59 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, and the mRNA expression levels of cdk5 were 1.27±0.19, 1.54±0.04, and 1.79±0.21 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, which were significantly higher than those in the untreated group (0.72±0.15 and 1.77±0.87) and those in the DMSO solvent control group (0.96±1.23 and 1.18±0.09) (P < 0.05). The mRNA expression levels of p35 in the 1 and 5 µmol/L As2O3 treated groups were 2.17±0.59 and 2.51±0.51, respectively, which were significantly higher than that in the untreated control group (1.26±0.37) (P < 0.05). The protein expression levels of calpain 1 were 0.37±0.10, 0.42±0.13, and 0.51±0.18 in the 1, 5, and 10 µmol/L As2O3 treated groups, respectively, which were significantly higher than those in the untreated control group (0.11±0.08) and DMSO solvent control group (0.13±0.07) (P < 0.05). In the 5 and 10 µmol/L As2O3 treated groups, the protein expression levels of cdk5 were 0.34±0.12 and 0.37±0.21, while the protein expression levels of p25 were 0.31±0.23 and 0.55±0.16, all of which were significantly higher than those in the untreated control group and DMSO solvent control group (P < 0.05). The protein expression levels of p35 were reduced in the 5 µmol/L As2O3 treated group (0.31±0.23) and 10 µmol/L As2O3 treated group (0.26±0.16), as compared with those in the untreated control group and DMSO solvent control group (P < 0.05). The mRNA and protein expression of calpain 2 showed no significant differences between all groups (P > 0.05).

CONCLUSIONThe calpain 1-cdk5/p25 pathway may be involved in the process of As2O3-induced neuronal cell apoptosis.

Animals ; Apoptosis ; drug effects ; Arsenic Poisoning ; Arsenicals ; Calpain ; metabolism ; Cells, Cultured ; Cyclin-Dependent Kinase 5 ; metabolism ; Neurons ; drug effects ; metabolism ; Oxides ; toxicity ; Rats

Alcoholism ; metabolism ; Animals ; Cyclin-Dependent Kinase 5 ; metabolism ; Glucosides ; pharmacology ; Male ; Prefrontal Cortex ; drug effects ; metabolism ; Rats ; Rats, Sprague-Dawley ; Stilbenes ; pharmacology

Cyclin-dependent kinase 5 (CDK5) is a member of cyclin-dependent kinase family, which does not directly regulate cell cycle. Through phosphorylation of target protein, CDK5 plays an irreplaceable role in the development, reparation and degeneration of neurons. Brain injury refers to the organic injury of brain tissue caused by external force hit on the head. Owing to the stress and repair system activated by our body itself after injury, various proteins and enzymes of the brain tissues are changed quantitatively, which can be used as indicators for estimating the time of injury. This review summarizes the progress on the distribution, the activity mechanism and the physiological effects of CDK5 after brain injury and its corresponding potential served as a marker for brain injury determination.
Brain/physiopathology* ; Brain Injuries/physiopathology* ; Cyclin-Dependent Kinase 5/metabolism* ; Nerve Tissue Proteins/metabolism* ; Neurons ; Neuroprotective Agents/pharmacology* ; Phosphorylation/drug effects* ; Time Factors

OBJECTIVETo investigate the effect of monoamine oxidase inhibitor phenelzine on proliferation, apoptosis and histone modulation in acute lymphoblastic leukemia cell line Molt-4 cells.

METHODSThe effect of Phenelzine on cell proliferation was detected by MTT. Apoptotic rate was measured by flow cytometry. The variation of apoptosis associated proteins Caspase-3, Bcl-2 and Bax, cyclin-dependent kinase inhibitor p21, tumor suppressor protein p15, and the expression level of histone methylation of H3K4, H3K9 and histone acetylation of H3, DNMT1 were detected by Western Blot.

RESULTS① Molt-4 cell proliferation rates were (87.68±3.54)%, (67.84±3.24)%, (51.48±3.37)%, (28.72±2.56)% respectively after exposured to phenelzine at 5, 10, 15, 20 μmol/L for 24 h, P<0.05. ② After 10 μmol/L of phenelzine exposure for 24, 48, 72 h, cell proliferation rates were (67.84±3.24)%, (50.24±2.01)%, (40.31±2.25)%, P<0.05. ③ The apoptotic rates were (13.64±2.58)%, (31.24±3.42)%, (56.37±4.26)% after phenelzine treatment at 5, 10, 20 μmol/L for 24 h, which was concentration dependent. ④ Phenelzine could upregulate the expression of Bax, caspase-3, p21, and downregulate Bcl-2 expression. Phenelzine upregulated the methylation level of histone H3K4me1, H3K4me2 and histone acetylated H3, while it didn't change the level of histone H3K4me3, H3K9me1, H3K9me2. ⑤ Phenelzine inhibited DNMT1 expression and promoted p15 expression.

CONCLUSIONSPhenelzine increased the methylation of histone H3K4me1, H3K4me2, acetylation of histone H3 and p21, and decreased the expression of DNMT1 and p15, and ultimately inhibited the proliferation and apoptosis of Molt-4 cells.

Acetylation ; Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Cyclin-Dependent Kinase Inhibitor p15 ; metabolism ; Cyclin-Dependent Kinase Inhibitor p21 ; metabolism ; DNA (Cytosine-5-)-Methyltransferase 1 ; DNA (Cytosine-5-)-Methyltransferases ; metabolism ; Histones ; metabolism ; Humans ; Methylation ; Phenelzine ; pharmacology