To explore the effect of different concentrations of corticosterone (CORT) on primary cultured hippocampal neurons and their Ca2+/CaMK II expression and possible mechanism, the changes of hippocampal neurons were observed in terms of morphology, activity of cells, cell death, concentrations of cytosolic free calcium, and the expression of CaMK II by using MTT assay, flow cytometry, fluorescent labeling of Fura-2/AM and Western blotting after 10(-7), 10(-6) and 10(-5) mol/L of CORT was added to culture medium, The evident effect of 10(-6) and 10(-5) mol/L of CORT on the morphology of hippocampal neuron was found. Compared with control neurons, the activity of the cells was markedly decreased and [Ca2+]i increased in the neurons treated with 10(-6) and 10(-5) mol/L of CORT, but no change was observed in the neuron treated with 10(-7) mol/L of CORT. The death was either by way of apoptosis or necrosis in the cells treated with 10(-6) and 10(-5) mol/L of CORT respectively. The correlation analysis showed that a reverse correlation existed between [Ca2+]i and the expression of CaMK II. Either apoptosis or necrosis occurs in the hippocampal neurons treated with CORT. The increased hippocampal [Ca2+]i is both the result of CORT impairing the hippocampal neurons and the cause of the apoptosis of hippocampal neurons and the decreased CaMK II expression.
Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; biosynthesis ; genetics ; Cells, Cultured ; Corticosterone ; pharmacology ; Hippocampus ; cytology ; enzymology ; Neurons ; cytology ; Rats ; Rats, Wistar

AIMTo investigate the resting [Ca2]i level and expression of calmodulin (CaM), calmodulin-dependent protein kinase II (CaMPKII) mRNA in hippocampal neurons of the mice with vascular dementia (VD) and their roles in the pathogenesis of VD.

METHODSThe mice were subjected for ischemia/reperfusion repeatedly on bilateral common carotid arteries by knots to establish the VD models. Animals with the sham-operation were taken as control group. The changes of behavior were observed through the step-down avoidance test and water maze test on the day 29, 30 after the operations. The hippocampal neurons were obtained immediately after mice were sacrificed and the resting [Ca2+]i was measured using laser scanning confocal microscopy with Fluo-3/AM as fluorescence indicator. RT-PCR technique was used to measure the mRNA expression of CaM, CaMPKII in hippocampal neurons.

RESULTS(1) The abilities of learning and memorizing in model group were inferior to those of sham-operation group( P < 0.05). (2) The resting [Ca2]i level in model group was significantly higher than sham-operation group (P < 0.05), while the expression of CaMmRNA, CaMPKIImRNA in VD group was significantly reduced than sham-operation group (P < 0.01).

CONCLUSIONOur study indicates that excessive resting[Ca2+ ]i level and lower CaM, CaMPKII expression in hippocampal neurons might participate in the pathogenesis of VD.

Animals ; Calcium ; analysis ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Calmodulin ; genetics ; metabolism ; Dementia, Vascular ; metabolism ; Hippocampus ; cytology ; metabolism ; Male ; Mice ; Mice, Inbred Strains ; Neurons ; metabolism ; RNA, Messenger ; genetics

Melanoma is the most aggressive cutaneous tumor. Neuropilin and tolloid-like 2 (NETO2) is closely related to tumorigenesis. However, the functional significance of NETO2 in melanoma progression remains unclear. Herein, we found that NETO2 expression was augmented in melanoma clinical tissues and associated with poor prognosis in melanoma patients. Disrupting NETO2 expression markedly inhibited melanoma proliferation, malignant growth, migration, and invasion by downregulating the levels of calcium ions (Ca²⁺) and the expression of key genes involved in the calcium signaling pathway. By contrast, NETO2 overexpression had the opposite effects. Importantly, pharmacological inhibition of CaMKII/CREB activity with the CaMKII inhibitor KN93 suppressed NETO2-induced proliferation and melanoma metastasis. Overall, this study uncovered the crucial role of NETO2-mediated regulation in melanoma progression, indicating that targeting NETO2 may effectively improve melanoma treatment.
Humans ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism* ; Cell Line, Tumor ; Cell Proliferation ; Melanoma/genetics* ; Membrane Proteins/genetics* ; Phosphorylation ; Signal Transduction

OBJECTIVETo explore effect of curcumin in different concentrations on learning and memory of senescence-accelerated mice (SAM) and their possible mechanisms.

METHODSMice were randomly divided into six groups: the SAMR1 normal control group, the SAMP8 model control group, the SAMP8 + solvent (the peanut oil) control group, SAMP8 + low, middle and high dose curcumin groups. Mice were gastrogavage for 25 successive days. On the next day of ending the experiment, changes of learning and memory in mice of each group were observed by Morris water maze. The hippocampal [Ca2+] was determined. Expressions of hippocampal calmodulin-dependent protein kinase II (CaMK II) and Calmodulin (CaM) mRNA were detected using Western blot and reverse transcription polymerase chain reaction (RT-PCR) respectively.

RESULTSThe latency to find the hidden platform was remarkably prolonged, the hippocampal [Ca2+]i was markedly increased, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA were significantly reduced in the SAMP8-model control group (P < 0.05, P < 0.01). The latency to find the hidden platform was remarkably shortened in the SAMP8 + middle dose curcumin and the SAMP8 + high dose curcumin groups (P < 0.01). The hippocampal [Ca2+]i was markedly lowered, the expression of CaMK II in the hippocampal membrane and the level of hippocampal CaM mRNA obviously increased in the SAMP8 + low, middle and high dose curcumin groups (P < 0.05, P < 0.01).

CONCLUSIONCurcumin could improve learning and memory Ca2+/capacities of SAM by lowering hippocampal [Ca2+] overload, increase the hippocampal CaM mRNA level and CaMK II expression in the hippocampal dose-dependently.

Aging ; drug effects ; metabolism ; Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Calmodulin ; metabolism ; Curcumin ; pharmacology ; Hippocampus ; drug effects ; metabolism ; Maze Learning ; drug effects ; Memory ; drug effects ; Mice ; RNA, Messenger ; genetics

BACKGROUNDThe effect of chronic stress on cognitive functions has been one of the hot topic in neuroscience. But there has been much controversy over its mechanism. Such single stressor applied in the past could not simulate complicated living circumstances that people confronted with. The aim of this study was to investigate the effects of chronic multiple-stress on learning and memory as well as on the levels of calcium/calmodulin-dependent protein kinase II (CaMKII), calmodulin (CaM) mRNA, and cAMP-response element binding protein (CREB) mRNA in the hippocampus of rats.

METHODSThe rats were divided randomly into stressed and control groups. The stressed group was given chronic multiple-stress for 6 weeks to set up a chronic multiple-stressed model. The rats' performance of spatial learning and memory was tested using Morris Water Maze (MWM) and Y-maze. Meanwhile, the expressions of CaMKII, CaM mRNA and CREB mRNA of rats' hippocampus were detected by immunohistochemistry, Western blot and reverse transcription-polymerase chain reaction (RT-PCR), respectively. In addition, the width of synaptic cleft and the thickness of post-synaptic densities (PSD) were observed in the hippocampal CA3 region of rats by electron microscopy.

RESULTSAfter exposure to chronic multiple-stress for 6 weeks, the ability of learning and memory of the stressed group was higher than that of the control group (P < 0.05, P < 0.01). The width of synaptic cleft was smaller and the thickness of PSD was larger in the hippocampal CA3 region of the stressed group than in that of the control group (P < 0.01). The CaMK II immunostaining of the stressed group was stronger than that of the control group in the stratum radiatum and oriens of the hippocampal CA1 and CA3, especially in the stratum oriens. Quantitative analysis indicated that the expression of CaMK II, CaM mRNA, and CREB mRNA in the hippocampus of the stressed group was higher than that of the control group (P < 0.05, P < 0.01).

CONCLUSIONSThe capacity of learning and memory can be enhanced after chronic multiple-stress. The increased levels of CaMK II, CaM mRNA, and CREB mRNA may contribute to the enhancing effect of chronic multiple-stress on learning and memory.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; Calmodulin ; genetics ; Chronic Disease ; Cyclic AMP Response Element-Binding Protein ; genetics ; Hippocampus ; metabolism ; ultrastructure ; Learning ; Male ; Memory ; Microscopy, Electron ; RNA, Messenger ; analysis ; Rats ; Rats, Wistar ; Stress, Physiological ; metabolism ; psychology ; Synapses ; ultrastructure

The Cre/LoxP system is a well-established approach to spatially and temporally control genetic inactivation. The calcium/calmodulin-dependent protein kinase II alpha subunit (CaMKIIalpha) promoter limits expression to specific regions of the forebrain and thus has been utilized for the brain-specific inactivation of the genes. Here, we show that CaMKIIalpha-Cre can be utilized for simultaneous inactivation of genes in the adult brain and in male germ cells. Double transgenic Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice generated by crossing CaMKIIalpha-Cre(+/Cre) mice with floxed ROSA26 lacZ reporter (Rosa26(+/stop-lacZ)) mice exhibited lacZ expression in the brain and testis. When these mice were mated to wild-type females, about 27% of the offspring were whole body blue by X-gal staining without inheriting the Cre transgene. These results indicate that recombination can occur in the germ cells of male Rosa26(+/stop-lacZ)::CaMKIIalpha-Cre(+/Cre) mice. Similarly, when double transgenic Gnao(+/f)::CaMKIIalpha-Cre(+/Cre) mice carrying a floxed Go-alpha gene (Gnao(f/f)) were backcrossed to wild-type females, approximately 22% of the offspring carried the disrupted allele (Gnao(Delta)) without inheriting the Cre transgene. The Gnao(Delta/Delta) mice closely resembled conventional Go-alpha knockout mice (Gnao(-/-)) with respect to impairment of their behavior. Thus, we conclude that CaMKIIalpha-Cre mice afford recombination for both tissue- and time-controlled inactivation of floxed target genes in the brain and for their permanent disruption. This work also emphasizes that extra caution should be exercised in utilizing CaMKIIalpha-Cre mice as breeding pairs.
Animals ; Brain/*metabolism ; Calcium-Calmodulin-Dependent Protein Kinase Type 2/genetics ; Female ; GTP-Binding Protein alpha Subunits, Gi-Go/*genetics ; *Gene Deletion ; Gene Knockout Techniques/*methods ; Male ; Mice ; RNA, Untranslated/genetics ; Recombination, Genetic ; Spermatozoa/*metabolism

OBJECTIVETo observe the effects of PSD95 gene specific siRNAs on neuropathic pain relief, neuron viability, and postsynaptic calcium/calmodulin-dependent protein kinase IIα (CaMKIIα) phosphorylation in vitro and in vivo.

METHODSGene-specific siRNAs of rat PSD95 were synthesized chemically for transfection. Adult male Sprague-Dawley (SD) rats were randomly divided into 3 groups: naïve group (n=6), sham group (n=6), and sciatic nerve chronic constriction injury (CCI) group (n=24). The CCI group was further divided into 4 groups (n=6 in each group), which were pretreated with normal saline, transfection vehicle, negative control siRNAs, and PSD95 gene specific siRNAs respectively. All the subgroups received corresponding agents intrathecally for 3 days, started one day before the CCI of sciatic nerve. Both mechanical allodynia and thermal hyperalgesia were measured on post-operative day 3 and 7. PSD95 gene silenced NG108-15 cells were further stimulated by glutamate, with the cell viability and the expression/phosphorylation of CaMKIIα measured by MTT cell proliferation assay and Western blot, respectively.

RESULTSThe siRNAs decreased PSD95 mRNA level significantly both in vivo and in vitro. Neuropathic pain rats pretreated with PSD95 gene specific siRNAs exhibited significant elevation in the mechanical withdrawal threshold and paw withdrawal thermal latency, without affecting the baseline nociception. PSD95 gene silencing enhanced neuronal tolerance against the glutamate excitotoxicity, meanwhile the phosphorylation of CaMKIIα Thr286 was attenuated.

CONCLUSIONPre-emptive administration of PSD95 gene specific siRNAs may attenuate the central sensitization CaMKIIα-related signaling cascades, leading to the relief of neuropathic pain.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Cells, Cultured ; Disks Large Homolog 4 Protein ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; genetics ; Neuralgia ; therapy ; Neurons ; physiology ; Phosphorylation ; RNA, Small Interfering ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo study the effects of methionine and choline on the expression levels of CaMKII and CREB mRNA and proteins in hippocampus of rats exposed to lead.

METHODSMale SD rats were divided into five groups. (1) control group, (2) group exposed to lead+2 by drinking water with 0.40 g/L lead acetate, (3) group exposed to methionine and choline (1:1, 400 mg/kg), (4) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 100 mg/kg), (5) group exposed to 0.40 g/L lead acetate plus methionine and choline (1:1, 400 mg/kg). In 8 weeks after exposure, all rats were killed. Then CREB mRNA and CaMK II mRNA expression levels in hippocampus were detected by real-time PCR, CREB and CaMK II protein expression levels in hippocampus were measured by western blot assay.

RESULTSThe expression levels (0.743 ± 0.185 and 0.729 ± 0.199) of CaMKII mRNA and CREB mRNA in the hippocampus of lead group were significantly lower than those (0.950 ± 0.238 and 0.901 ± 0.232) of control group (P < 0.05), also the expression levels (0.271 ± 0.045 and 0.212 ± 0.058) of CREB protein and pCREB protein in the hippocampus of lead group were significantly lower than those (0.319 ± 0.058 and 0.506 ± 0.125) of control group (P < 0.05). The expression levels (1.014 ± 0.210 and 1.126 ± 0.379) of CaMKII mRNA and the expression levels (1.029 ± 0.335 and 0.932 ± 0.251) of CREB mRNA in the hippocampus of 2 groups exposed to lead acetate plus methionine and choline were significantly higher than those of lead group (P < 0.05). The expression levels (0.407 ± 0.951 and 0.563 ± 0.178) of CREB protein and pCREB protein in the hippocampus of group exposed to lead acetate plus 400 mg/kg methionine and choline were significantly higher than those of lead group (P < 0.05).

CONCLUSIONMethionine and choline could decrease the inhibition effects of lead on the expression of CaMKII and CREB mRNA or CREB and pCREB proteins in the hippocampus of rats.

Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; metabolism ; Choline ; pharmacology ; Cyclic AMP Response Element-Binding Protein ; metabolism ; Hippocampus ; drug effects ; metabolism ; Lead ; toxicity ; Male ; Methionine ; pharmacology ; RNA, Messenger ; genetics ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the effects of the γ isoform of Ca(2+)/calmodulin-dependent protein kinase II (CaMKIIγ) on colorectal cancer (CRC) cell growth in vitro and in vivo and explore the mechanisms.

METHODSThe mRNA levels of CaMKIIγ in 5 CRC cell lines, tumor tissues and matched adjacent tissues from 20 CRC patients were examined by semi-quantitative RT-PCR. The lentiviral vector pLenti6.3-MCS-IRES2-eGFP was used to generate the lentivirus particle Lenti-CaMKIIγ for transfecting SW620 cells. The proliferation ability of the transfected SW620-CaMKIIγ cells was assessed by growth curve and colony formation assay. The expression of IKKα, IKKβ, IKKγ, p-IKKα/β, p-IκB andIκB of the transfected cells were determined by Western blotting, and the expression and localization of nuclear factor-κB (NF-κB) p65 were detected by immunofluorescence. In nude mouse models bearing the transfected SW620-CaMKIIγ cell xenograft, the tumor volume was measured twice a week.

RESULTSCaMKIIγ mRNA showed high expressions in the 5 colorectal cancer cell lines. Eighteen of the 20 tumor tissues showed higher expressions of CaMKIIγ than the adjacent non-tumor tissues. The proliferation of transfected SW620-CaMKIIγ cells was enhanced significantly. CaMKIIγ activated NF-κB signaling pathway and led to NF-κB p65 nuclear translocation. In the tumor-bearing mouse model, the volume of the tumors generated by the transfected SW620-CaMKIIγ cells was 1.46- and 1.68-fold higher than that of the tumors with the control cells at the 8th and 12th day, respectively.

CONCLUSIONCaMKIIγ can effectively promote the growth of colorectal cancer cells in vitro and in vivo by activating NF-κB signaling pathway.

Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; genetics ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Colorectal Neoplasms ; metabolism ; pathology ; Humans ; NF-kappa B ; metabolism ; RNA, Messenger ; genetics ; Signal Transduction ; Up-Regulation

To have more insight into the mechanism of neuronal injury in phenylketonuria patients, gene expression profiles were studied in cell culture of embryonic rat cortical neurons induced by phenylalanine. Randomly chosen cortical cultures for 3 d were treated by 0.9 mmol/L phenylalanine for 12 h. Control group of the same batch was treated with the same volume of medium. Total RNA was extracted and hybridized with the Affymetrix gene chip U34 according to the protocol provided by the Affymetrix Company. Real-time PCR was used to further confirm the result. We found that the hybridization signals of 167 genes were increased among the total 1323 probes plotted on the chip. The 167 increased genes could be functionally categorized into signal transduction, neuron related, cytoskeleton, metabolism, ion channels, transcription factors, cytokines, and apoptosis related. Signals of 7 probes were decreased, which accounted to 0.5% of the total number. A series of genes that were not reported before were up-regulated by phenylalanine, including Ca(2+)/calmodulin-dependent protein kinase, Brain type II (CaMK II), Ras, P38 MAP kinase, L-voltage dependent calcium channel, some genes related to vesicle formation and transmitter release, some glutamate receptor subunits and glutamate transporters. According to the gene expression profile, it is likely that multi-processes are involved in the neuronal injury induced by high phenylalanine, such as the activation of the NMDR-Ca(2+)- CaMK II - Ras- P38 axis, the abnormality in neurotransmitter release. Our study also suggests that the excitatory neurotransmitter glutamate may play a role in the neural pathology of phenylketonuria.
Animals ; Calcium-Calmodulin-Dependent Protein Kinase Type 2 ; Calcium-Calmodulin-Dependent Protein Kinases ; genetics ; Cells, Cultured ; Cerebral Cortex ; metabolism ; pathology ; Embryo, Mammalian ; Gene Expression Profiling ; Neurons ; metabolism ; pathology ; Oligonucleotide Array Sequence Analysis ; Phenylalanine ; toxicity ; Phenylketonurias ; genetics ; pathology ; Random Allocation ; Rats ; Rats, Sprague-Dawley