Levetiracetam Protects Against Cognitive Impairment of Subthreshold Convulsant Discharge Model Rats by Activating Protein Kinase C (PKC)-Growth-Associated Protein 43 (GAP-43)-Calmodulin-Dependent Protein Kinase (CaMK) Signal Transduction Pathway.

BACKGROUND Subclinical epileptiform discharges (SEDs) are defined as epileptiform electroencephalographic (EEG) discharges without clinical signs of seizure in patients. The subthreshold convulsant discharge (SCD) is a frequently used model for SEDs. This study aimed to investigate the effect of levetiracetam (LEV), an anti-convulsant drug, on cognitive impairment of SCD model rats and to assess the associated mechanisms. MATERIAL AND METHODS A SCD rat model was established. Rats were divided into an SCD group, an SCD+ sodium valproate (VPA) group, and an SCD+ levetiracetam (LEV) group. The Morris water maze was used to evaluate the capacity of positioning navigation and space exploration. The field excitatory post-synaptic potentials (fEPSPs) were evaluated using a bipolar stimulation electrode. NCAM, GAP43, PS95, and CaMK II levels were detected using Western blot and RT-PCR, respectively. PKC activity was examined by a non-radioactive method. RESULTS LEV shortens the latency of platform seeking in SCD rats in positioning navigation. fEPSP slopes were significantly lower in the SCD group, and LEV treatment significantly enhanced the fEPSP slopes compared to the SCD group (P<0.05). The NCAM and GAP-43 levels were increased and PSD-95 levels were increased in SCD rats (P<0.05), which were improved by LEV treatment. The PKC activity and CaMK II levels were decreased in SCD rats and LEV treatment significantly enhanced PKC activity and increased CaMK II levels. CONCLUSIONS Cognitive impairment in of SCD model rats may be caused by decreased PKC activity, low expression of CaMK II, and inhibition of LTP formation. LEV can improve cognitive function by activating the PKC-GAP-43-CaMK signal transduction pathway.

Trichostatin A sensitizes cisplatin-resistant A549 cells to apoptosis by up-regulating death-associated protein kinase.

AIM: To investigate the apoptosis-inducing effect of trichostatin A (TSA) in the human lung adenocarcinoma cisplatin-resistant cell line (A549/CDDP) and to examine whether TSA can enhance sensitivity to cisplatin treatment and the underlying molecular mechanisms of such an enhancement. METHODS: Cell viability was evaluated using the Neutral Red assay. Apoptosis was assessed using Hoechst 33258 staining and flow cytometry analysis. Protein expression was detected by Western blotting. To determine the role of Death-associated protein kinase (DAPK) in TSA-induced apoptosis in the A549/CDDP cell line, cells were transfected with pcDNA3.1(+)-DAPK, which has a higher expression level of DAPK compared to endogenous expression, and DAPK activity was inhibited by both over-expression C-terminal fragment of DAPK which may competitive binding DAPK substrates to inhibit the function of DAPK and RNA interference. RESULTS: TSA induced apoptosis in both A549 cells and A549/CDDP cells. TSA enhanced the sensitivity of A549/CDDP cells to cisplatin, along with concomitant DAPK up-regulation. When DAPK was over-expressed, A549/CDDP cells became sensitive to cisplatin and the cytotoxicity of TSA could be increased. Moreover, the cytotoxicity of TSA could be alleviated by inhibition of DAPK activity by the expression of a recombinant C-terminal fragment of DAPK or RNA interference. CONCLUSION: TSA induced sensitivity to cisplatin treatment in cisplatin-resistant A549 cells. The up-regulation of DAPK is one of the mechanisms mediating sensitization to TSA-induced apoptosis in cisplatin-resistant cells.

Promoter hypermethylation in tumour suppressor genes and response to interleukin-2 treatment in bladder cancer: a pilot study.

PURPOSE: Non-muscle invasive bladder cancer (BC) is a highly recurrent disease, with the first recurrences arising shortly after transurethral resection of the bladder (TURB). Topical administration of interleukin-2 (IL-2) has been shown as an effective adjuvant therapy for BC; however, predictive biomarkers that may identify suitable subgroups of patients are lacking. In this pilot study we sought to determine the prognostic value of epigenetic and genetic inactivation of tumour suppressor genes (TSGs) among BC patients treated with IL-2. METHODS: After complete TURB, patients with multifocal superficial BC were treated with five daily intravesical instillations of IL-2. Promoter hypermethylation in six TSGs and the TP53 gene mutations were prospectively assessed by methylation-specific PCR and automated capillary single-strand conformation polymorphism in 21 primary bladder cancer specimens and ten bladder wall biopsies collected during follow-up. RESULTS: After IL-2 treatment, 9 out of 21 (43%) patients did not develop recurrent tumour within the 1 year of follow-up period. The mean duration of recurrence-free survival in the rest of the study group was 112 days. In the current pilot study, BC with p16 gene hypermethylation had a lower risk of recurrence after treatment with IL-2, as compared to IL-2 treated BC without p16 hypermethylation (p = 0.02). Significant associations were observed between tumour grade and the mean methylation index (p = 0.003), as well as the hypermethylation of the RARbeta gene (p = 0.048). CONCLUSION: Our preliminary data suggest that DNA methylation biomarkers may assist in selection of BC patients for efficient IL-2 therapy.

Negative regulation of multifunctional Ca2+/calmodulin-dependent protein kinases: physiological and pharmacological significance of protein phosphatases.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) play pivotal roles in intracellular Ca2+ signaling pathways. There is growing evidence that CaMKs are involved in the pathogenic mechanisms underlying various human diseases. In this review, we begin by briefly summarizing our knowledge of the involvement of CaMKs in the pathogenesis of various diseases suggested to be caused by the dysfunction/dysregulation or aberrant expression of CaMKs. It is widely known that the activities of CaMKs are strictly regulated by protein phosphorylation/dephosphorylation of specific phosphorylation sites. Since phosphorylation status is balanced by protein kinases and protein phosphatases, the mechanism of dephosphorylation/deactivation of CaMKs, corresponding to their 'switching off', is extremely important, as is the mechanism of phosphorylation/activation corresponding to their 'switching on'. Therefore, we focus on the regulation of multifunctional CaMKs by protein phosphatases. We summarize the current understanding of negative regulation of CaMKs by protein phosphatases. We also discuss the biochemical properties and physiological significance of a protein phosphatase that we designated as Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP), and those of its homologue CaMKP-N. Pharmacological applications of CaMKP inhibitors are also discussed. These compounds may be useful not only for exploring the physiological functions of CaMKP/CaMKP-N, but also as novel chemotherapies for various diseases.

Roles of hippocampal N-methyl-D-aspartate receptors and calcium/calmodulin-dependent protein kinase II in amphetamine-produced conditioned place preference in rats.

This study investigates the roles of hippocampal N-methyl-D-aspartate (NMDA) glutamate receptors and CaMKII (calcium/calmodulin-dependent protein kinase II) in amphetamine-produced conditioned place preference (AMPH-CPP) in rats. An earlier report showed that AMPH-CPP resulted in the enhancement of hippocampal CaMKII activity. In this study, AMPH-CPP significantly increased hippocampal GluR1 receptors, though AMPH-CPP was impaired by either blockade of NMDA receptors (AP5) or inhibition of CaMKII (KN-93) during conditioning. These treatments also impaired CPP if administered before testing, but CPP recovered during the next testing session. Therefore, these treatments had no effect on the extinction of CPP. If the conditioned rats were, however, reexposed to AMPH-CPP after a hippocampal-infusion of AP5 or KN-93, the extinction of the original CPP was greater than that seen in the controls. The hippocampal-infusion of D-cycloserine before CPP testing enhanced the extinction of CPP. These results, taken together, indicate that NMDA receptor activation and CaMKII activity are essential for the AMPH-CPP. AMPH-CPP reexposure is similar to the memory reconsolidation process, being disrupted by either a blockade of the NMDA receptor or an inhibition of CaMKII. Furthermore, the extinction of CPP resembles new learning, which is an active process and is facilitated by a partial NMDA agonist.

Distinct developmental expression of two isoforms of Ca2+/calmodulin-dependent protein kinase kinases and their involvement in hippocampal dendritic formation.

Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) are upstream protein kinases that phosphorylate and activate CaMKI and CaMKIV, both of which are involved in a variety of neuronal functions. Here, we first demonstrated that the two isoforms of CaMKK were differentially expressed during neural development by in situ hybridization. We also demonstrated that both dominant negative and pharmacological interference with CaMKK inhibitor, STO-609 resulted in a significant decrease in the number of primary dendrites of cultured hippocampal neurons. Our present findings provide the detailed anatomical information on the developmental expression of CaMKKs and the functional involvement of CaMKK in the formation of primary dendrites.

alpha-CaMKII controls the growth of human osteosarcoma by regulating cell cycle progression.

Osteosarcoma is the most frequent type of primary bone cancer in children and adolescents. These malignant osteoid forming tumors are characterized by their uncontrolled hyperproliferation. Here, we investigate the role of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) in the growth of human osteosarcoma. We show that alpha-CaMKII is expressed in human osteosarcoma cell lines and in primary osteosarcoma tissue derived from patients. The pharmacologic inhibition of CaMKII in MG-63 and 143B human osteosarcoma cells by KN-93 resulted in an 80 and 70% decrease in proliferation, respectively, and induced cell cycle arrest in the G(0)/G(1) phase. The in vivo administration of KN-93 to mice xenografted with human osteosarcoma cells significantly decreased intratibial and subcutaneous tumor growth. Mechanistically, KN-93 and alpha-CaMKII siRNA increased p21((CIP/KIP)) gene expression, protein levels, and decreased the phosphorylation of retinoblastoma protein and E2F transactivation. Furthermore, the inhibition of CaMKII decreased membrane-bound Tiam1 and GTP-bound Rac1, which are known to be involved in p21 expression and tumor growth in a variety of solid malignant neoplasms. Our results suggest that CaMKII plays a critical role in the growth of osteosarcoma, and its inhibition could be an attractive therapeutic target to combat conventional high-grade osteosarcoma in children.

Estrogen-modulated frontal cortical CaMKII activity and behavioral supersensitization induced by prolonged cocaine treatment in female rats.

RATIONALE: Females have been demonstrated repeatedly to be more sensitive to cocaine. The role of the frontal cortex (FCX) in mediating behavioral sensitization and the underlying signaling pathways are unclear. OBJECTIVE: The study was designed to characterize the role of FCX calcium/calmodulin-dependent protein kinase II (CaMKII) activity in the behavioral supersensitization observed in female rats after prolonged cocaine exposure. MATERIALS AND METHODS: Intact female rats that received cocaine for 9 days followed by 7 days of drug withdrawal constituted the model used for studying the mechanism of supersensitization. RESULTS: This cocaine withdrawal treatment resulted in behavioral supersensitization in intact female rats as indicated by an enhanced behavioral response to cocaine challenge assessed on day 16 (7-day withdrawal) and compared to the response on day 9 of cocaine treatment. This treatment regimen did not lead to supersensitization in male or in ovariectomized (OVX) rats. Administration of estrogen to OVX rats restored behavioral supersensitivity to repeated cocaine. FCX CaMKII activity was significantly altered by cocaine in females, and this effect was related to estrogen's presence; cocaine-induced changes in striatal CaMKII activity were, however, less estrogen-sensitive. Furthermore, estrogen-modulated FCX CaMKII activity in cocaine-supersensitized rats was dependent on D(1) dopamine receptor activation. CONCLUSION: Estrogen-modulated D(1) dopamine receptor activity mediates the effects of prolonged cocaine exposure on FCX CaMKII, and this, in turn, may contribute to the development of behavioral supersensitivity to repeated cocaine treatment in intact female rats.

Impaired long-term depression in P2X3 deficient mice is not associated with a spatial learning deficit.

The hippocampus is a brain region critical for learning and memory processes believed to result from long-lasting changes in the function and structure of synapses. Recent findings suggest that ATP functions as a neurotransmitter or neuromodulator in the mammalian brain, where it activates several different types of ionotropic and G protein-coupled ATP receptors that transduce calcium signals. However, the roles of specific ATP receptors in synaptic plasticity have not been established. Here we show that mice lacking the P2X3 ATP receptor (P2X3KO mice) exhibit abnormalities in hippocampal synaptic plasticity that can be restored by pharmacological modification of calcium-sensitive kinase and phosphatase activities. Calcium imaging studies revealed an attenuated calcium response to ATP in hippocampal neurons from P2X3KO mice. Basal synaptic transmission, paired-pulse facilitation and long-term potentiation are normal at synapses in hippocampal slices from P2X3KO. However, long-term depression is severely impaired at CA1, CA3 and dentate gyrus synapses. Long-term depression can be partially rescued in slices treated with a protein phosphatase 1-2 A activator or by postsynaptic inhibition of calcium/calmodulin-dependent protein kinase II. Despite the deficit in hippocampal long-term depression, P2X3KO mice performed normally in water maze tests of spatial learning, suggesting that long-term depression is not critical for this type of hippocampus-dependent learning and memory.

Dual regulation of translation initiation and peptide chain elongation during BDNF-induced LTP in vivo: evidence for compartment-specific translation control.

Protein synthesis underlying activity-dependent synaptic plasticity is controlled at the level of mRNA translation. We examined the dynamics and spatial regulation of two key translation factors, eukaryotic initiation factor 4E (eIF4E) and elongation factor-2 (eEF2), during long-term potentiation (LTP) induced by local infusion of brain-derived neurotrophic factor (BDNF) into the dentate gyrus of anesthetized rats. BDNF-induced LTP led to rapid, transient phosphorylation of eIF4E and eEF2, and enhanced expression of eIF4E protein in dentate gyrus homogenates. Infusion of the extracellular signal-regulated kinase (ERK) inhibitor U0126 blocked BDNF-LTP and modulation of the translation factor activity and expression. Quantitative immunohistochemical analysis revealed enhanced staining of phospho-eIF4E and total eIF4E in dentate granule cells. The in vitro synaptodendrosome preparation was used to isolate the synaptic effects of BDNF in the dentate gyrus. BDNF treatment of synaptodendrosomes elicited rapid, transient phosphorylation of eIF4E paralleled by enhanced expression of alpha-calcium/calmodulin-dependent protein kinase II. In contrast, BDNF had no effect on eEF2 phosphorylation state in synaptodendrosomes. The results demonstrate rapid ERK-dependent regulation of the initiation and elongation steps of protein synthesis during BDNF-LTP in vivo. Furthermore, the results suggest a compartment-specific regulation in which initiation is selectively enhanced by BDNF at synapses, while both initiation and elongation are modulated at non-synaptic sites.

[Ras-MAPK signaling pathway activated via brain-derived neurotrophic factor].

Brain-derived neurotrophic factor (BDNF)is the richest neurophin in brain tissue and may act as an activity-dependent neuronal survival factor. In vitro, BDNF plays an important role in preventing cortical neurons from hypoxia-induced neurotoxicity. It could induce a variety of cellular responses such as cell growth, survival, differentiation, and anti-apoptosis mainly via activating mitogen-activated protein kinase (MAPK) and Ca2+/calmodulin-dependent kinase (CaMK) signaling pathways. And among these multiple signaling pathways there is growing evidence of complicated cross talk.

Leptin facilitates learning and memory performance and enhances hippocampal CA1 long-term potentiation and CaMK II phosphorylation in rats.

Leptin, an adipocytokine encoded by an obesity gene and expressed in adipose tissue, affects feeding behavior, thermogenesis, and neuroendocrine status via leptin receptors distributed in the brain, especially in the hypothalamus. Leptin may also modulate the synaptic plasticity and behavioral performance related to learning and memory since: leptin receptors are found in the hippocampus, and both leptin and its receptor share structural and functional similarities with the interleukin-6 family of cytokines that modulate long-term potentiation (LTP) in the hippocampus. We therefore examined the effect of leptin on (1) behavioral performance in emotional and spatial learning tasks, (2) LTP at Schaffer collateral-CA1 synapses, (3) presynaptic and postsynaptic activities in hippocampal CA1 neurons, (4) the intracellular Ca(2+) concentration ([Ca(2+)](i)) in CA1 neurons, and (5) the activity of Ca(2+)/calmodulin protein kinase II (CaMK II) in the hippocampal CA1 tissue that exhibits LTP. Intravenous injection of 5 and/or 50mug/kg, but not of 500mug/kg leptin, facilitated behavioral performance in passive avoidance and Morris water-maze tasks. Bath application of 10(-12)M leptin in slice experiments enhanced LTP and increased the presynaptic transmitter release, whereas 10(-10)M leptin suppressed LTP and reduced the postsynaptic receptor sensitivity to N-methyl-d-aspartic acid. The increase in the [Ca(2+)](i) induced by 10(-10)M leptin was two times greater than that induced by 10(-12)M leptin. In addition, the facilitation (10(-12)M) and suppression (10(-10)M) of LTP by leptin was closely associated with an increase and decrease in Ca(2+)-independent activity of CaMK II. Our results show that leptin not only affects hypothalamic functions (such as feeding, thermogenesis, and neuroendocrine status), but also modulates higher nervous functions, such as the behavioral performance related to learning and memory and hippocampal synaptic plasticity.

Molecular studies on the protective effect of nicotine in adult-onset hypothyroidism-induced impairment of long-term potentiation.

We have recently shown that chronic nicotine treatment reverses hypothyroidism-induced learning and memory impairment. Chronic nicotine treatment also reverses the hypothyroidism-induced impairment of long-term potentiation (LTP). Analysis of LTP associated key signaling molecules revealed that chronic nicotine treatment prevented the hypothyroidism-induced reduction of the basal phosphotransferase activity of CaMKII and protein levels of P-CaMKII. In addition, the failure of high frequency stimulation to increase the levels of P-CaMKII in hypothyroid rats was reversed by nicotine treatment, suggesting that the neuroprotective effect of nicotine during hypothyroidism involved activation of CaMKII. Furthermore, chronic nicotine treatment reverses the hypothyroidism-induced elevated phosphatase activity and protein levels of calcineurin, a phosphatase that regulates CaMKII activation. We conclude that the neuroprotective effects of nicotine in adult-onset hypothyroidism may result from restoration of CaMKII and calcineurin activity.

Lithium-induced activation of Akt and CaM kinase II contributes to its neuroprotective action in a rat microsphere embolism model.

Lithium used in bipolar mood disorder therapy protects neurons from brain ischemic cell death. Here, we documented that lithium administration under microsphere-embolism (ME)-induced brain ischemia restored decreased protein kinase B (Akt) and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) activities 24 h after ischemia in rat brain. Akt activation was associated with increased phosphorylation of its potential targets forkhead transcription factor (FKHR) and glycogen synthase kinase-3beta (GSK-3beta). In parallel with decreased CaMKII autophosphorylation, we also found marked dephosphorylation of tau proteins 24-72 h after ME. Increased protein phosphatase 2A (PP2A) activity was found 24 h after ME. Inhibition of increased PP2A activity by lithium treatment apparently mediated restored tau phosphorylation. Taken together, activation of Akt and CaMKII by lithium was associated with neuroprotective activity in ME-induced neuronal injury.

Effects of neonatal dexamethasone treatment on hippocampal synaptic function.

OBJECTIVE: Synthetic glucocorticoid dexamethasone (DEX) is frequently used as a therapeutic agent to lessen the morbidity of chronic lung disease in premature infants. Surprisingly, little is known about the long-term neurodevelopmental outcomes of this therapy. METHODS: Using a schedule of tapering doses of DEX similar to that used in premature infants, we examined the consequences of neonatal DEX treatment on hippocampal synaptic plasticity of infants and associative memory later in their lives. RESULTS: Neonatal DEX treatment changed the direction of synaptic plasticity, favoring low-frequency, stimulation-induced, long-term depression and opposing the induction of long-term potentiation by high-frequency stimulation in adolescent (5-week-old) rats, but these alterations disappeared in young adult (8-week-old) rats. The effects of DEX on long-term depression and long-term potentiation were found to correlate with an increase in the autophosphorylation of Ca(2+)/calmodulin-dependent protein kinase II and a decrease in the protein phosphatase 1 activity. Neonatal DEX treatment also disrupted memory retention in 5-week-old (but not 8-week-old) rats subjected to passive avoidance learning tasks. INTERPRETATION: These results suggest that neonatal DEX treatment alters hippocampal synaptic plasticity and contextual fear memory formation in later life, but these impairments apparently are not permanent.

Inhibition of protein synthesis by activation of NMDA receptors in cultured retinal cells: a new mechanism for the regulation of nitric oxide production.

The synthesis of nitric oxide (NO) is limited by the intracellular availability of L-arginine. Here we show that stimulation of NMDA receptors promotes an increase of intracellular L-arginine which supports an increase in the production of NO. Although L-[3H]arginine uptake measured in cultured chick retina cells incubated in the presence of cycloheximide (CHX, a protein synthesis inhibitor) was inhibited approximately 75% at equilibrium, quantitative thin-layer chromatography analysis showed that free intracellular L-[3H]arginine was six times higher in CHX-treated than in control cultures. Extracellular L-[3H]citrulline levels increased threefold in CHX-treated groups, an effect blocked by NG-nitro-L-arginine, a NO synthase (NOS) inhibitor. NMDA promoted a 40% increase of free intracellular L-[3H]arginine in control cultures, an effect blocked by the NMDA antagonist 2-amino 5-phosphonovaleric acid. In parallel, NMDA promoted a reduction of 40-50% in the incorporation of 35[S]methionine or L-[3H]arginine into proteins. Western blot analysis revealed that NMDA stimulates the phosphorylation of eukaryotic elongation factor 2 (eEF2, a factor involved in protein translation), an effect inhibited by (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imine maleate (MK801). In conclusion, we have shown that the stimulation of NMDA receptors promotes an inhibition of protein synthesis and a consequent increase of an intracellular L-arginine pool available for the synthesis of NO. This effect seems to be mediated by activation of eEF2 kinase, a calcium/calmodulin-dependent enzyme which specifically phosphorylates and blocks eEF2. The results raise the possibility that NMDA receptor activation stimulates two different calmodulin-dependent enzymes (eEF2 kinase and NOS) reinforcing local NO production by increasing precursor availability together with NOS catalytic activity.

Chronic psychosocial stress-induced impairment of hippocampal LTP: possible role of BDNF.

Electrophysiological recording reveals that chronic nicotine treatment prevents stress-induced impairment of long-term potentiation (LTP) in the CA1 region of the hippocampus of anesthetized rats. We investigated the molecular mechanism of this action of nicotine in the CA1 region. Immunoblot analysis showed that chronic nicotine treatment (1 mg/kg, 2 times/day) normalized the stress-induced decrease in the basal levels of BDNF, CaMKII (total and phosphorylated; P-CaMKII), and calmodulin. Additionally, nicotine reversed the stress-induced increase in calcineurin basal levels. Chronic nicotine treatment also markedly increased the basal levels of BDNF in naïve rats. Furthermore, high-frequency stimulation (HFS), which increased the levels of P-CaMKII in control as well as nicotine-treated stressed rats, failed to increase P-CaMKII levels in untreated stressed rats. Compared to unstimulated control, the levels of both total CaMKII and calcineurin were increased after HFS in all groups including the stressed, but no changes were detected after HFS in the levels of BDNF and calmodulin. These results indicate that normalization by nicotine of the stress-induced changes in the levels of signaling molecules including BDNF may contribute to the recovery of LTP.

Tobacco cembranoids protect the function of acute hippocampal slices against NMDA by a mechanism mediated by alpha4beta2 nicotinic receptors.

Nicotine has been reported to be neuroprotective in experimental and epidemiological studies. In addition to nicotine, tobacco and cigarette smoke contain cembranoids, which are antagonists of neuronal nicotinic receptors (nAChR). Exposure of hippocampal slices to N-methyl-D-aspartate (NMDA) decreases the population spikes (PS). This parameter has been used as a measure of excitotoxicity. Surprisingly, both nicotine and tobacco cembranoids protected against NMDA and this neuroprotection was not blocked by methyllycaconitine (MLA), an antagonist of alpha7 nAChR. On the contrary, MLA had a neuroprotective effect of its own. We examined the effect of the tobacco cembranoid (1S,2E,4R,6R,7E,11E)-cembra-2,7,11-triene-4,6-diol (4R) on the neuroprotection against NMDA. DHbetaE, a selective antagonist of alpha4beta2 nAChR, inhibited the neuroprotection by nicotine, 4R, and MLA, suggesting the involvement of alpha4beta2 nAChRs in the neuroprotection. The cell-signaling pathways underlying the neuroprotection by 4R and by nicotine are different. The activity of phosphatidylinositol-3 kinase (PI3K) was required in both cases; however, 4R required the activity of L-type calcium channels and CAM kinase, whereas nicotine required the extracellular signal regulated kinase-1,2 (ERK) and protein kinase C (PKC). In addition, 4R did not enhance total phospho-ERK-1/2 but increased the amount of total Akt/PKB phosphorylated on the activation site and of glycogen synthase kinase 3-beta phosphorylated on the inhibitory site. Total levels of phosphoenzymes are presented instead of the ratio of phospho- over total enzyme because in preliminary experiments total ERK-1/2 levels were slightly increased by 4R. In conclusion, these findings demonstrate that there are two different nicotinic neuroprotective mechanisms mediated by alpha4beta2.

The role of regulatory domain interactions in UNC-43 CaMKII localization and trafficking.

Calcium and calmodulin-dependent protein kinase II (CaMKII) plays a fundamental role in the synaptic plasticity events that underlie learning and memory. Regulation of CaMKII kinase activity occurs through an autoinhibitory mechanism in which a regulatory domain of the kinase occupies the catalytic site and calcium/calmodulin activates the kinase by binding to and displacing this regulatory domain. A single putative ortholog of CaMKII, encoded by unc-43, is present in the Caenorhabditis elegans nervous system. Here we examined UNC-43 subcellular localization in the neurons of intact animals and show that UNC-43 is localized to clusters in ventral cord neurites, as well as to an unlocalized pool within these neurites. A mutation that mimics autophosphorylation within the regulatory domain results in an increase in the levels of UNC-43 in the unlocalized neurite pool. Multiple residues of CaMKII facilitate the interaction between the catalytic domain and the regulatory domain, thereby keeping the kinase inactive. Whereas most mutations in these residues result in an increased neurite pool of UNC-43, we have identified two residues that result in the opposite effect when mutated: a decreased neurite pool of UNC-43. The activity of UNC-2, a voltage-dependent calcium channel, is also required for UNC-43 to accumulate in the neurites, suggesting that neural activity regulates the localization of UNC-43. Our results suggest that the activation of UNC-43 by calcium/calmodulin displaces the autoinhibitory domain, thereby exposing key residues of the catalytic domain that allow for protein translocation to the neurites.

Effect of post-hypoxic MgSO(4) administration in utero on Ca(2+)-influx and Ca(2+)/calmodulin kinase IV activity in cortical neuronal nuclei.

Previously we have demonstrated that in utero hypoxia results in increased nuclear Ca(2+)-influx and increased CaM kinase IV activity in neuronal nuclei of the guinea pig fetus. The present study tests the hypothesis that maternal treatment with magnesium sulfate (MgSO(4)) following in utero hypoxia will attenuate the hypoxia-induced increase in Ca(2+)-influx and CaM kinase IV activity in neuronal nuclei of the fetal guinea pig brain during recovery. Pregnant guinea pigs at 60 days of gestation were divided into four groups: normoxic (Nx=5), hypoxic (Hx, n=4), untreated post-hypoxic 24h recovery (Rec, n=8) and Mg(2+)-treated post-hypoxic 24h recovery (Mg(2+)-Rec, n=8). Maternal hypoxia was induced by decreasing FiO(2) to 8% for 1h. Recovery groups received either saline or 300 mg/kg MgSO(4) (i.p.) followed by 100mg/kg/h i.p. for 4h. Fetal cerebral tissue hypoxia was documented by ATP and phosphcreatine (PCr) levels. Neuronal nuclei were isolated and nuclear Ca(2+)-influx as well as CaM kinase activity was determined. Nuclear Ca(2+) influx (pmol/mg protein) was 4.84+/-0.83 in Nx, 12.50+/-2.97 (p<0.05) in Hx, 7.83+/-1.78 in Rec group (p<0.05 versus Nx and Hx) and 5.02+/-1.77 in Mg(2+)-Rec group (p<0.05 versus Rec group, p<0.05 versus Hx, p=NS versus Nx). CaM kinase IV activity (pmol/mg protein/min) was 1197+/-62 in Nx, 2524+/-132 (p<0.05 versus Nx) in Hx, 1830+/-141 (p<0.05 versus Nx and Hx) in Rec and 1938+/-118 in Mg(2+)-Rec group (p<0.05 versus Hx and Nx, p=n.s. versus Rec). The data show that MgSO(4) administration following in utero hypoxia prevents hypoxia-induced increase in neuronal nuclear Ca(2+)-influx but has no effect on CaM kinase activity in the guinea pig fetus during recovery. We conclude that post-hypoxic administration of Mg(2+) prevents hypoxia-induced modification of neuronal nuclear membrane function.