Inhibitor 1 of Protein Phosphatase 1 Regulates Ca2+/Calmodulin-Dependent Protein Kinase II to Alleviate Oxidative Stress in Hypoxia-Reoxygenation Injury of Cardiomyocytes.

Ca2+/calmodulin-dependent protein kinase II (CaMKII), regulated by inhibitor 1 of protein phosphatase 1 (I1PP1), is vital for maintaining cardiovascular homeostasis. However, the role and mechanism of I1PP1 against hypoxia-reoxygenation (H/R) injury in cardiomyocytes remain a question. In our study, after I1PP1 overexpression by adenovirus infection in the neonatal cardiomyocytes followed by hypoxia for 4 h and reoxygenation for 12 h, the CaMKIIδ alternative splicing subtype, ATP content, and lactate dehydrogenase (LDH) release were determined. CaMKII activity was evaluated by phosphoprotein phosphorylation at Thr17 (p-PLB Thr17), CaMKII phosphorylation (p-CaMKII), and CaMKII oxidation (ox-CaMKII). Reactive oxygen species (ROS), mitochondrial membrane potential, dynamin-related protein 1 (DRP1), and optic atrophy 1 (OPA1) expressions were assessed. Our study verified that I1PP1 overexpression attenuated the CaMKIIδ alternative splicing disorder; suppressed PLB phosphorylation at Thr17, p-CaMKII, and ox-CaMKII; decreased cell LDH release; increased ATP content; attenuated ROS production; increased mitochondrial membrane potential; and decreased DRP1 expression but increased OPA1 expression in the cardiomyocytes after H/R. Contrarily, CaMKIIδ alternative splicing disorder, LDH release, ATP reduction, and ROS accumulation were aggravated after H/R injury with the I1PP1 knockdown. Collectively, I1PP1 overexpression corrected disorders of CaMKIIδ alternative splicing, inhibited CaMKII phosphorylation, repressed CaMKII oxidation, suppressed ROS production, and attenuated cardiomyocyte H/R injury.

Prostaglandin E2 inhibits profibrotic function of human pulmonary fibroblasts by disrupting Ca2+ signaling.

We have shown that calcium (Ca2+) oscillations in human pulmonary fibroblasts (HPFs) contribute to profibrotic effects of transforming growth factor-ß (TGF-ß) and that disruption of these oscillations blunts features of pulmonary fibrosis. Prostaglandin E2 (PGE2) exerts antifibrotic effects in the lung, but the mechanisms for this action are not well defined. We thus sought to explore interactions between PGE2 and the profibrotic agent TGF-ß in pulmonary fibroblasts (PFs) isolated from patients with or without idiopathic pulmonary fibrosis (IPF). PGE2 inhibited TGF-ß-promoted [Ca2+] oscillations and prevented the activation of Akt and Ca2+/calmodulin-dependent protein kinase-II (CaMK-II) but did not prevent activation of Smad-2 or ERK. PGE2 also eliminated TGF-ß-stimulated expression of collagen A1, fibronectin, and α-smooth muscle actin and reduced stress fiber formation in the HPFs. RNA sequencing revealed that HPFs preferentially express EP2 receptors relative to other prostanoid receptor subtypes: EP2 expression is ~10-fold higher than that of EP4 receptors; EP1 and EP3 receptors are barely detectable; and EP2-receptor expression is ~3.5-fold lower in PFs from IPF patients than in normal HPFs. The inhibitory effects of PGE2 on synthetic function and stress fiber formation were blocked by selective EP2 or EP4 antagonists and mimicked by selective EP2 or EP4 agonists, the phosphodiesterase inhibitor isobutylmethylxanthine and forskolin, all of which elevate cellular cAMP concentrations. We conclude that PGE2, likely predominantly via EP2 receptors, interferes with Ca2+ signaling, CaMK-II activation, and Akt activation in IPF-HPFs and HPFs treated with TGF-ß. Moreover, a decreased expression of EP2 receptors in pulmonary fibroblasts from IPF patients may contribute to the pathophysiology of this disease.

Inhibition of miR-219 Alleviates Arsenic-Induced Learning and Memory Impairments and Synaptic Damage Through Up-regulating CaMKII in the Hippocampus.

Epidemiological investigations and experimental studies indicate that chronic arsenic exposure can reduce learning and memory function. However, the underlying mechanism of this effect remains largely unknown. Emerging evidence suggests that microRNA (miRNA) play an important role in toxicant exposure and a regulatory role in cognitive function. In this study, we observed that subchronic arsenic exposure induced impairment of learning and memory and significantly up-regulated miRNA-219 (miR-219) expression in the mouse hippocampus. Furthermore, the expression of CaMKII, an experimentally validated target of miR-219, was decreased in the mice exposed to arsenic. Suppression of miR-219 by adeno-associated viral (AAV)-delivered anti-miR-219 prevented the arsenic-induced impairment of learning and memory and relieved the pathological changes in the synaptic structure of the hippocampus. Furthermore, we observed that the NMDA receptor subunit 2 (NR2) and the memory-related proteins c-Fos and c-Jun were up-regulated by inhibition of miR-219 in the mouse hippocampus. Taken together, the results of this study indicate that inhibition of miR-219 regulates arsenic-induced damage in the structure of the hippocampus and impairment of learning and memory, possibly by targeting CaMKII. Suppression of miR-219 may be a potential strategy to ameliorate arsenic-induced neurotoxicity.

In vitro neurotoxicity by ropivacaine is reduced by silencing Cav3.3 T-type calcium subunits in neonatal rat sensory neurons.

Neurotoxicity of local anaesthetics has been alerted by more and more peoples. Cav3.1 and Cav3.2 T-type calcium channels were closely related with local anaesthetics toxicity. However, the role of Cav3.3, another subtype of the T-type calcium channel, on the neurotoxicity induced by local anaesthetics remains unclear. CaMKIIγ is a kind of multifunctional kinase and associated with a variety of physiological and pathological process. T-type calcium channel is closely related with CaMKIIγ. Up-regulation CaMKIIγ can increase T-type currents at the dorsal root ganglia (DRG). On the contrary, down-regulation results in the T-type currents decrease. Is the relation between Cav3.3 T-type channel calcium and CaMKIIγ involved with the ropivacaine hydrochloride neurotoxicity? In this study, we generated pAd-Cav3.3 and pAd-shRNA adenovirus vector to up-regulate and down-regulate Cav3.3 mRNA expression of the DRG. The cells treated or untreated with ropivacaine hydrochloride (3 mM) for 4 h were used to evaluate the neurotoxicity. Cell viability, cell death rate and apoptosis rate, Cav3.3 and CaMKIIγ expression were detected with MTT method, Hoechst-PI, flow cytometry, qRT-PCR and western blotting. Results showed that the cell viability of the DRG treated with ropivacaine hydrochloride markedly decreased, death rate and apoptosis rate, Cav3.3 and CaMKIIγ mRNA and protein expression significantly increased. Cav3.3 overexpression aggravated DRG injury induced by ropivacaine hydrochloride and inhibition of Cav3.3 expression improved the cell damages. Cav3.3 can regulate CaMKIIγ mRNA and protein expression. In conclusion, Cav3.3 regulated CaMKIIγ in DRG, which was involved with the cell injury induced by ropivacaine hydrochloride.

Fingolimod (FTY720) attenuates social deficits, learning and memory impairments, neuronal loss and neuroinflammation in the rat model of autism.

AIMS: To investigate the effect of FTY720 on the valproic acid (VPA) rat model of autism. MAIN METHODS: As an animal model of autism, we used intraperitoneal injection of VPA on embryonic day 12.5 in Wistar rats. The pups were given FTY720 orally at doses of 0.25, 0.5 and 1mg/kg daily from postnatal day 15 to 35. Social behavior, spatial learning and memory were assessed at the end of FTY720 treatment. The histological change, oxidative stress, neuroinflammatory responses, and apoptosis-related proteins in the hippocampus were evaluated. KEY FINDINGS: FTY720 (1mg/kg) administration to VPA-exposed rats (1) improved social behavior, spatial learning and memory impairment; (2) resulted in a reduction in neuronal loss and apoptosis of pyramidal cells in hippocampal CA1 regions; (3) inhibited activation of microglial cells, in turn lowering the level of pro-inflammatory cytokines interleukin-1ß (IL-1ß) and IL-6 in the hippocampus; (4) changed Malondialdehyde (MDA) levels, Glutathione (GSH) levels, superoxide dismutase (SOD) activity and Glutathione Peroxidase (GSH-Px) activity in the hippocampus; (6) inhibited the elevated Bax and caspase-3 protein levels and enhanced the relative expression level of Bcl-2 in the hippocampus; and (7) increased phospho-Ca2+/calmodulin-dependent protein kinase II (p-CaMKII), phospho-cAMP-response element binding protein (p-CREB) and Brain Derived Neurotrophic Factor (BDNF) protein expression in the hippocampus. SIGNIFICANCE: FTY720 rescues social deficit, spatial learning and memory impairment in VPA-exposed rats. FTY720 exerts both a direct protection for neurons and an indirect modulation of inflammation-mediated neuron loss as a possible mechanism of neuroprotection.

One cell model establishment to inhibit CaMKIIγ mRNA expression in the dorsal root ganglion neuron by RNA interfere.

CaMKIIγ in dorsal root ganglion neurons is closely related to the neuropathic pain, neuron injury induced by local anesthetics. To get great insight into the function of CaMKIIγ in dorsal root ganglion neurons, we need one cell model to specially inhibit the CaMKIIγ mRNA expression. The present study was aimed to establish one cell model to specially inhibit the CaMKIIγ mRNA expression. We designed the CaMKIIγ shRNA sequence and connected with pYr-1.1 plasmid. The ligation product of the CaMKIIγshRNA and pYr-1.1 plasmid was recombined with pAd/PL-DEST vector into pAD-CaMKIIγ-shRNA. adenovirus vector. pAD-CaMKIIγ-shRNA. adenovirus vector infected the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA expression in vitro. The pAD-CaMKIIγ-shRNA adenovirus vector was verified to be correct by the digestion, sequence. And pAD-CaMKIIγ-shRNA. adenovirus vector can infect the DRG cells to inhibit the CaMKIIγ mRNA or protein expression by the real-time polymerase chain reaction (PCR) or western blotting. Those results showed that we successfully constructed one adenovirus vector that can infect the dorsal root ganglion neuron to inhibit the CaMKIIγ mRNA and protein expression. That will supply with one cell model for the CaMKIIγ function study.

Bryonolic Acid, a Triterpenoid, Protect Against N-methyl-d-Aspartate-Induced Neurotoxicity in PC12 Cells.

Calcium overload is considered to be one of the mechanisms of cerebral ischemia. Ca(2+) influx and Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and cAMP response element-binding protein (CREB) phosphorylation are considered to be involved in N-Methyl-d-aspartate (NMDA)-induced apoptosis process. This study investigated the neuroprotective effects of bryonolic acid (BA) in an NMDA-induced rat adrenal pheochromocytoma cell line (PC12) cells and the potential mechanism. PC12 was treated by NMDA to establish an excitotoxicity model. BA (110,100 and 1000 µM final concentration) was added to the medium 24 h prior to the addition of NMDA. Subsequently, a methyl thiazolyl tetrazolium (MTT) assay and a lactate dehydrogenase (LDH) release were performed. Ca(2+) concentration was demonstrated using a scanning-dual wavelength fluorimetric method. In addition, protein and mRNA levels were determined via Western blot and real-time PCR. In the presence of BA, MTT assay and LDH assay showed that more cells were viable in comparison with the NMDA group. Moreover, the concentration of Ca(2+) decreased with the addition of BA in culture. Furthermore, BA could upregulate protein expressions of Bcl-2, p-CREB, and p-CaMKII and downregulate protein expression of Bax. The mRNA results showed that the pattern of mRNA expression were similar to their respective protein levels. All these results indicate that BA protected PC12 cells against NMDA-induced apoptosis by inhibiting Ca(2+) influx and regulating gene expression in the Ca(2+)-CaMKII-CREB signal pathway. Therefore, the present study supports the notion that BA may be a promising neuroprotective agent for the treatment of cerebral ischemia disease.

Effects of acute and chronic sunitinib treatment on cardiac function and calcium/calmodulin-dependent protein kinase II.

BACKGROUND AND PURPOSE: Calcium/calmodulin-dependent protein kinase IIδ (CaMKIIδ) is an important regulator of cardiac contractile function and dysfunction and may be an unwanted secondary target for anti-cancer drugs such as sunitinib and imatinib that have been reported to alter cardiac performance. This study aimed to determine whether anti-cancer kinase inhibitors may affect CaMKII activity and expression when administered in vivo. EXPERIMENTAL APPROACH: Cardiovascular haemodynamics in response to acute and chronic sunitinib treatment, and chronic imatinib treatment, were assessed in guinea pigs and the effects compared with those of the known positive and negative inotropes, isoprenaline and verapamil. Parallel studies from the same animals assessed CaMKIIδ expression and CaMKII activity following drug treatments. KEY RESULTS: Acute administration of sunitinib decreased left ventricular (LV) dP/dtmax. Acute administration of isoprenaline increased LVdP/dtmax dose-dependently, while LVdP/dtmax was decreased by verapamil. CaMKII activity was decreased by acute administration of sunitinib and was increased by acute administration of isoprenaline, and decreased by acute administration of verapamil. CaMKIIδ expression following all acute treatments remained unchanged. Chronic imatinib and sunitinib treatments did not alter fractional shortening; however, both CaMKIIδ expression and CaMKII activity were significantly increased. Chronic administration of isoprenaline and verapamil decreased LV fractional shortening with parallel increases in CaMKIIδ expression and CaMKII activity. CONCLUSIONS AND IMPLICATIONS: Chronic sunitinib and imatinib treatment increased CaMKIIδ expression and CaMKII activity. As these compounds are associated with cardiac dysfunction, increased CaMKII expression could be an early indication of cellular cardiotoxicity marking potential progression of cardiac contractile dysfunction.

ROS-Induced Nuclear Translocation of Calpain-2 Facilitates Cardiomyocyte Apoptosis in Tail-Suspended Rats.

Isoproterenol (ISO) induced nuclear translocation of calpain-2 which further increased susceptibility of cardiomyocyte apoptosis in tail-suspended rats. The underlying mechanisms remain elusive. In the present study, the results showed that ISO (10 nM) significantly elevated NADPH oxidases (NOXs) activity and NOXs-derived ROS productions which induced nuclear translocation of calpain-2 in cardiomyocytes of tail-suspended rats. In contrast, the inhibition of NADPH oxidase or cleavage of ROS not only reduced ROS productions, but also resisted nuclear translocation of calpain-2 and decreased ISO-induced apoptosis of cardiomyocyte in tail-suspended rats. ISO also increased the constitutive binding between calpain-2 and Ca(2+)/calmodulin-dependent protein kinase II δB (CaMK II δB) in nuclei, concomitant with the promotion of CaMK II δB degradation and subsequent down-regulation of Bcl-2 mRNA expression and the ratio of Bcl-2 to Bax protein in tail-suspended rat cardiomyocytes. These effects of ISO on cardiomyocytes were abolished by a calpain inhibitor PD150606. Inhibition of calpain significantly reduced ISO-induced loss of the mitochondrial membrane potential, cytochrome c release into the cytoplasm, as well as the activation of caspase-3 and caspase-9 in mitochondrial apoptotic pathway. In summary, the above results suggest that ISO increased NOXs-derived ROS which activated nuclear translocation of calpain-2, subsequently nuclear calpain-2 degraded CaMK II δB which reduced the ratio of Bcl-2 to Bax, and finally the mitochondria apoptosis pathway was triggered in tail-suspended rat cardiomyocytes. Therefore, calpain-2 may represent a potentially therapeutic target for prevention of oxidative stress-associated cardiomyocyte apoptosis.

Omega-3 fatty acids prevent LPS-induced passive avoidance learning and memory and CaMKII-α gene expression impairments in hippocampus of rat.

BACKGROUND: Neuroinflammation is considered to be a major factor in several neurodegenerative diseases. Recently, the polyunsaturated fatty acid omega-3 has been shown to have anti-inflammatory effects and might play an effective role in improving memory impairment due to inflammation. In order to test this, we stimulated neuroinflammation in an animal model and induced memory dysfunction as measured by reduced retention of passive avoidance learning (PAL) and altered expression of CaMKII-α, a gene known to be crucial for memory formation. We then investigated whether treatment with dietary omega-3 prevents inflammation-induced memory dysfunction in this model. METHODS: Male wistar rats (200-220 g) were fed either a control diet or a diet containing omega-3 (400mg/kg, po) for 1 month prior. Rats then received injection of either saline or LPS (500 µg/kg, ip) and were subjected to the PAL acquisition task. The retention test was performed 24h later, and animals were sacrificed immediately. Hippocampi were dissected and stored at -80°C. Finally, TNF-α levels and CaMKII-α gene expression were measured by ELISA and qRT-PCR, respectively. RESULTS: We found that LPS treatment significantly impaired PAL and memory, increased TNF-α levels and impaired CaMKII-α gene expression. In control and LPS-injected animals, pre-treatment with omega-3 improved performance on the PAL task and increased CAMKII-α gene expression. CONCLUSION: Taken together, these data suggest that dietary omega-3 may improve cognitive function and provide a potential therapy for memory impairment due to neuroinflammation.

Modification of hippocampal markers of synaptic plasticity by memantine in animal models of acute and repeated restraint stress: implications for memory and behavior.

Stress is any condition that impairs the balance of the organism physiologically or psychologically. The response to stress involves several neurohormonal consequences. Glutamate is the primary excitatory neurotransmitter in the central nervous system, and its release is increased by stress that predisposes to excitotoxicity in the brain. Memantine is an uncompetitive N-methyl D-aspartate glutamatergic receptors antagonist and has shown beneficial effect on cognitive function especially in Alzheimer's disease. The aim of the work was to investigate memantine effect on memory and behavior in animal models of acute and repeated restraint stress with the evaluation of serum markers of stress and the expression of hippocampal markers of synaptic plasticity. Forty-two male rats were divided into seven groups (six rats/group): control, acute restraint stress, acute restraint stress with Memantine, repeated restraint stress, repeated restraint stress with Memantine and Memantine groups (two subgroups as positive control). Spatial working memory and behavior were assessed by performance in Y-maze. We evaluated serum cortisol, tumor necrotic factor, interleukin-6 and hippocampal expression of brain-derived neurotrophic factor, synaptophysin and calcium-/calmodulin-dependent protein kinase II. Our results revealed that Memantine improved spatial working memory in repeated stress, decreased serum level of stress markers and modified the hippocampal synaptic plasticity markers in both patterns of stress exposure; in ARS, Memantine upregulated the expression of synaptophysin and brain-derived neurotrophic factor and downregulated the expression of calcium-/calmodulin-dependent protein kinase II, and in repeated restraint stress, it upregulated the expression of synaptophysin and downregulated calcium-/calmodulin-dependent protein kinase II expression.

Implications of the Wnt5a/CaMKII pathway in retinoic acid-induced myogenic tongue abnormalities of developing mice.

Although proper tongue development is relevant to other structures in the craniofacial region, the molecular details of muscle development in tongue remain poorly understood. Here, we report that pregnant mice treated with retinoic acid (+RA) produce embryos with tongue malformation and a cleft palate. Histological analyses revealed that at E14.5, the tongues of +RA fetuses failed to descend and flatten. Ultrastructural analysis showed that at perinatal stage E18.5, the myofilaments failed to form normal structures of sarcomeres, and arranged disorderly in the genioglossus. The proliferation and levels of myogenic determination markers (Myf5 and MyoD) and myosin in the genioglossus were profoundly reduced. Wnt5a and Camk2d expressions were down-regulated, while levels of Tbx1, Ror2, and PKCδ were up-regulated in the tongues of +RA fetuses. In mock- and Wnt5a-transfected C2C12 (Wnt5a-C2C12) cells, Wnt5a overexpression impaired proliferation, and maintained Myf5 at a relative high level after RA treatment. Furthermore, Wnt5a overexpression positively correlated with levels of Camk2d and Ror2 in C2C12 cells after RA exposure. These data support the hypothesis that the Wnt5a/CaMKII pathway is directly involved in RA-induced hypoplasia and disorder of tongue muscles.

Berbamine inhibits the growth of liver cancer cells and cancer-initiating cells by targeting Ca²âº/calmodulin-dependent protein kinase II.

Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here, we report that berbamine and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca(2+)/calmodulin-dependent protein kinase II (CAMKII). Furthermore, berbamine inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice and downregulated the self-renewal abilities of liver cancer-initiating cells. Chemical inhibition or short hairpin RNA-mediated knockdown of CAMKII recapitulated the effects of berbamine, whereas overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to berbamine treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peritumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of berbamine for liver cancer therapies. Our data suggest that berbamine and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII. Mol Cancer Ther; 12(10); 2067-77. ©2013 AACR.

Genome wide array analysis indicates that an amyotrophic lateral sclerosis mutation of FUS causes an early increase of CAMK2N2 in vitro.

Mutations in the RNA binding protein FUS (fused in sarcoma) have been linked to a subset of familial amyotrophic lateral sclerosis (ALS) cases. The mutations are clustered in the C-terminal nuclear localization sequence (NLS). Various FUS mutants accumulate in the cytoplasm whereas wild-type (WT) FUS is mainly nuclear. Here we investigate the effect of one ALS causing mutant (FUS-ΔNLS, also known as R495X) on pre-mRNA splicing and RNA expression using genome wide exon-junction arrays. Using a non-neuronal stable cell line with inducible FUS expression, we detected early changes in RNA composition. In particular, mutant FUS-ΔNLS increased calcium/calmodulin-dependent protein kinase II inhibitor 2 (CAMK2N2) at both mRNA and protein levels, whereas WT-FUS had no effect. Chromatin immunoprecipitation experiments showed that FUS-ΔNLS accumulated at the CAMK2N2 promoter region, whereas promoter occupation by WT-FUS remained constant. Given the loss of FUS-ΔNLS in the nucleus through the mutation-induced translocation, this increase of promoter occupancy is surprising. It indicates that, despite the obvious cytoplasmic accumulation, FUS-ΔNLS can act through a nuclear gain of function mechanism.

Calcium mediates high glucose-induced HIF-1α and VEGF expression in cultured rat retinal Müller cells through CaMKII-CREB pathway.

AIM: To investigate the effects of high glucose (HG) medium on expression of hypoxia-inducible factor-1α (HIF-1α) and vascular endothelial growth factor (VEGF) in cultured rat retinal Müller cells and to determine the signaling pathways mediating the effects. METHODS: Primary cultures of retinal Müller cells were prepared from Sprague-Dawley rats, and incubated in a medium containg HG (30 mmol/L) in the presence of the membrane-permeable Ca(2+) chelator BAPTA-AM (10 µmol/L) or the CaMKII inhibitor KN93 (10 µmol/L). The levels of CaMKII, p-CaMKII, CREB, p-CREB, HIF-1α, and VEGF proteins were measured with Western blotting, while HIF-1á and VEGF mRNA levels were determined using real-time RT-PCR. RESULTS: The stimulation of retinal Müller cell with HG for 24 h remarkably increased the expression levels of HIF-1α and VEGF. These responses were significantly inhibited in the presence of BAPTA-AM or KN93. Both BAPTA-AM and KN93 also significantly inhibited HG-induced phosphorylation of CaMKII and CREB in the cultured retinal Müller cells. Transfection of the cultured retinal Müller cells with antisense CREB oligonucleotide (300 nmol/L) was similarly effective in blocking the HG-induced increase of HIF-1α and VEGF. CONCLUSION: HG-induced HIF-1α and VEGF expression in cultured rat retinal Müller cells depends on intracellular free Ca(2+) and activation of CaMKII-CREB pathway. The activation of CaMKII-CREB pathway by HG may be a possible mechanism underlying the pathogenesis of diabetic retinopathy.

The differential profiles of withdrawal symptoms induced by morphine and beta-endorphin administered intracerebroventricularly in mice.

In the present study, withdrawal symptoms induced by morphine or ß-endorphin administered intracerebroventricularly (i.c.v.) were compared in ICR mice. Naloxone (10mg/kg) was post-treated intraperitoneally (i.p.) 3h after either a single or repeated (1 time/day for 3 days) i.c.v. injections with opioids. Withdrawal symptoms such as jumping frequency, diarrhea, weight loss, rearing, penile licking and paw tremor were observed for 30 min immediately after naloxone treatment. Withdrawal symptoms (jumping, diarrhea, weight loss, rearing, penile licking and paw tremor) observed in the group treated with morphine was persistently increased during 3 days. On the other hand, withdrawal symptoms such as diarrhea, weight loss and rearing in ß-endorphin-treated group were increased after a single injection with ß-endorphin, but gradually decreased after the repeated injection. Furthermore, no jumping behavior, penile licking and paw tremor in ß-endorphin-treated group were observed throughout the whole period of time. In addition, the hypothalamic changes of several signal molecules such as pERK, pCaMK-IIα, c-FOS and pCREB expression were observed during the presence or absence of withdrawal responses induced by morphine or ß-endorphin administered once or repeatedly. Both hypothalamic pCaMK-IIα and c-FOS expressions were increased by naloxone treatment in acutely administered morphine group, whereas only pCaMK-IIα expression was elevated by naloxone treatment in repeatedly administered morphine group. In contrast with the findings in morphine-treated group, only pCaMK-IIα expression was decreased by naloxone treatment in repeatedly administered ß-endorphin group. Our results suggest that profiles of the withdrawal symptoms induced by morphine and ß-endorphin administered supraspinally appear to be differentially regulated. The pCaMK-IIα and the c-FOS protein expression may play important roles for the regulation of naloxone-precipitated withdrawal symptoms such as jumping, diarrhea, weight loss, rearing, penile licking and paw tremor induced by morphine-treated group, whereas the phosphorylation of hypothalamic pCaMK-IIα appears to be involved only in the regulation of naloxone-precipitated withdrawal symptoms such as diarrhea, weight loss and rearing in ß-endorphin-treated group.

Visualizing long-term memory formation in two neurons of the Drosophila brain.

Long-term memory (LTM) depends on the synthesis of new proteins. Using a temperature-sensitive ribosome-inactivating toxin to acutely inhibit protein synthesis, we screened individual neurons making new proteins after olfactory associative conditioning in Drosophila. Surprisingly, LTM was impaired after inhibiting protein synthesis in two dorsal-anterior-lateral (DAL) neurons but not in the mushroom body (MB), which is considered the adult learning and memory center. Using a photoconvertible fluorescent protein KAEDE to report de novo protein synthesis, we have directly visualized cyclic adenosine monophosphate (cAMP) response element-binding protein (CREB)-dependent transcriptional activation of calcium/calmodulin-dependent protein kinase II and period genes in the DAL neurons after spaced but not massed training. Memory retention was impaired by blocking neural output in DAL during retrieval but not during acquisition or consolidation. These findings suggest an extra-MB memory circuit in Drosophila: LTM consolidation (MB to DAL), storage (DAL), and retrieval (DAL to MB).

Spatial memory is improved by aerobic and resistance exercise through divergent molecular mechanisms.

A growing body of scientific evidence indicates that exercise has a positive impact on human health, including neurological health. Aerobic exercise, which is supposed to enhance cardiovascular functions and metabolism, also induces neurotrophic factors that affect hippocampal neurons, thereby improving spatial learning and memory. Alternatively, little is known about the effect of resistance exercise on hippocampus-dependent memory, although this type of exercise is increasingly recommended to improve muscle strength and bone density and to prevent age-related disabilities. Therefore, we evaluated the effects of resistance training on spatial memory and the signaling pathways of brain-derived neurotrophic factor (BDNF) and insulin-like growth factor 1 (IGF-1), comparing these effects with those of aerobic exercise. Adult male Wistar rats underwent 8 weeks of aerobic training on a treadmill (AERO group) or resistance training on a vertical ladder (RES group). Control and sham groups were also included. After the training period, both AERO and RES groups showed improved learning and spatial memory in a similar manner. However, both groups presented distinct signaling pathways. Although the AERO group showed increased level of IGF-1, BDNF, TrkB, and ß-CaMKII (calcium/calmodulin-dependent kinase II) in the hippocampus, the RES group showed an induction of peripheral and hippocampal IGF-1 with concomitant activation of receptor for IGF-1 (IGF-1R) and AKT in the hippocampus. These distinct pathways culminated in an increase of synapsin 1 and synaptophysin expression in both groups. These findings demonstrated that both aerobic and resistance exercise can employ divergent molecular mechanisms but achieve similar results on learning and spatial memory.

Inhibition of calmodulin-dependent kinase kinase blocks human cytomegalovirus-induced glycolytic activation and severely attenuates production of viral progeny.

Viruses depend on the host cell to provide the energy and biomolecular subunits necessary for production of viral progeny. We have previously reported that human cytomegalovirus (HCMV) infection induces dramatic changes to central carbon metabolism, including glycolysis, the tricarboxylic acid (TCA) cycle, fatty acid biosynthesis, and nucleotide biosynthesis. Here, we explore the mechanisms involved in HCMV-mediated glycolytic activation. We find that HCMV virion binding and tegument protein delivery are insufficient for HCMV-mediated activation of glycolysis. Viral DNA replication and late-gene expression, however, are not required. To narrow down the list of cellular pathways important for HCMV-mediated [corrected] activation of glycolysis, we utilized pharmaceutical inhibitors to block pathways reported to be both involved in metabolic control and activated by HCMV infection. We find that inhibition of calmodulin-dependent kinase kinase (CaMKK), but not calmodulin-dependent kinase II (CaMKII) or protein kinase A (PKA), blocks HCMV-mediated activation of glycolysis. HCMV infection was also found to target calmodulin-dependent kinase kinase 1 (CaMKK1) expression, increasing the levels of CaMKK1 mRNA and protein. Our results indicate that inhibition of CaMKK has a negligible impact on immediate-early-protein accumulation yet severely attenuates production of HCMV viral progeny, reduces expression of at least one early gene, and blocks viral DNA replication. Inhibition of CaMKK did not affect the glycolytic activation induced by another herpes virus, herpes simplex virus type 1 (HSV-1). Furthermore, inhibition of CaMKK had a much smaller impact on HSV-1 replication than on that of HCMV. These data suggest that the role of CaMKK during the viral life cycle is, in this regard, HCMV specific. Taken together, our results suggest that CaMKK is an important factor for HCMV replication and HCMV-mediated glycolytic activation.

The role of CaMKII in calcium-activated death pathways in bone marrow B cells.

Calcium is an essential signaling molecule in developing B cells, thus altering calcium dynamics represents a potential target for toxicant effects. GW7845, a tyrosine analog and potent peroxisome proliferator-activated receptor γ agonist, induces rapid mitogen-activated protein kinase (MAPK)-dependent apoptosis in bone marrow B cells. Changes in calcium dynamics are capable of mediating rapid initiation of cell death; therefore, we investigated the contribution of calcium to GW7845-induced apoptosis. Treatment of a nontransformed murine pro/pre-B cell line (BU-11) with GW7845 (40 µM) resulted in intracellular calcium release. Multiple features of GW7845-induced cell death were suppressed by the calcium chelator BAPTA, including MAPK activation, loss of mitochondrial membrane potential, cytochrome c release, caspase-3 activation, and DNA fragmentation. A likely mechanism for the calcium-mediated effects is activation of CaMKII, a calcium-dependent MAP4K. We observed that three CaMKII isoforms (ß, γ, and δ) are expressed in lymphoid tissues and bone marrow B cells. Treatment with GW7845 increased CaMKII activity. All features of GW7845-induced cell death, except loss of mitochondrial membrane potential, were suppressed by CaMKII inhibitors (KN93 and AIP-II), suggesting the activation of multiple calcium-driven pathways. To determine if CaMKII activation is a common feature of early B cell death following perturbation of Ca(2+) flux, we dissected tributyltin (TBT)-induced death signaling. High-dose TBT (1 µM) is known to activate calcium-dependent death. TBT induced rapid apoptosis that was associated with intracellular calcium release, CaMKII activation and MAPK activation, and was inhibited by AIP-II. Thus, we show that early B cells are susceptible to calcium-triggered cell death through a CaMKII/MAPK-dependent pathway.