Synaptic tagging and capture : differential role of distinct calcium/calmodulin kinases in protein synthesis-dependent long-term potentiation

Weakly tetanized synapses in area CA1 of the hippocampus that ordinarily display long-term potentiation lasting ~3 h (called early-LTP) will maintain a longer-lasting change in efficacy (late-LTP) if the weak tetanization occurs shortly before or after strong tetanization of an independent, but convergent, set of synapses in CA1. The synaptic tagging and capture hypothesis explains this heterosynaptic influence on persistence in terms of a distinction between local mechanisms of synaptic tagging and cell-wide mechanisms responsible for the synthesis, distribution, and capture of plasticity-related proteins (PRPs). We now present evidence that distinct CaM kinase (CaMK) pathways serve a dissociable role in these mechanisms. Using a hippocampal brain-slice preparation that permits stable long-term recordings in vitro for >10 h and using hippocampal cultures to validate the differential drug effects on distinct CaMK pathways, we show that tag setting is blocked by the CaMK inhibitor KN-93 (2-[N-(2-hydroxyethyl)]-N-(4-methoxybenzenesulfonyl)amino-N-(4-chlorocinnamyl)-N-methylbenzylamine) that, at low concentration, is more selective for CaMKII. In contrast, the CaMK kinase inhibitor STO-609 [7H-benzimidazo(2,1-a)benz(de)isoquinoline-7-one-3-carboxylic acid] specifically limits the synthesis and/or availability of PRPs. Analytically powerful three-pathway protocols using sequential strong and weak tetanization in varying orders and test stimulation over long periods of time after LTP induction enable a pharmacological dissociation of these distinct roles of the CaMK pathways in late-LTP and so provide a novel framework for the molecular mechanisms by which synaptic potentiation, and possibly memories, become stabilized

Identification of novel human tumor cell-specific CaMK-II variants

AbstractCaMK-II (the (type II) multifunctional Ca2+/CaM-dependent protein kinase) has been implicated in diverse neuronal and non-neuronal functions, including cell growth control. CaMKII expression was evaluated in a variety of human tumor cell lines using RT-PCR (reverse transcriptase coupled polymerase chain reaction). PCR primers which flanked the CaMK-II variable domain were used so that all possible variants of the four mammalian CaMK-II genes (α, β, γ and δ) could be identified. 8 distinct CaMK-II isozymes were identified from human mammary tumor and neuroblastoma cell cDNA, each of which represented a variant of β, γ or δ CaMK-II. They included 2 β isozymes (βe, βe′), 4 γ isozymes (γB, γC, γG, γH) and 2 δ isozymes (δC, δE) This is the first report of human β and δ CaMK-II sequences.A panel of human cell types was then screened for these CaMK-II isozymes. As expected, cerebral cortex predominately expressed α, β and δA CaMK-II. In contrast, tumor cells, including those of neuronal origin, expressed an entirely different spectrum of CaMK-II isozymes than adult neuronal tissue. Tumor cells of diverse tissue origin uniformly lacked α CaMK-II and expressed 1–2 β isozymes, at least 3 γ isozymes and 1–2 δ isozymes. When compared to undifferentiated fibroblasts, βe, βe′, γG and γH were preferentially expressed in tumor cells. CaMK-II immunoblots also indicated that neuroblastoma and mammary tumor cells express isozymes of CaMK-II not present in their non-transformed cell or tissue counterpart. The identification of these new, potential tumor-specific CaMK-II variants supports previous indications that CaMK-II plays a role in growth control. In addition, these results provide insight into both splice variant switching and variable domain structural similarities among all CaMK-II isozymes

Anti-epileptic effect of Ganoderma lucidum polysaccharides by inhibition of intracellular calcium accumulation and stimulation of expression of CaMKII a in epileptic hippocampal neurons

Purpose: To investigate the mechanism of the anti-epileptic effect of Ganoderma lucidum polysaccharides (GLP), the changes of intracellular calcium and CaMK II a expression in a model of epileptic neurons were investigated. Method: Primary hippocampal neurons were divided into: 1) Control group, neurons were cultured with Neurobasal medium, for 3 hours; 2) Model group I: neurons were incubated with Mg2+ free medium for 3 hours; 3) Model group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with the normal medium for a further 3 hours; 4) GLP group I: neurons were incubated with Mg2+ free medium containing GLP (0.375 mg/ml) for 3 hours; 5) GLP group II: neurons were incubated with Mg2+ free medium for 3 hours then cultured with a normal culture medium containing GLP for a further 3 hours. The CaMK II a protein expression was assessed by Western-blot. Ca2+ turnover in neurons was assessed using Fluo-3/AM which was added into the replacement medium and Ca2+ turnover was observed under a laser scanning confocal microscope. Results: The CaMK II a expression in the model groups was less than in the control groups, however, in the GLP groups, it was higher than that observed in the model group. Ca2+ fluorescence intensity in GLP group I was significantly lower than that in model group I after 30 seconds, while in GLP group II, it was reduced significantly compared to model group II after 5 minutes. Conclusion: GLP may inhibit calcium overload and promote CaMK II a expression to protect epileptic neuron

The Role of Ca2+/Calmodulin Dependent Protein Kinase II, CaMK-II, in Kidney Morphogenesis

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common heritable diseases in the world, characterized by the development of large fluid filled cyst. Treatments for this disease are limited due to a lack of understanding of disease pathogenesis. Ca2+/calmodulin dependent protein kinase II (CaMK-II) is necessary for kidney morphogenesis in zebrafish as well as being a downstream effector of pkd2, one of the genes most commonly mutated in ADPKD patients. The roles of CaMK-II during zebrafish kidney development include regulation of kidney cell migration as well as cloacal cilia stability. The influence of CaMK-II on these pathways is partially dependent on its regulation of HDAC4 localization. Inhibition of CaMK-II caused the translocation of HDAC4 from the cytosol to the nucleus, leading to the model that CaMK-II activation via PKD2 retains HDAC4 in the cytosol allowing for transcription of its target genes. Further studies attempted with a zebrafish camk2g1 mutant proved inconclusive as mutants displayed none of the phenotypic defects seen in morphant embryos. Analysis of CaMK-II gene expression in camk2g1 mutants identified the capability of genetic compensation between CaMK-II family members. In camk2g1 mutants, the paralog gene camk2g2 is upregulated over 3 fold, compensating for the loss of its paralog gene. In conclusion, this study has furthered the understanding of the roles of CaMK-II during kidney development and demonstrated the ability of CaMK-II family members to compensate for one other. In addition, this study also further validates the uses of knockdown methods in developmental studies

The Role of CaMK-II in the Development of Leukemia/Lymphoma in Danio Rerio

Acute lymphocytic leukemia (ALL) is a blood disorder characterized by aberrant proliferation of immature lymphocytes. ALL is the most common cancer in children and can result from external influences, such as radiation, or internal influences, such as genetic mutation. Additionally, Ca2+/calmodulin-dependent kinase II (CaMK-II) is a serine/threonine protein kinase whose increased expression has been found in various leukemias. Zebrafish and human immune cells are analogous and both species have conserved hematopoietic stem cell specification mechanisms. In this study, the constitutively active form of CaMK-II, resulting from a threonine to aspartic acid point mutation at the 287 base-pair location (T287D), was paired with the EGFP transgene using Tol2 Gateway technology and injected into zebrafish at the one-cell stage. The zebrafish expressing the transgene were outcrossed to wild type and mutant p53 zebrafish and then monitored for leukemic development using flow cytometry and pathology. Based on FACS results, the mutant p53 bearing the T287D mutation were found to have increased levels of lymphocytes compared to the mutant p53 zebrafish without the T287D mutation. Furthermore, hematologists confirmed the development of B-cell leukemia/lymphoma from histological slides prepared from the mutant p53 with the T287D mutation. The mutant p53 without the T287D mutation did not yield such results. These findings highlight a potential role of CaMK-II in the abnormal development of lymphocytes and provide a useful model, from which, drug studies can be performed for potential treatment options.https://scholarscompass.vcu.edu/uresposters/1244/thumbnail.jp

Crosstalk Between Brain-Derived Neurotrophic Factor And N-Methyl-D-Aspartate Receptor Signaling In Neurons

Glutamate is the major excitatory neurotransmitter in brain exerting prosurvival effect on neurons via N-methyl-D-aspartate receptor (NMDAR) signaling under physiological conditions. However in pathological circumstances such as ischemia, NMDARs might have proapoptotic excitotoxic activity. In contrast brain-derived neurotrophic factor (BDNF) signaling via TrkB receptors has been largely considered to promote neuronal differentiation, plasticity and survival during normal development, and protect neurons in pathophysiological conditions antagonizing the NMDAR-mediated excitotoxic cell death. In this review we summarize recent evidence for the existent crosstalk and positive feedback loops between the BDNF and NMDAR signaling and point out some of the important specific features of each signaling pathway

Colon cancer cell-derived 12(S)-HETE induces the retraction of cancer-associated fibroblast via MLC2, RHO/ROCK and Ca2+ signalling

Retraction of mesenchymal stromal cells supports the invasion of colorectal cancer cells (CRC) into the adjacent compartment. CRC-secreted 12(S)-HETE enhances the retraction of cancer-associated fibroblasts (CAFs) and therefore, 12(S)-HETE may enforce invasivity of CRC. Understanding the mechanisms of metastatic CRC is crucial for successful intervention. Therefore, we studied pro-invasive contributions of stromal cells in physiologically relevant three-dimensional in vitro assays consisting of CRC spheroids, CAFs, extracellular matrix and endothelial cells, as well as in reductionist models. In order to elucidate how CAFs support CRC invasion, tumour spheroid-induced CAF retraction and free intracellular Ca2+ levels were measured and pharmacological-or siRNA-based inhibition of selected signalling cascades was performed. CRC spheroids caused the retraction of CAFs, generating entry gates in the adjacent surrogate stroma. The responsible trigger factor 12(S)-HETE provoked a signal, which was transduced by PLC, IP3, free intracellular Ca2+, Ca(2+)calmodulin-kinase-II, RHO/ROCK and MYLK which led to the activation of myosin light chain 2, and subsequent CAF mobility. RHO activity was observed downstream as well as upstream of Ca2+ release. Thus, Ca2+ signalling served as central signal amplifier. Treatment with the FDA-approved drugs carbamazepine, cinnarizine, nifedipine and bepridil HCl, which reportedly interfere with cellular calcium availability, inhibited CAF-retraction. The elucidation of signalling pathways and identification of approved inhibitory drugs warrant development of intervention strategies targeting tumour-stroma interaction

Inhibition of phosphoinositide 3-kinase/protein kinase B signaling hampers the vasopressin-dependent stimulation of myogenic differentiation

Arginine-vasopressin (AVP) promotes muscle differentiation, hypertrophy, and regeneration through the combined activation of the calcineurin and Calcium/Calmodulin-dependent Protein Kinase (CaMK) pathways. The AVP system is impaired in several neuromuscular diseases, suggesting that AVP may act as a physiological factor in skeletal muscle. Since the Phosphoinositide 3-kinases/Protein Kinase B/mammalian Target Of Rapamycin (PI3K/Akt/mTOR) signaling plays a significant role in regulating muscle mass, we evaluated its role in the AVP myogenic effect. In L6 cells AKT1 expression was knocked down, and the AVP-dependent expression of mTOR and Forkhead box O3 (FoxO) was analyzed by Western blotting. The effect of the PI3K inhibitor LY294002 was evaluated by cellular and molecular techniques. Akt knockdown hampered the AVP-dependent mTOR expression while increased the levels of FoxO transcription factor. LY294002 treatment inhibited the AVP-dependent expression of Myocyte Enhancer Factor-2 (MEF2) and myogenin and prevented the nuclear translocation of MEF2. LY294002 also repressed the AVP-dependent nuclear export of histone deacetylase 4 (HDAC4) interfering with the formation of multifactorial complexes on the myogenin promoter. We demonstrate that the PI3K/Akt pathway is essential for the full myogenic effect of AVP and that, by targeting this pathway, one may highlight novel strategies to counteract muscle wasting in aging or neuromuscular disorders

CASK regulates CaMKII autophosphorylation in neuronal growth, calcium signaling, and learning

Calcium (Ca2+)/calmodulin (CaM)-dependent kinase II (CaMKII) activity plays a fundamental role in learning and memory. A key feature of CaMKII in memory formation is its ability to be regulated by autophosphorylation, which switches its activity on and off during synaptic plasticity. The synaptic scaffolding protein CASK (calcium (Ca2+)/calmodulin (CaM) associated serine kinase) is also important for learning and memory, as mutations in CASK result in intellectual disability and neurological defects in humans. We show that in Drosophila larvae, CASK interacts with CaMKII to control neuronal growth and calcium signalling. Furthermore, deletion of the CaMK-like and L27 domains of CASK (CASK β null) or expression of overactive CaMKII (T287D) produced similar effects on synaptic growth and Ca2+ signalling. CASK overexpression rescues the effects of CaMKII overactivity, consistent with the notion that CASK and CaMKII act in a common pathway that controls these neuronal processes. The reduction in Ca2+ signalling observed in the CASK β null mutant caused a decrease in vesicle trafficking at synapses. In addition, the decrease in Ca2+ signalling in CASK mutants was associated with an increase in Ether-à-go-go (EAG) potassium (K+) channel localisation to synapses. Reducing EAG restored the decrease in Ca2+ signalling observed in CASK mutants to the level of wildtype, suggesting that CASK regulates Ca2+ signalling via EAG. CASK knockdown reduced both appetitive associative learning and odour evoked Ca2+ responses in Drosophila mushroom bodies, which are the learning centres of Drosophila. Expression of human CASK in Drosophila rescued the effect of CASK deletion on the activity state of CaMKII, suggesting that human CASK may also regulate CaMKII autophosphorylation