This laboratory investigates the roles of protein phosphorylation and oxidation involved in synaptic transmission and plasticity. Modifications of protein by phosphorylation and oxidation are important mechanisms for the modulation of a plethora of cellular responses. Protein phosphorylation catalyzed by a variety of protein kinases and oxidation by many endogenously produced oxidants have been linked to the regulation of cellular processes as diverse as ion channels, cellular metabolism, synaptic plasticity, and growth and differentiation. Studies of these neural processes are essential to understanding the complex problem related to neurodegenerative diseases and learning and memory. Our approach is to generate genetically modified mice by deletion or expression of a gene specifically expressed in the brain. These efforts have led us to generate a strain of mice devoid of a neural-specific protein, neurogranin (Ng). This protein is normally expressed at high levels in the neurons within cerebral cortex, hippocampus, and amygdala and has been implicated in the modulation of synaptic plasticity. Ng binds calmodulin (CaM) in an inverse Ca2+-sensitive manner; namely, its binding affinity for CaM decreases with increasing Ca2+ concentration. It is believed that at basal levels of Ca2+ all CaM is sequestered by Ng, whose cellular concentration is much higher than that of CaM. Upon synaptic stimulation, the influxed Ca2+ will displace Ng from the Ng/CaM complex to form Ca2+/CaM. The buffering of CaM by Ng serves as a mechanism to regulate neuronal free Ca2+ and Ca2+/CaM concentrations. Furthermore, Ng is readily phosphorylated by protein kinase C (PKC) and oxidized by nitric oxide (NO) and other oxidants. Both the phosphorylated and oxidized Ng exhibit lower affinities for CaM than the unmodified protein; thus, these modifications of Ng extend the availability of CaM even after the intracellular level of Ca2+ is reduced to basal levels. Synaptic responses triggering long-term potentiation (LTP) or long-term depression (LTD) depend on the amplitude of Ca2+ influx and the sensitivity of the transduction machinery to amplify the signal. Based on our previous finding that the autophosphorylation of Ca2+/CaM-dependent protein kinase II (CaMKII) was enhanced by Ng, we speculate that other Ca2+- and Ca2+/CaM-regulated pathways are also upregulated by Ng. The multifarious effects of Ng could account for the positive impact of this protein on the behavior of mice. Investigation of the regulatory mechanism of synaptic transmission and plasticity by Ng will help us to design novel therapeutic approaches with the potential to improve memory in human and alleviate symptoms related to dementia. ? ? The concentration of hippocampal Ng in adult wild type mice was estimated to be at least twice as much as CaM; at this level, Ng is one of the most abundant CaM-binding proteins in the neurons of the brain. Induction of LTP caused a rapid phosphorylation of this protein in the hippocampal CA1 region. Testing with the Morris water maze showed significant relationships between the levels of hippocampal Ng among heterozygous mice and their performances; however, such relationships were less significant among the wild type mice. These findings suggest that the wild type mice contain supra-threshold levels of Ng for proficient performance of these tasks. Ng knockout (KO) mice performed poorly in all spatial and contextual fear-conditioning tasks and exhibited deficits in LTP and concurrent CaMKII autophosphorylation. Provision of environmental enrichment (EE) and increased physical activity to the mutant mice did not improve their performance, even though it is beneficial to the wild type and heterozygous mice, whose hippocampal Ng levels and high frequency stimulation (HFS)-induced LTP in the CA1 region were elevated under EE as compared to the control mice housed in regular cages. Results from in situ hybridization also showed that Ng mRNA levels in frontal cortex, caudate putamen, hippocampal CA1, CA3, and dentate gyrus of the wild type and heterozygous mice were significant increased among EE groups as compared to the control. Interestingly, for Ng KO mice, EE caused negligible effect on their LTP in spite of the fact that other important signaling components for synaptic plasticity, including CaMKII and cAMP responsive element-binding protein, were elevated to the same levels as the wild type and heterozygous mice. These findings suggest that Ng gates the neuronal signaling reactions involved in learning and memory and during EE, those Ng-regulated reactions are critical for the enhancement of synaptic plasticity and cognitive functions. Electrophysiological experiments showed that the tetanus-frequency response curve of Ng KO mice was shifted to the right compared to that of the wild type mice; low frequency stimulation (5-10 Hz) induced LTD in the former and modest LTP in the latter. Measurement of intracellular Ca2+ induced by HFS confirmed our hypothesis that Ng is involved in the potentiation of Ca2+-transient amplitude through a mass-action mechanism, which predicts that increasing Ng levels can potentiate the synaptic responses by raising free Ca2+ at any given Ca2+ influx. An increase in Ca2+ favors the activation of PKC, which is involved in the phosphorylation of Ng. Several Ca2+-dependent PKC isozymes were found to be active in the phosphorylation of Ng. Activation of group 1 metabotropic glutamate receptors by an agonist, dihydroxyphenylglycine (DHPG), is known to stimulate phospholipase C and PKC; however, it induces dephosphorylation of Ng. These findings suggest that DHPG-mediated response also triggers the activation of protein phosphatase, which preferentially dephosphorylates Ng and causes LTD. As Ng contains redox-active thiols, it may serve as an intracellular reductant for defense against oxidative stress. Indeed, oxidants trigger modifications of Ng in mouse brain slices to form intramolecular disulfides and mixed disufides. Oxidants, such as hydrogen peroxide, diamide, and sodium nitroprusside, also induced oxidation of CaMKII in mouse brain synaptosomes to form aggregates. Formation of CaMKII aggregates, which deposit at postsynaptic density, has been shown previously as a result of synaptic stimulation. We have identified two potent thionylating agents, glutathione disulfide S-monoxide and S-dioxide that could modify CaMKII by thionylation and formation of aggregates. It seems that these compounds may serve as proximal mediators for oxidants to modify proteins near the location where oxidants are generated.
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