Signal transduction in synaptic transmission and plasticity

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

This laboratory investigates the signal transduction in synaptic transmission and plasticity by biochemical, behavioral, and electrophysiological approaches using a genetically modified mice model. A strain of mutant mice was established by deleting the gene coding for a neural-specific protein, neurogranin (Ng). This protein is expressed at high levels in cerebral cortex, hippocampus, and amygdala, and has been implicated in the modulation of synaptic plasticity. Ng is a small molecular weight protein, which binds calmodulin (CaM) at low level of calcium under basal physiological conditions. The binding of Ng and CaM is weakened upon neuronal stimulation that results in an increase in calcium. When dissociated from CaM, Ng is phosphorylated by PKC and/or oxidized by nitric oxide and other oxidants. The phosphorylated and oxidized Ngs are poor binding partners of CaM. These multifaceted regulations of Ng provide a fine tune mechanism to set the levels of free calcium and calcium-CaM depending on the strength of stimulation to the neurons and, thus, gate the output response.[unreadable] [unreadable] Synaptic responses triggering long-term potentiation (LTP) or long-term depression (LTD) depend on the amplitude of calcium influx and the sensitivity of the transduction machinery to amplify the signal. Ng knockout (KO) mice performed poorly in cognitive behavior tasks and exhibited deficits in LTP and activation of CaMKII. Provision of short-term (3 weeks) environmental enrichment (SEE) for the mutant mice did not improve their performance, even though it was beneficial to the wild type and heterozygous mice, whose hippocampal Ng levels and LTP were elevated under SEE as compared to the control mice. Interestingly, for Ng KO mice, SEE caused a 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 (CREB), were elevated to the same levels as the wild type and heterozygous mice. In contrast, a long-term environmental enrichment (LEE) for the aging mice was beneficial to Ng KO as well as wild type and heterozygous mice in preventing age-related cognitive decline. LEE also caused an increase in the hippocampal CREB level of all three genotypes and Ng level of wild type and heterozygous mice, but not that of CaMKII or ERK. Hippocampal slices of these enriched aging Ng KO mice, unlike those of wild type and heterozygous mice didnt show enhancement in LTP. It appears that the learning and memory processes in these enriched aging Ng KO mice do not correlate with the LTP, which is facilitated by Ng. These results indicate that LEE for the aging Ng KO mice may improve their cognitive function through an Ng-independent plasticity pathway. To improve the behavioral deficits of young adult Ng KO mice, they were treated with Ritalin that has been shown to be beneficial for human patients suffering from ADHD. Short-term treatment (up to 1 month) of the mutant mice with Ritalin in combination with EE appeared to improve their cognitive function and reduced their anxiety and hyperactivity behaviors. These Ng mutant mice may serve as a useful model to investigate the cause and treatment strategy for ADHD. [unreadable] [unreadable] Neuronal stimulation that triggers the enhancement of synaptic plasticity requires the activation of calcium and calcium/CaM-dependent signaling pathways, which are modulated by Ng through its interaction with CaM. Immuno-staining showed that Ng was localized in the soma and dendrites of the principle neurons in the hippocampus, whereas CaM was concentrated in the nucleus with relative low level in the dendrites. However, following high frequency stimulation (HFS) of the hippocampal CA1 region, CaM became concentrated in the dendritic spines where it co-localized with Ng. The accumulation of Ng and CaM in the spines may serve as tags for the stimulated neuronal network to increase synaptic efficacy. Measurement of intracellular calcium induced by HFS confirmed our hypothesis that Ng is involved in the potentiation of calcium-transients amplitude through a mass-action mechanism, which predicts that increasing Ng levels can potentiate the synaptic responses by raising free calcium at any given calcium influx. An increase in calcium favors the activation of PKC-, cAMP/PKA-, and calcium-CaM-regulated signaling pathways. Because Ng KO mice exhibit deficits in the HFS-induced LTP, which is mediated by the calcium influx through NMDA receptors, we investigated if direct stimulation of the down-stream signaling components of these pathways could rescue their deficits. Bath application of activator of PKC, phorbol 12,13-dibutyrate (PDBu), or cAMP/PKA, forskolin, to the hippocampal slices was effective to induce LTP in the Ng KO mice, albeit, less effective than their wild type counterpart. Similarly, application of a histone deacetylase inhibitor, trichostatin A, could augment the HFS-induced LTP in Ng KO mice, likely resulting from chromatin remodeling-mediated gene transcription. These findings suggest that drugs that stimulate these signaling components will be beneficial to correct the behavioral deficits of the mutant mice.[unreadable] [unreadable] Synaptic stimulation results in the generation of several oxidants, including nitric oxide, superoxide, and hydrogen peroxide, and these oxidants serve as messenger molecules under normal physiological conditions. However, during oxidative stress resulting from excessive synaptic stimulation associated with seizure, ischemia, and neurodegenerative diseases, high levels of these oxidants can cause cellular damage by modification of the various cellular components. CaMKII, a dodecameric enzyme highly concentrated in the neurons, was shown to be very susceptible to oxidative modulation. Treatment of mouse brain synaptosomes with hydrogen peroxide, diamide, and sodium nitroprusside caused aggregation of CaMKII through formation of disulfide and non-disulfide linkages, and partial inhibition of the kinase activity. These CaMKII aggregates were found to associate with the post synaptic density. However, treatment of purified CaMKII with these oxidants did not replicate those effects observed in the synaptosomes. Using two previously identified potential mediators of oxidants in the brain, glutathione disulfide S-monoxide (GS-DSMO) and glutathione disulfide S-dioxide (GS-DSDO), we showed that they oxidized and inhibited CaMKII in a manner partly related to those of the oxidant-treated synaptosomes as well as the ischemia-elicited oxidative stress in the acutely prepared hippocampal slices. Interestingly, the autophosphorylated and activated CaMKII was relatively refractory to GS-DSMO and GS-DSDO mediated aggregation. Short-term ischemia (10 min) caused a depression of basal synaptic response of the hippocampal slices and re-oxygenation (after 10 min) reversed the depression. However, oxidation of CaMKII remained at above the pre-ischemic level throughout the treatment. Oxidation of CaMKII also prevented full recovery of CaMKII autophosphorylation after re-oxygenation. Subsequently, the high frequency stimulation-mediated synaptic potentiation in the hippocampal CA1 region was significantly reduced compared to the control without ischemia. Thus, ischemia-evoked oxidation of CaMKII, probably via the action of glutathione disulfide S-oxides or their analogues, may be involved in the suppression of synaptic plasticity.