SIGNAL TRANSDUCTION IN SYNAPTIC TRANSMISSION AND PLASTICITY
, Head, Section on Metabolic Regulation
Freesia L. Huang, PhD, Staff Scientist
Guo-Chiuan Hung, PhD, Postdoctoral Fellow
Catherine Boucheron, PhD, Visiting Fellow
Pavan K. Shetty, PhD, Visiting Fellow
Suzanne J. Lo, BS, Postbaccalaureate Fellow
We investigate the signal transduction mechanisms involved in synaptic transmission and plasticity. Studies of these neural processes are essential for understanding cognition and behavioral disturbances. Our approach is to generate genetically modified mice by deletion or expression of a gene expressed specifically in the brain. We have generated a strain of mice devoid of neurogranin (Ng), a neural-specific protein that is normally highly expressed in specific neurons in the forebrain and that has been implicated in the regulation of reactions dependent on Ca2+/calmodulin (CaM). Ng binds to apoCaM, and its binding affinity is attenuated following an increase in Ca2+ concentration, phosphorylation by PKC, and/or oxidation of the protein’s sulfhydryl groups. In the neuronal soma and dendrites, Ng levels are very high; there, Ng sequesters apoCaM at basal physiological Ca2+. Upon synaptic stimulation, the influxed Ca2+ displaces Ng from the Ng/apoCaM complex to form Ca2+/CaM and free Ng. The buffering of CaM by Ng is a fundamental mechanism in the regulation of neuronal free Ca2+ and Ca2+/CaM concentrations. We aim to define the regulatory functions of Ng in neuronal signaling and to design therapeutic approaches to remediate the cognitive deficits and behavioral abnormality of Ng-devoid mice.
Regulation of synaptic plasticity by neurogranin
Ng is a neuronal protein that binds to apoCaM under basal physiological conditions and dissociates from CaM upon synaptic stimulation, thereby raising intracellular Ca2+. Ng is localized in the neuronal soma and dendrites and is present at high concentrations in the dendritic spines. It has been suggested that Ng modulates synaptic responses by buffering and sequestering CaM to regulate the level of free Ca2+ and Ca2+/CaM complexes. The binding affinity of Ng for CaM is reduced by increasing Ca2+, phosphorylation by PKC, or oxidation by oxidants. The Ng concentration in the hippocampus of adult mice is one of the highest among all neuronal CaM-binding proteins. Among Ng+/− mice, but less so in Ng+/+ mice, we observed a significant relationship between hippocampal levels of Ng and performance in several cognitive function tasks. Ng−/− mice performed poorly in all tasks; they also displayed deficits in high-frequency stimulation– (HFS) induced long-term potentiation (LTP) in area CA1 of hippocampal slices but enhancement of low-frequency–induced long-term depression (LTD). Measurements of Ca2+ transients in CA1 pyramidal neurons following weak and strong tetanic stimulations revealed a significantly greater [Ca2+]i-response in Ng+/+ than in Ng−/− mice. The diminished Ca2+ dynamics in Ng−/− mice is a likely cause of their decreased propensity to undergo LTP and their deficit in cognitive function.
Given that HFS-induced LTP is initiated by Ca2+ influx through N-methyl-d-aspartic acid (NMDA) receptors, we reasoned that stimulation of postsynaptic downstream signaling components or increased presynaptic transmitter release may rescue the deficits of Ng−/− mice. We focused on the PKC and PKA signaling pathways as well as on gene transcription. Treatment of hippocampal slices of Ng−/− mice with phorbol ester, which enhances presynaptic responses by increasing neurotransmitter release, induced robust LTP in Ng−/− mice. Short-term (5-minute) application of forskolin (adenylate cyclase activator), rolipram (phosphodiesterase inhibitor), and picrotoxin (GABAA inhibitor) to the Ng−/− hippocampal slices also induced LTP. By stimulating cAMP/PKA signaling and suppressing the GABAA-mediated inhibitory pathway, the drugs bypassed the requirement of Ng to induce LTP. Histone deacetylase (HDAC) inhibitors are known to enhance both memory and synaptic plasticity at the level of chromatin remodeling. Bath application of the HDAC inhibitor trichostatin A also augmented the HFS-induced LTP in the Ng−/− hippocampal slices. Using these chemically induced LTP protocols, we have established potential drug treatment regimens to ameliorate the behavioral deficits of Ng−/− mice. To confirm the critical role of Ng in the enhancement of synaptic plasticity, we prepared Ng-expressing DNA constructs for injection into the brain of Ng−/− mice. The transgene is packaged in non-pathogenic recombinant adeno-associated virus (rAAV) and driven by a hybrid chicken B-actin/CMV promoter with the addition—to increase expression—of the cis-acting woodchuck post-transcriptional regulatory element. The transgene consists of Ng, IRES (internal ribosome entry site), and green fluorescent protein in a bi-cistronic–expressing cassette for co-expression of both proteins. We will stereotaxically inject the rAAV constructs into the hippocampus and amygdala of Ng−/− mice to determine if expression of Ng in these brain regions can correct their electrophysiological and behavioral deficits.
Effect of environmental enrichment on the cognitive behaviors of neurogranin knockout mice
Environmental enrichment (EE) can increase neurogenesis, alter neural plasticity, and improve cognitive performances. Ng, a specific substrate of PKC, is abundantly expressed in brain regions important for cognitive functions. Deletion of Ng in mice causes severe deficits in learning and memory of spatial tasks, induction and maintenance of LTP, and amplification of Ca2+-mediated signaling in the hippocampus. To determine if an enriched environment and an increase in the physical activities of the mutant mice can improve performance of memory tasks, we housed groups of mutant mice and their wild-type littermates of different age groups in roomier cages for three weeks; we outfitted each cage with several toys and a running wheel for voluntary exploration and exercise. We housed the control groups in the regular home cages without any toys. For young adults, both Ng+/+ and Ng+/− mice housed in the enriched environment (EE) performed significantly better than their controls in several learning and memory tasks. However, the short-term EE (SEE) had minimal effect on the performances of Ng−/− mice on the same tasks. In Ng+/+ and Ng+/− mice, hippocampal LTP in the CA1 region induced by high-frequency stimulation was also higher in the enriched group than in controls, whereas we observed no significant change among Ng−/− mice. Quantitative immunoblot analyses, however, showed that enriched mice of all three groups had elevated hippocampal levels of Ca2+/CaM-dependent protein kinase II (CaMKII) and CREB, but not of ERK. Interestingly, enrichment resulted in an increase in hippocampal Ng levels in Ng+/+ and Ng+/− mice, which seemingly contributed to improved LTP and behavioral performances.
As SEE nominally benefits only young adult Ng−/− mice, we used long-term EE (LEE) to test for a beneficial effect for the older mice. In contrast to SEE, LEE benefited the Ng−/− as well as the Ng+/+ and Ng+/− mice by preventing age-related cognitive decline. LEE also caused an increase in the hippocampal CREB levels of all three genotypes and of the Ng levels of Ng+/+ and Ng+/− mice, but not those of a CaMKII or ERK. Interestingly, hippocampal slices of the enriched aging Ng−/− mice, unlike those of Ng+/+ and Ng+/− mice, did not show enhancement in HFS-induced LTP in the CA1 region. It appears that the learning and memory processes in the enriched aging Ng−/− mice do not correlate with the HFS-induced LTP, which is facilitated by Ng. The results demonstrated that LEE may improve the cognitive function of aging Ng−/− mice through an Ng-independent plasticity pathway. Results from in situ hybridization also showed that Ng mRNA levels in frontal cortex, hippocampal CA1, CA3, and dentate gyrus were significantly higher among LEE groups than in the controls.
Huang FL, Huang K-P, Boucheron C. Long-term enrichment enhances the cognitive behavior of the aging neurogranin null mice without affecting their hippocampal LTP. Learn Mem 2007;14:512-9.
Huang FL, Huang K-P, Wu J, Boucheron C. Environmental enrichment enhances neurogranin expression and hippocampal learning and memory but fails to rescue the impairments of neurogranin null mutant mice. J Neurosci 2006;26:6230-7.
Modification of protein by reactive sulfur species: disulfide S-monoxide and S-dioxide
Generation of reactive oxygen and nitrogen species during oxidative stress has been linked to aging and many human diseases. In the central nervous system, neurotransmission under normal and pathological conditions generates a variety of oxidants that, if not properly neutralized, are detrimental to cell survival. Numerous neural disorders, including Alzheimer’s and Parkinson’s diseases, are believed to result, in part, from excessive damage to neurons by the endogenous oxidants. Glutathione (GSH), at its cellular concentrations of 1 to 10 mM in mammalian cells, is one of the major low molecular weight reductants to counter the assault from oxidative stress; it also serves as an abundant precursor of reactive sulfur species. We found that several oxidized forms of GSH with higher oxidative states, including glutathione disulfide S-monoxide (GS-DSMO) and disulfide S-dioxide (GS-DSDO), could be generated in oxidant-treated rat brain slices. These two compounds are much more reactive than glutathione disulfide (GSSG) for the thionylation of protein. Glutathionylation or thionylation of proteins has been shown to regulate the activities of enzymes, transcriptional factors, cell surface receptors, and cytoskeletal proteins. We proposed that these reactive disulfide S-oxides (DSOs) may be involved in oxidant-mediated signaling cascades to modify proteins at locations where oxidants are generated.
To investigate the biochemical characteristics of these reactive sulfur species, we devised a method to synthesize the DSOs by oxidation of thiol with H2O2 in the presence of a metal catalyst. The DSOs exhibited high reactivity toward thiol to form a mixed disulfide. The compounds are highly reactive toward the sulfhydryl group of proteins. Using PKC as a substrate for the DSOs, we found that DSOs derived from different thiols modified the kinase with various efficacies and specificities. The stoichiometries of thionylation of PKCb mediated by GS-DSMO and GS-DSDO were approximately 1 and 5 mol/mol, respectively, and we identified at least four glutathionylation sites in the GS-DSDO–treated kinase. Modification of PKC by GS-DSDO and captopril-DSDO (CPS-DSDO) rendered the kinase highly susceptible to limited proteolysis; the former preferentially caused the degradation of the catalytic and the latter of the regulatory domain of the kinase. Furthermore, CPS-DSDO–mediated modification of PKC increased autonomous kinase activity, which was not the case for GS-DSDO–mediated modification. Given that DSOs of different oxidative states as well as those derived from different thiols exert different effects on a target protein, these molecules could cause distinct cellular responses if derived from endogenous cellular reactions or even if they arise from exogenous sources. The unique specificity and diverse effects of various reactive sulfur species toward PKC may serve as a prototype for designing drugs to target a specific group of proteins for therapeutic application.
Huang K-P, Huang FL, Shetty PK, Yergey AL. Modification of protein by disulfide S-monoxide and disulfide S-dioxide: distinctive effects on PKC. Biochemistry 2007;46:1961-71.
Oxidative modulation of CaMKII in the brain
CaMKII is one of the major Ca2+-sensing enzymes and is important in transducing neuronal, hormonal, and electrical signals in brain, heart, and other tissues. In the CNS, CaMKII plays a pivotal role in the facilitation of synaptic plasticity, learning, and memory as well as in activity-dependent developmental processes. The CaMKII holoenzyme is a dodecamer composed of two stacked hexameric rings, and the kinase undergoes intersubunit autophosphorylation in the presence of Ca2+/CaM, which converts the kinase into an activator-independent autonomous enzyme. Autophosphorylation also leads to increased affinity of the kinase for several proteins near the sites of elevated Ca2+ with functional consequences. In the brain, translocation and aggregation of CaMKII have been implicated in NMDA receptor–dependent enhancement of synaptic plasticity as well as in neurological disorders associated with ischemic injury and seizure. Others have proposed that, during ischemic stress and seizure-induced neuronal excitation, clustering and inactivation of CaMKII within soma and neuronal processes provide a neuroprotective mechanism, which would limit excessive kinase activity during episodes of Ca2+ overload. The mechanism for this activity-dependent aggregation of CaMKII is not entirely clear. We hypothesize that excessive oxidants generated during ischemic and excitotoxic stress may trigger aggregation of CaMKII.
Previously, we showed that activation of NMDA receptors or direct administration of oxidants to brain slices could trigger thionylation of neurogranin and formation of intramolecular disulfide. We therefore hypothesized that the oxidant-induced modification of protein results from oxidation by the endogenously produced GS-DSOs. Treatment of mouse brain synaptosomes with H2O2, diamide, and sodium nitroprusside caused aggregation of CaMKII with partial inhibition of the kinase activity. The CaMKII aggregates were found to associate with the postsynaptic density. However, treatment of purified CaMKII by these oxidants failed to replicate the effects observed in the synaptosomes. Using GS-DSMO and GS-DSDO, we demonstrated the disulfides’ efficacy in inhibiting CaMKII and forming disulfide-linked aggregates. Interestingly, the autophosphorylated CaMKII was relatively refractory to GS-DSMO– and GS-DSDO–mediated aggregation. Oxidation of purified CaMKII by these two glutathione disulfide S-oxides mimicked more closely the oxidant-induced modification of CaMKII in the synaptosomes as well as the ischemia-induced oxidative stress in the acutely prepared hippocampal slices. The results suggest that these disulfide oxides or compounds with similar chemical properties mediate oxidative modification of CaMKII. Because ischemia could induce rapid oxidation of CaMKII in the hippocampal slices, we infer that this could also occur in vivo. Ischemia causes oxidation of CaMKII and suppression of basal synaptic transmission in the hippocampal CA1 region. Although reperfusion with oxygenated solution restored basal synaptic transmission, the extent of HFS-induced LTP in tissues undergoing an ischemia/reperfusion cycle was significantly lower than that of non-ischemic controls. These findings suggest that ischemia has a deleterious effect on the signaling components for the enhancement of synaptic plasticity.
COLLABORATOR
Alfred L. Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD
For further information, contact huangk@mail.nih.gov.
