Studies on the signal transduction mechanisms involved in synaptic transmission and plasticity are essential to understanding learning and memory. We generate genetically modified mice by deletion or expression of a gene specifically expressed in the brain; one such mouse is devoid of neurogranin (Ng), a neural-specific protein that is normally expressed at a high level in specific neurons in the forebrain and has been implicated in the regulation of Ca2+/calmodulin (CaM)-dependent reactions. Ng binds to CaM in an inverse Ca2+-sensitive manner, i.e., its binding affinity for CaM decreases with increasing Ca2+ concentration. Furthermore, the binding affinity is attenuated by phosphorylation with PKC and by oxidation of Ng’s sulfhydryl groups. It is believed that, at basal physiological levels of Ca2+, all CaM is sequestered by Ng and that, upon synaptic stimulation, the influxed Ca2+ displaces Ng from the Ng/CaM complex to form Ca2+/CaM and free Ng. Ng’s buffering of CaM regulates neuronal free Ca2+ and Ca2+/CaM concentrations. Furthermore, Ng is a potent reductant to counteract oxidants generated during synaptic transmission. We aim to define the regulatory functions of Ng and to design therapeutic approaches to improve memory in an aging human population and patients suffering from dementia.
Regulation of synaptic plasticity by neurogranin
Huang K-P, Huang F, Li2; in collaboration with Balschun, Jäger, Reymann
Ng is localized in the neuronal soma and dendrites and is present in high concentrations in the dendritic spines. It has been suggested that Ng modulates synaptic responses by buffering and sequestering CaM in order to regulate the levels of free Ca2+ and Ca2+/CaM complexes. Increasing Ca2+ concentrations, phosphorylation by PKC, or oxidation by oxidants reduces the binding affinity of Ng for CaM. The Ng concentration in the hippocampus of adult mice varies broadly; in fact, the concentration in Ng+/+ mice is one of the highest among all neuronal CaM-binding proteins. Moreover, in Ng+/– mice, a significant relationship exists between hippocampal levels of Ng and performance on several cognitive functional tests, a relationship that is less apparent in Ng+/+ mice. Compared with Ng+/+ mice, Ng–/– mice performed poorly on all the tests; they also displayed deficits in high frequency–induced long-term potentiation (LTP) in area CA1 of hippocampal slices while demonstrating enhanced low frequency–induced long-term depression (LTD). Paired-pulse facilitation and synaptic fatigue during prolonged stimulation at 10 Hz (900 pulses) showed no changes in Ng–/– slices, indicating a normal presynaptic function. Measurement of Ca2+ transients in CA1 pyramidal neurons following weak and strong tetanic stimulations revealed a significantly greater [Ca2+]i-response in Ng+/+ than in the pyramidal neurons of Ng–/– mice, but the decay time constants did not differ. The diminished Ca2+ dynamics in Ng–/– mice is a likely cause of their decreased propensity to undergo LTP. Thus, Ng may promote high [Ca2+]i by a “mass action” mechanism; namely, the higher the Ng concentration, the greater the number of Ng/CaM complexes that form, effectively raising [Ca2+]i at any given Ca2+ influx. The mechanism provides potent signal amplification in enhancing synaptic plasticity as well as learning and memory.
Huang K-P, Huang FL, Jäger T, Li J, Reymann KG, Balschun D. Neurogranin/RC3 enhances long-term potentiation and learning by promoting calcium-mediated signaling. J Neurosci 2004;24:10660-10669.
Wu J, Huang K-P, Huang FL. Participation of NMDA-mediated phosphorylation and oxidation of neurogranin in the regulation of Ca2+- and Ca2+/calmodulin-dependent neuronal signaling in the hippocampus. J Neurochem 2003;86:1524-1533.
Role of neurogranin in the regulation of metabotropic glutamate receptor–mediated long-term depression
Huang F, Huang K-P
Calcium entry into post-synaptic sites is essential for triggering the signaling cascade for synaptic plasticity; a higher level of post-synaptic Ca2+ induces LTP while a lower level induces LTD. Two mechanistically distinct forms of LTD coexist in the hippocampus; one depends on activation of NMDA receptors and post-synaptic protein phosphatases and the other on activation of post-synaptic group 1 metabotropic glutamate receptors (mGluRs) and the local translation of dendritic mRNA. Low-frequency tetanic stimulation induces LTD that is dependent on NMDA, which is expressed normally in Ng–/– mice. Similarly, brief treatment of hippocampal slices from Ng–/– mice with DHPG, a group 1 mGluR agonist, induces LTD to a similar extent as in Ng+/+ mice. DHPG is known to activate mGluR5, the major post-synaptic group 1 mGluR in the hippocampal area CA1; MPEP, a noncompetitive antagonist of mGluR5, blocks DHPG-induced LTD. However, Ng–/– mice were insensitive to MPEP-mediated antagonism. Recognizing that group 1 mGluR agonists activate the phospholipase C cascade, including stimulation of PKC, we monitored the phosphorylation of Ng. Unexpectedly, we found that Ng was dephosphorylated following DHPG treatment, probably owing to the activation of calcineurin by low post-synaptic Ca2+ transients. Pretreatment with MPEP, which blocked LTD in Ng+/+ mice, prevented DHPG-induced dephosphorylation of Ng. The signaling cascade of DHPG-induced LTD requires rapid translation of pre-existing dendritic mRNA, which results from the activation of several upstream components, including Gq-type G proteins, MAP kinases, tyrosine phosphatases, PI3-kinase, PDK1, Akt, mTOR kinase, and S6 kinase. Given that MPEP does not directly inhibit mGluR5 receptors, the antagonistic action of MPEP on DHPG-induced LTD could be attributed to interference with any of the steps leading to translation of dendritic mRNA. Lacking Ng, Ng–/– mice are unresponsive to MPEP-mediated blockade of DHPG-induced LTD, suggesting that Ng is required for the action of MPEP to antagonize the group1 mGluR–mediated synaptic plasticity.
Effect of environmental enrichment on the cognitive behaviors of neurogranin knockout mice
Huang F, Huang K-P, Boucheron
Environmental enrichment and exercise (EEE) can increase neurogenesis and alter neural plasticity and improve cognitive performances. As described above, neurogranin knockout mice exhibit 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 increase in the physical activities of the mutant mice can improve their performances of memory tasks, groups of mutant mice and their wild-type littermates of different age groups were housed for a period of three weeks in relatively roomier cages, each with several toys and a running wheel for voluntary exploration and exercise. The control groups were housed in their regular home cages without toys. In the enriched environment, both Ng+/+ and Ng+/– mice performed significantly better than their controls in both the hidden platform and probe trial versions of the Morris water maze as well as in the step-down inhibitory avoidance fear conditioning task. However, EEE had minimal effect on the performances of Ng–/– mice on these tasks. Hippocampal LTP in the CA1 region induced by high-frequency stimulation also higher in the enriched group than the control Ng+/+ and Ng+/– mice, whereas we observed no significant change in Ng–/– mice. Quantitative immunoblot analyses, however, showed that enriched mice in all three groups had elevated hippocampal levels of CaMKII and CREB, but not of ERK. Interestingly, enrichment results in a greater increase in hippocampal Ng levels in Ng+/+ than in Ng+/– mice. Quantification of Ng mRNA from the control and EEE groups among young (about three months old), adult (three to six months old), and aged (greater than 18 months) Ng+/+, Ng+/–, and Ng–/– mice showed that EEE enhanced the hippocampal Ng mRNA levels among Ng+/+ and Ng+/– mice but had a negligible effect on CaMKII-alpha mRNA. In situ hybridization also showed that Ng mRNA levels within frontal cortex, caudate putamen, hippocampal CA1, CA3, and dentate gyrus were significantly higher in aged EEE groups than in the control group. The findings demonstrate that environmental enrichment can augment the hippocampal level of Ng, the extent of LTP, and performance on cognitive tasks. Without Ng, as in the case of Ng–/–, increased physical activity has minimal effect on neural plasticity and cognitive behavior. The observed positive correlation between the level of Ng and neural functions after exposure of the Ng+/+ and Ng+/– mice to an enriched environment recapitulates the pivotal role of Ng in enhancing the efficacy of signaling to improve cognitive behaviors.
Thionylation of protein by reactive sulfur species: modification of PKC by disulfide S–monoxide and S–dioxide
Huang K-P, Huang F
Accumulation of reactive oxygen and nitrogen species produced by oxidative stress has been linked to aging and many human diseases. In the central nervous system, neurotransmission under normal or pathological conditions generates a variety of oxidants, which, if not neutralized, are detrimental to cell survival. Several 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 is also an abundant precursor of reactive sulfur species. Recently, 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 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 the reactive disulfide S–oxides (DSOs) may be involved in the oxidant-mediated signaling cascade in order to modify proteins at locations where oxidants are generated. In addition to endogenous thiols, numerous thiol-containing drugs and food stuffs might participate in the signaling system.
To characterize further the reactions mediated by DSOs, we devised methods to synthesize highly pure compounds in bulk. Thus, we synthesized both DSMO and DSDO by iron- or methyltrioxorhenium (VII)-catalyzed oxidation of thiols, e.g., glutathione (GSH) and captopril (CPSH), with H2O2. In these metal-catalyzed reactions, thiols were better substrates for forming DSOs than their corresponding disulfides. The DSOs exhibited high reactivity toward thiol to form mixed disulfides. The compounds are highly reactive toward the sulfhydryl groups of proteins, including those of PKC. GS-DSDO was a 10-fold more potent activator of PKC than CPS-DSDO; both GS- and CPS-DSDO were more potent inhibitors of PKC than their corresponding DSMOs. We verified thionylation of PKC by GS-DSOs by immunoblot with antibody against GSH and by incorporation of GS-moiety from [35S]GS-DSOs into the kinase. Thionylation of PKC by GS-DSDO and CPS-DSDO rendered the kinase highly susceptible to proteolysis. Interestingly, GS-DSDO and CPS-DSDO preferentially targeted PKC at the catalytic and regulatory domains, respectively, as revealed by their susceptibilities to limited proteolysis. Furthermore, thionylation of PKC mediated by CPS-DSDO, but not that by GS-DSDO, increased autonomous kinase activity. The results show that the reactive sulfur species, especially DSDOs, generated from various thiols are potent thionylating agents and possess unique specificity toward PKC and probably other proteins. Thus, DSOs generated from thiols during oxidative stress can modulate the activity of PKC and tag the kinase for proteolysis. The unique specificity and diverse effects of various reactive sulfur species toward PKC may provide a prototype for the design of drugs to target a specific group of proteins for therapeutic application.
Huang K-P, Huang FL. Glutathionylation of proteins by glutathione disulfide S-oxide. Biochem Pharmacol 2002;64:1049-1056.
Inhibition of autophosphorylation by oxidative modifications of CaMKII
Shetty, Huang F, Huang K-P
CaMKII is one of the most abundant kinases in neurons, and its autophosphorylation has been linked to the enhancement of synaptic plasticity. Stimulus-induced autophosphorylation of CaMKII-alpha at Thr286 converts the kinase into a constitutively active form that is independent of Ca2+/CaM for activity. During synaptic stimulation, reactive oxygen species are generated at post-synaptic sites where CaMKII is a potential target of the oxidants mentioned above. In vitro treatment of rat brain CaMKII with glutathione disulfide S-monoxide (GS-DSMO) and S-dioxide (GS-DSDO) resulted in thionylation and aggregation of the kinase, forming intermolecular disulfide bonds as revealed by incorporation of glutathione moiety into the kinase from 35S-labeled GS-DSMO and GS–DSDO and by immunoblot analyses with antibodies against CaMKII-alpha and glutathione. Treatment with the reactive sulfur species dose-dependently reduced CaMKII activity assayed with autocamtide as a substrate. The inactivated kinase exhibited reduced Vmax but similar Ka for Ca2+/CaM as the native kinase, suggesting that thionylation of CaMKII occurs primarily at the Cys residues affecting the catalytic domain. The glutathione disulfide S–oxides could completely inhibit autophosphorylation of CaMKII. Treatment of rat brain synaptosomes with oxidants, such as H2O2, diamide, and sodium nitroprusside, caused glutathionylation of several proteins detectable by immunoblot; however, CaMKII primarily formed aggregates that were poorly resolved by nonreducing SDS-PAGE. Depolarization-induced autophosphorylation of CaMKII in these oxidant-treated synaptosome preparations was significantly attenuated. The findings suggest that oxidants generated during normal synaptic stimulation or under pathological conditions could modulate CaMKII activity through oxidative modification by thionylation as well as by forming intersubunit disulfide bonds.
1Now at the Université Bordeaux I, Talence, France
2Jungfa Li, MD, PhD, former Postdoctoral Fellow
COLLABORATORS
Detlef Balschun, PhD, Leibniz Institut für Neurobiologie, Magdeburg, Germany
Tino Jäger, PhD, Leibniz Institut für Neurobiologie, Magdeburg, Germany
Klaus Reymann, PhD, Leibniz Institut für Neurobiologie, Magdeburg, Germany
For further information, contact huangk@mail.nih.gov.