SCS | SHR | SME | SMR
| UMSPC | UMST | SSR
| Main Page
SIGNAL TRANSDUCTION IN SYNAPTIC TRANSMISSION
AND PLASTICITY
|
Kuo-Ping Huang, PhD, Head, Section on Metabolic Regulation Freesia L. Huang, PhD, Staff Scientist Pavan K. Shetty, PhD, Visiting Fellow |
|
| We investigate signal transduction mechanisms involved in the enhancement of synaptic transmission and plasticity. Studies of these neural processes are essential to understanding the complexities of 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 resulted in a strain of mice lacking a neural-specific protein, neurogranin (Ng), which is normally expressed at high levels in selective neurons within cerebral cortex, hippocampus, and amygdala and has been implicated in the modulation of synaptic plasticity. Ng binds to 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 physiological levels of Ca2+, all Cam is sequestered by Ng and, upon synaptic stimulation, the influxed Ca2+ will displace Ng from the Ng/Cam complex to form Ca2+/Cam and free Ng. The buffering of Cam by Ng serves as a mechanism to regulate neuronal free Ca2+ and Ca2+/Cam concentrations. The goals of our project are to define the regulatory function of Ng and to design therapeutic approaches for improving memory in the aging human population and patients suffering from dementia. Neurogranin enhances hippocampus-dependent learning and memory by promoting calcium-mediated signaling F. Huang, Wu, K. Huang, Li* ; in collaboration with Balschun, Jäger, Reymann The neuronal Cam-binding protein Ng, which binds to Cam at the low concentrations of Ca2+ normally found under basal physiological conditions, is localized in the neuronal soma and dendrites and found in particularly 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 hippocampal levels of Ng in adult Ng+/+ mice range from 160 to 330 pmol/mg, which is one of the highest levels among neuronal Cam-binding proteins, whereas, in Ng+/- mice, they range between 60 and 240 pmol/mg protein. Induction of long-term potentiation (LTP) caused rapid phosphorylation of the protein by PKC in the hippocampal CA1 region. When tested in the Morris water maze, Ng+/- mice showed significant relationships between the levels of hippocampal Ng and their performance; however, such relationships were less significant among Ng+/+ mice whose Ng levels apparently were near the threshold value for optimal performance of such tasks. Ng-/- mice performed poorly in all spatial tasks for which they were tested and exhibited deficits in LTP and concurrent CaMKII autophosphorylation. The frequency response curve of Ng-/- mice was shifted to the right compared with that of the Ng+/+ mice; low- frequency stimulations (5 to 10 Hz) induced LTD in the former and modest LTP in the latter. Tetanic stimulation that evoked strong LTP (100 Hz, 1s) displayed a significantly greater Ca2+-transient amplitude in the CA1 pyramidal neurons of Ng+/+ than those of Ng-/- mice. However, the [Ca2+]i decay time constants were similar. The Ca2+-transient amplitudes following weak LTP (100Hz, 400 ms) and long-term depression (LTD) (5 Hz, 3 min) were also greater in Ng+/+ than in Ng-/- mice, albeit less significantly so. The diminished Ca2+ dynamics in the Ng-/- mice is a likely cause for the animals' decreased propensity to undergo LTP. Thus, the higher the Ng concentration, the greater is the number of Ng/CaM complexes that will form, effectively raising [Ca2+]i at any given Ca2+ influx to promote a more potent signal amplification to enhance synaptic plasticity and learning and memory. Watson JB, Khorasani H, Person A, Huang K-P, Huang FL, O'Dell TJ. Age-related deficits in long-term potentiation are insensitive to hydrogen peroxide: coincidence with enhanced autophosphorylation of Ca2+/calmodulin-dependent protein kinase II. J Neurosci Res 2002;70:298-308. Participation of NMDA-mediated phosphorylation and oxidation of neurogranin in the regulation of Ca2+- and Ca2+/calmodulin-dependent neuronal signaling in the hippocampus Wu, K. Huang, F. Huang Activation of the N-methyl-D-asparate (NMDA) receptor is one of the critical steps leading to the enhancement of synaptic plasticity that underlie learning and memory. Calcium influx induced by the activation of this receptor triggers the stimulation of PKC, Ca2+/CaM-dependent protein kinases, adenylyl cyclases, and nitric oxide synthase. It is the activation of these Ca2+- and Ca2+/CaM-dependent enzymes and their downstream targets that contributes to the NMDA receptor-dependent plasticity. Stimulation of PKC causes phosphorylation of Ng, which promotes the release of Ca2+ from intracellular stores through G protein-coupled phosphoinositide second-messenger pathways. In addition, Ng is susceptible to oxidant-mediated modification, which causes thionylation and/or formation of intramolecular disulfide bonds. The latter modification, resembling PKC-mediated phosphorylation, also results in a reduction of binding affinity of CaM. Using mouse hippocampal slices, we demonstrated that NMDA induced rapid and transient phosphorylation and oxidation of Ng. NMDA also caused activation of PKCs, as evidenced by their phosphorylation, whereas such activations were greatly reduced in Ng knock-out mice. A higher degree of phosphorylation of Ca2+/CaM-dependent kinase II and activation of cAMP-dependent protein kinase were also in greater evidence in the wild-type than the knock-out mice. Phosphorylation of downstream targets, including mitogen-activated protein kinases and cAMP response element-binding protein, were significantly attenuated in the knock-out mice. The results suggest that, by its inverse Ca2+-sensitive CaM-binding feature and through its phosphorylation and oxidation, Ng regulates the Ca2+- and Ca2+/ CaM-dependent signaling pathways subsequent to the stimulation of NMDA receptor. We hypothesize that the derangement of hippocampal signal transduction cascades in Ng knock-out mice causes the deficit in synaptic plasticity and the defects in cognitive behaviors in these mice. Wu J, Huang K-P, Huang F. 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.
Effect of environmental enrichment on the cognitive behaviors of neurogranin knockout mice F. Huang, Wu, Kolker, Park, K. Huang Environmental enrichment and exercise can alter neural plasticity and improve cognitive performances of rodents as a result of increasing neurogenesis. Neurogranin (Ng) knockout mice exhibit severe deficits in learning and memory of spatial tasks, induction and maintenance of LTP, and amplification of Ca2+-mediated signaling in hippocampus. To determine if increasing physical activities of these mutant mice can ameliorate their deficits, groups of mutant mice and their wild-type littermates were housed for three weeks in roomier cages, each with several toys and a running wheel for voluntary exploration and exercise. The control groups of both genotypes were housed in the same type of cages without any toy. The wild-type mice in the enriched environment performed significantly better than their controls in both the hidden platform and probe trial versions of the Morris water maze. However, environmental enrichment had no effect on the performance of the mutant mice on these tasks. Hippocampal LTP in the CA1 region induced by high-frequency stimulation was also increased in the enriched group as compared with the control group, whereas no significant change was observed among the mutants. Quantification of the hippocampal levels of Ng by immunoblot revealed that the Ng levels among the wild-type mice housed in the enriched environment were significantly greater than those of the controls. These findings show that environmental enrichment can augment the hippocampal level of Ng, the extent of LTP, and performance in cognitive tasks. Without Ng, as in the case of Ng knockouts, an increase in physical activities has minimal effects on neural plasticity and cognitive behavior. Role of PKC gamma in the phosphorylation of neurogranin: studies with PKC gamma knock-out mice K. Huang, Wu, F. Huang PKC gamma is one of the major Ca2+/phospholipid/diacylglycerol-activated isozymes and is expressed at a high level in the neurons of mammalian brain. Subcellularly, this kinase is localized in the cell bodies, axons, and dendrites but not in the presynaptic terminals. Among all the PKCs, the cellular localization and patterns of developmental expression of PKC gamma resemble those of Ng more closely than those of other PKC isoforms. In the hippocampus, PKC gamma is associated with the NMDA receptor-PSD-95 complexes and is believed to participate in NMDA receptor-mediated signal transduction and enhancement of synaptic plasticity. This brain-specific PKC gamma was believed to be the kinase responsible for the phosphorylation of Ng, an event that has been implicated in signal amplification during synaptic transmission. However, deletion of PKC gamma in mice resulted in only a mild modification of hippocampal LTP induced by high-frequency stimulation and had no effect on synaptic transmission, paired-pulse facilitation, long-term depression, or phorbol-induced LTP. To determine the specificity of the various PKCs in the phosphorylation of Ng and the role of Ng phosphorylation in signal transduction, we investigated the phosphorylation of Ng by PKCs in vitro and in hippocampal slices from the wild-type and PKC gamma null mutant mice. In vitro, Ng is a substrate of the Ca2+-activated the PKC alpha, beta, and gamma group of isoforms but is poorly phosphorylated by the Ca2+-insensitive delta, epsilon, zeta, and eta group of isoforms; the activities of the latter group of PKCs were less than one-third those of the former. Both the glutamate- and NMDA-stimulated phosphorylations of Ng in hippocampal slices of PKC gamma knockout mice were lower than those of the wild type. In contrast, the phorbol ester- and high-frequency-tetanus-induced phosphorylations of Ng were comparable between the PKC gamma knock-out and wild-type mice. The findings suggest that activation of PKC gamma is closely coupled to glutamate receptors, whereas phorbol ester and high-frequency tetanic stimulation cause a global activation of multiple PKCs to phosphorylate Ng. The compensatory effects of other PKCs provide a logical explanation for the mild deficits in synaptic plasticity and cognitive behaviors observed in PKC gamma knock-out mice. signal transduction in the neurogranin knockout mice. J Biol Chem 2002;277:19498-19505. Physiological implication of generating potent thionylating agents, disulfide S-monoxide, and disulfide S-dioxide from oxidation of sulfhydryl compounds by Fenton's reaction K. Huang, F. Huang Redox regulation through modification of proteins has emerged as one of the major cellular responses to oxidative and nitrosative stresses. In the CNS, neurotransmission under normal or pathological condition generates a variety of oxidants. Cellular sulfhydryl compounds are the major reductants to protect against oxidative stress, which is detrimental to the survival of cells if not properly suppressed. Numerous neural diseases, including Alzheimer's and Parkinson's diseases, are believed to result in part from excessive damage to neurons by the endogenously produced oxidants in the presence of iron. It has been suggested that the pivotal role of iron in pathogenesis derives from its participation in Fenton's reaction, which generates a highly reactive and toxic hydroxyl radical via reaction of Fe2+ and hydrogen peroxide. Oxidation of sulfhydryl compounds by oxidants generated from Fenton's reaction is generally thought to result in disulfides. Detailed analysis of the oxidation products of glutathione and the commonly prescribed sulfhydryl-containing antihypertensive drug captopril by hydrogen peroxide and iron revealed that, in addition to each of their respective disulfides, both the disulfide S-monoxide and disulfide S-dioxide of these two compounds were formed. We purified the oxidized products by reverse-phase HPLC and identified them by mass spectrometry. The disulfide oxides can also be generated by oxidation of sulfhydryl compounds with methyltrioxorhenium (CH3ReO3). Both disulfide S-monoxide and S-dioxide are highly reactive toward free sulfhydryl groups to form mixed disulfide; the reaction rate of the latter is much faster than the former when tested by reacting with 5-mercapto-2-nitro benzoate. Both compounds are potent thionylating agents for proteins, which cause reversible modifications of Ng and PKC in vitro. Thionylation of Ng attenuates its phosphorylation by PKC, and thionylation of PKC inhibits the kinase activity, suggesting that oxidants may reduce the phosphorylation of Ng through concerted thionylation of both Ng and PKC. Treatment of rat brain slices with oxidants resulted in a rapid thionylation of Ng, which contains four reactive cysteine residues susceptible to modification The findings indicate that sulfhydryl compounds can protect cells from damage caused by Fenton's reaction through generation of disulfide oxides for reversible modification of proteins. Furthermore, thionylation of proteins serves as a mechanism to transduce the oxidant-mediated signal by modulation of enzymatic activity. Huang K-P, Huang FL. Glutathionylation of proteins by glutathione disulfide S-oxide. Biochem Pharmacol 2002;64:1049-1056. Li J, Huang FL, Huang K-P. Glutathiolation of proteins by glutathione disulfide S-oxide derived from S-nitrosoglutathione. Modifications of rat brain neurogranin/RC3 and neuromodulin/GAP-43. J Biol Chem 2001;276:3098-3105. 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 Joseph B. Watson, PhD, University of California Los Angeles, CA *Jungfa Li, MD, PhD, former Postdoctoral Fellow For further information, contact kphuang@helix.nih.gov |
|
