We investigate the signal transduction mechanisms involved in the enhancement of synaptic transmission and plasticity. Studies of these neural processes are essential to understanding the complex problems of learning and memory. Our approach is to generate genetically modified mice by deletion or expression of a gene specifically expressed in the brain. We have generated a strain of mice devoid of a neural-specific protein, neurogranin (Ng). Ng is normally expressed at a high level in the neurons within the cerebral cortex, hippocampus, and amygdala; it has been implicated in the modulation of synaptic plasticity. The multiple effects of Ng could account for the positive impact of the protein on the behavior of mice.

Roles of Neurogranin in Enhancing the Signalling for Hippocampus-Dependent Learning and Memory
Huang F, Li, Wu, Huang K
Ng binds to calmodulin (CaM) in an inverse Ca2+-sensitive manner; that is, 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 at least twice that of CaM. Upon synaptic stimulation, the influxed Ca2+ displaces 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). Both the phosphorylated and oxidized Ng exhibit lower affinities for CaM than the unmodified protein; thus, the 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 was enhanced by Ng, we speculate that other Ca2+- and Ca2+/CaM-regulated pathways are also upregulated by neurogranin.

Ng is highly concentrated in the cell bodies and dendrites of selective neurons within the cerebral cortex, hippocampus, and amygdala. To examine the role of Ng in neural function, we generated mutant mice devoid of Ng. The Ng knock-out (KO) mice did not exhibit any obvious developmental or neuroanatomical abnormalities but showed impairment of spatial learning and memory. These deficits in the KO mice were accompanied by a defective mechanism in the activation of Ca2+/CaM-dependent protein kinase II, whose activation by autophosphoryla-tion has been linked to the postsynaptic mechanism for the induction of LTP and storage of long-term memory. The concentration of hippocampal Ng in adult wild-type mice is one of the highest of the known neuronal CaM-binding proteins. Induction of LTP caused a rapid phosphorylation of the protein in the hippocampal CA1 region. Testing with the Morris water maze showed significant relationships between the levels of hippocampal Ng among Ng+/- mice and their performances; however, such relationships were less significant among Ng+/+ subjects. These findings suggest that the wild-type mice contain supra-threshold levels of Ng for proficient performance of water maze tasks. Ng-/- mice performed poorly in all spatial tasks for which they were tested and exhibited deficits in LTP and concurrent CaMKII autophosphorylation. The tetanus-frequency response curve of Ng-/- mice is shifted to the right compared with that of the Ng+/+ mice; low-frequency stimulation (5–10 Hz) induced LTD in the former and modest LTP in the latter. Thus, Ng might regulate neuronal function by increasing the efficiency of Ca2+- and Ca2+/CaM-mediated signalling and thus improve the performance in behavioral tests.

Role of NMDA-Mediated Phosphorylation and Oxidation of Neurogranin in Ca2+- and Ca2+/CaM-Regulated Neuronal Signaling in the Hippocampus
Wu, Huang K, Huang F
Activation of the NMDA receptor is one of the critical steps leading to the enhancement of synaptic plasticity, which underlies learning and memory. Calcium influx induced by activation of the 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 contribute to NMDA receptor–dependent LTP. 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 glutathio-nylation 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 showed that NMDA induced a rapid and transient phosphorylation and oxidation of Ng. NMDA also caused activation of various PKC isozymes as evidenced by their phosphorylation, notably at the carboxyl terminal autophosphorylation sites; such activations were much reduced in the Ng knock-out mice. A high degree of phosphorylation of Ca2+/CaM-dependent kinase II and activation of cAMP-dependent protein kinase were also evident in the wild-type mice compared with the kinases of the knock-out mice. The findings demonstrate the functional role of Ng in Ca2+- and Ca2+/CaM-dependent signalling pathways subsequent to stimulation of the NMDA receptor, suggesting that both phosphorylation and oxidation of Ng may regulate neuronal signalling.

Glutathionylation of Proteins by Glutathione Disulfide S-Oxide
Huang K, Huang F
Redox regulation through modifications of proteins has emerged as one of the major cellular responses to oxidative and nitrosative stresses. In the central nervous system, neurotransmission under normal or pathological conditions generates a variety of oxidants. A wide range of modifications of the sulfhydryl group in proteins, including formation of S-nitrosocysteine, cysteine sulfenic acid (Cys-SOH), sulfinic acid (Cys-SO2H), sulfonic acid (Cys-SO3H), inter- and intramolecular disulfide, and mixed disulfide with glutathione (GSH) (Cys-S-SG), have been identified. Many of these modifications are potential sensors of the redox state’s response to changing environments that are induced by growth factors, hormones, neurotransmitters, and cytokines. Thionylation of protein is one of the mechanisms that can serve as protection against oxidative insults and can be involved in cell signalling. Resembling the well-characterized mechanism of protein phosphorylation in cellular regulation, protein S-glutathionylation adds ionic charges by introducing the g-glutamyl tri-peptide into a protein. The potential target proteins for thionylation are likely to be as abundant as those for phosphorylation. However, unlike protein phosphorylation, for which numerous protein kinases have been identified, there is no evidence for the involvement of a specific thionylating enzyme for each target protein. Thus, the specificity for the thionylation may be endowed on each protein by its affinity for the modifiers, namely, thionylating agents, and the accessibility of the sulfhydryl group. The thionylating agent must be highly reactive and preferably have a short half-life so that the reaction will be localized near the origin of the oxidant. Recently, we identified a highly reactive glutathionylating agent, glutathione disulfide S-oxide (GS(O)SG) from the aqueous solution of S-nitrosogluta-thione (GSNO), that fulfills some of these criteria. This compound, also called glutathione thiosulfinate, is the anhydride of glutathione sulfenic acid (GSOH). Introduction of an oxygen atom into a disulfide bond significantly decreases the bond energy and transforms it into a highly reactive agent toward thiol, resulting in the formation of mixed disulfides. The rate of reaction of GS(O)SG with 5-mercapto-2-nitro-benzoate was nearly 20-fold higher than that of GSNO. The mechanism for the formation of GS(O)SG is believed to involve the sulfenic acid (GSOH) and thiosulfinamide (GS(O)NH2) intermediates; the former under-goes self-condensation and the latter reacts with GSH to form GS(O)SG. Many reactive oxygen and nitrogen species are also capable of oxidizing GSH or GSSG to form GS(O)SG, which probably plays a central role in integrating the oxidative and nitrosative cellular responses through thionylation of thiols. Treatment of rat brain tissue slices with oxidants resulted in enhanced thionylation of proteins with a concomitant increase in the cellular level of GS(O)SG, suggesting that the compound may play a second messenger role for stimuli that produce a variety of oxidative species.