Signal Transduction in Synaptic Transmission and Plasticity

Kuo-Ping Huang, PhD, Head, Section on Metabolic Regulation
Freesia L. Huang, PhD, Staff Scientist
Guo-Chiuan Hung, PhD, Postdoctoral Fellow
Pavan K. Shetty, PhD, Postdoctoral Fellow

We investigate the signal transduction mechanisms involved in synaptic transmission and plasticity. Studies of these neural processes are essential to understanding the complex problems related to cognition and behavioral disturbances. Our approach is to generate genetically modified mice by deletion or expression of a gene specifically expressed in the brain. Our efforts have led us to generate a strain of mice devoid of neurogranin (Ng), a neural-specific protein. Ng 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 levels in the neuronal soma and dendrites are very high, and the protein sequesters apoCaM at basal physiological Ca²⁺. Upon synaptic stimulation, influxed Ca²⁺ displaces Ng from the Ng/apoCaM complex to form Ca²⁺/CaM and free Ng. The buffering of CaM by Ng functions as a mechanism to regulate 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 treat cognitive deficits and behavioral disturbances.

Regulation of synaptic plasticity by neurogranin

Huang K-P, Huang F

Ng binds to apoCaM under basal physiological conditions and dissociates from CaM upon synaptic stimulation that raises intracellular Ca²⁺. 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 phosphokinase C (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, a significant relationship exists between the hippocampal levels of Ng and performance in several cognitive tasks. Ng^−/− mice perform poorly in all such tasks; they also display deficits in high-frequency stimulation (HFS)–induced long-term potentiation (LTP) in area CA1 of hippocampal slices but enhanced 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 the animals’ decreased propensity to undergo LTP and of their deficits in cognitive function.

An immunohistochemical study showed that, in the wild-type mice, Ng was localized in the cell bodies and dendrites of the principal neurons in the hippocampus, whereas CaM concentrated in the nucleus, with a relatively low level in the dendrites. However, following HFS of the Schaffer/collateral fibers 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. Given that the HFS-induced LTP is initiated by Ca²⁺ influx through NMDA receptors, we reasoned that stimulation of postsynaptic downstream signaling components or increasing 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 (an adenylate cyclase activator), rolipram (a phosphodiesterase inhibitor), and picrotoxin (a GABA_A inhibitor) to Ng^−/− hippocampal slices also effectively induced LTP. By stimulating cAMP/PKA signaling and suppressing the GABA_A-mediated inhibitory pathway, these drugs could bypass the requirement of Ng to induce LTP. Histone deacetylase (HDAC) inhibitors are known to enhance both memory and synaptic plasticity at transcriptional level. Bath application of an HDAC inhibitor, trichostatin A, could also augment the HFS-induced LTP in the Ng^−/− hippocampal slices. Using these chemically induced LTP protocols, we have established a potential drug treatment regimen to ameliorate the behavioral deficits of Ng^−/− mice.

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.

Effect of environmental enrichment and drug treatment on the cognitive behaviors of neurogranin knockout mice

Huang F, Huang K-P

Synaptic responses triggering LTP or 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 (calcium/calmodulin-dependent protein kinase II) by autophosphorylation. More specifically, deletion of Ng in mice caused severe deficits in learning and memory of spatial tasks, in induction and maintenance of LTP, and in amplification of Ca²⁺-mediated signaling in the hippocampus. However, environmental enrichment (EE) can increase neurogenesis, alter neural plasticity, and improve cognitive performance. To that end, we attempted to employ EE and drug treatment to improve the cognition and behavioral abnormalities of the mutant mice.

To determine if an increase in the physical activities of the mutant mice can improve their performance of memory tasks, we transferred groups of mutant mice and their wild-type littermates of different age groups to 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 any toys. Provision of short-term environmental enrichment (SEE) (3 weeks for the mutant mice) did not improve their performance but did benefit the wild-type and heterozygous mice, whose hippocampal Ng levels and LTP were higher under SEE than in control mice. Interestingly, for Ng KO mice, SEE caused a negligible effect on their LTP even though other important signaling components for synaptic plasticity, including CaMKII and cAMP-responsive element–binding protein (CREB), were elevated to the same levels as in the wild-type and heterozygous mice. In contrast, a long-term environmental enrichment (LEE) for the aging mice benefited the Ng KO as well as the wild-type and heterozygous mice by preventing age-related cognitive decline. LEE also caused an increase in the hippocampal CREB level of all three genotypes and the Ng level of wild-type and heterozygous mice but not the levels of CaMKII or ERK (extracellular signal-regulated kinase). Hippocampal slices from the enriched aging Ng KO mice, unlike those from the wild-type and heterozygous mice, did not show enhancement in LTP. It appears that the learning and memory processes in the enriched aging Ng KO mice do not correlate with LTP, which is facilitated by Ng. The 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, we treated the animals with Ritalin®, which has proven 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 for investigating the cause and treatment strategy for ADHD.

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;14:512-519.
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;26:6230-6237.

Ischemia-elicited oxidative modulation of CaMKII in the brain

Shetty, Huang F, Huang K-P

CaMKII is one of the major Ca²⁺-sensing enzymes involved in transducing neuronal, hormonal, and electrical signals in brain, heart, and other tissues. In the central nervous system, CaMKII plays a pivotal role in facilitating synaptic plasticity, learning and memory, and activity-dependent developmental processes. The CaMKII holoenzyme is a dodecamer composed of two stacked hexameric rings, and the kinase undergoes inter-subunit autophosphorylation in the presence of Ca²⁺/CaM that converts the kinase into an activator-independent autonomous enzyme. The 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. 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 the aggregation of CaMKII.

Previously, we showed that activation of NMDA receptors or direct administration of oxidants to brain slices could trigger thionylation (oxidation) of neurogranin and formation of intramolecular disulfide. We hypothesized that the oxidant-induced modification of protein resulted from oxidation by the endogenously produced glutathione disulfide S-oxides (GS-DSO). To investigate the mechanism and physiological response of CaMKII oxidation, we treated mouse brain synaptosomes with H₂O₂, diamide, and sodium nitroprusside. These oxidants caused aggregation of CaMKII through formation of disulfide and non-disulfide linkages and partial inhibition of the kinase activity. We found that CaMKII aggregates associated with the postsynaptic density. However, treatment of purified CaMKII with the oxidants did not replicate the 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 similar to the oxidant-treated synaptosomes—reminiscent of oxidative stress of acutely prepared hippocampal slices elicited by ischemia. Interestingly, the autophosphorylated and activated CaMKII was relatively refractory to GS-DSMO– and GS-DSDO–mediated aggregation. Short-term ischemia (10 minutes) caused a depression of basal synaptic response of the hippocampal slices, and re-oxygenation (after 10 minutes) reversed the depression. However, oxidation of CaMKII remained above the pre-ischemic level throughout the treatment. Oxidation of CaMKII also prevented full recovery of CaMKII autophosphorylation after re-oxygenation. Subsequently, the HFS-mediated synaptic potentiation in the hippocampal CA1 region was significantly lower than in 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.

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;20:46:1961-1971.
Shetty PK, Huang FL, Huang K-P. Ischemia-elicited oxidative modulation of Ca²⁺/calmodulin-dependent protein kinase II. J Biol Chem 2008;283:5389-5401.

Collaborator

Alfred L. Yergey, PhD, Mass Spectrometry Core Facility, NICHD, Bethesda, MD

For further information, contact huangk@mail.nih.gov.