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Signal Transduction in Synaptic Transmission and Plasticity

Kuo-Ping Huang, PhD, Head, Section on Metabolic Regulation
Freesia L Huang, PhD, Staff Scientist

We investigate 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 abnormalities. We currently focus on genetically modified mice with deletion of Nrgn, the gene encoding neurogranin (Ng), which is specifically expressed in the brain. The Nrgn knockout (Nrgn-KO) mice exhibit cognitive deficits and several behavioral abnormalities, including hyperactivity, impulsivity, social withdrawal, and attention deficits. Ng is normally expressed at a high level in subsets of neurons in the forebrain. The protein has been implicated in the regulation of Ca²⁺- and Ca²⁺/calmodulin (CaM)–dependent cellular processes important for the enhancement of synaptic transmission and plasticity. In humans, mutation of the NRGN gene has been linked to behavioral abnormalities and cognitive deficits, including schizophrenia, bipolar disorder, and attention-deficit and hyperactivity disorder.

In the neuronal soma and dendrites, Ng levels are very high, and Ng sequesters apoCaM at basal physiological Ca²⁺ levels. Upon synaptic stimulation, the influxed Ca²⁺ displaces Ng from the Ng/apoCaM complex to form Ca²⁺/CaM and free Ng. Ng then becomes a substrate of protein kinase C. The resulting phosphorylated Ng no longer binds to CaM and thus prolongs the stimulation of CaM–dependent enzymes involved in synaptic responses. In addition, free Ng is readily oxidized by oxidants generated during synaptic stimulation, and the resulting oxidized Ng also exhibits a low affinity for CaM, with a functional consequence for synaptic responses similar to that of the phosphorylated Ng. The divers regulation of Ng by Ca²⁺, phosphorylation, and oxidation make this protein a critical player in the modulation of synaptic plasticity. The buffering of CaM by Ng serves as a mechanism to regulate neuronal free Ca²⁺ and Ca²⁺/CaM concentrations. The aim of this project is to delineate the fine-tuning regulatory functions of Ng in neuronal signaling and to design therapeutic approaches to treat cognitive deficits and behavioral disturbances in humans suffering from mutations in Nrgn.

Treatment of Nrgn knockout mice with methylphenidate (Ritalin) improves their cognition and behavioral abnormalities.

Deletion of Nrgn in mice (Nrgn-KO) caused dysfunction in Ca²⁺- and Ca²⁺/CaM–mediated signaling reactions in the brain and impairment of learning and memory and of high frequency stimulation (HFS)–induced long-term potentiation (LTP) in hippocampal slices. Further characterization revealed that such animals also exhibited behavioral abnormalities, including hyperactivity, impulsivity, and deficits in social interaction. The phenotypes likely result from disruption of Ng-regulated signaling; the behavioral deficits could not be improved by environmental enrichment (EE) alone. We treated the Nrgn-KO mice with Ritalin while keeping them under EE conditions. Ritalin is a psychostimulant drug known to increase extracellular dopamine. Experimental animals (including control and drug-treated wild-type and Nrgn-KO mice) were injected with Ritalin or saline for three weeks and then subjected to a battery of behavioral tests for two weeks. Treatment of Nrgn-KO mice with Ritalin improved their cognitive functions, as evidenced by a reduction of the latency time to locate the hidden platform in the water maze and an increase in the freezing time after fear-conditioning. Ritalin also reduced the hyperactivity of Nrgn-KO mice in the open field and increased immobility time in the forced-swim chamber. The drug-treated mutant mice also exhibited improvement of their social interactions and in recognition of novel ones. The drug treatment, however, only had a marginal effect on the performances of the wild-type mice. Measurement of the HFS–induced LTP in the hippocampal CA1 region in vitro showed a positive effect of the drug on the Nrgn-KO mice.

At the cellular level, treatment of Nrgn-KO mice with Ritalin increased glial fibrillary acidic protein (GFAP)–positive astrocytes in the hippocampus, especially prominent in the hilus of dentate gyrus and stratum radiatum of the CA1 region. In the hilus, many astrocytes were congregated at the subgranular zone, where subpopulations of these cells are known to be neural precursor cells. Indeed, the drug-treated Nrgn-KO mice exhibited an increase in the number of proliferative Ki-67–positive cells and doublecortin-positive neuronal precursor cells. We carried out separate experiments with the same drug treatment regimen for five weeks without behavioral testing to assess cell proliferation by injection of BrdU for immunohistochemical localization of the newly generated cells. For both wild-type and Nrgn-KO mice, EE caused an increase in the number of newborn cells, and treatment with Ritalin further elevated these in wild-type mice. Surprisingly, the drug-treated Nrgn-KO mice exhibited a reduction of both the Ki-67– and BrdU–positive cells in the dentate gyrus (DG) subgranular zone. Thus, drug treatment plus intensive behavioral testing are essential to promote neurogenesis in Nrgn-KO mice. Under these conditions, structural remodeling may underlie drug-mediated neurobehavioral responses. The results indicate that Ritalin, a drug commonly used for the treatment of attention-deficit hyperactivity disorder (ADHD), may exert different effects on individuals of different genetic backgrounds. The studies also suggest that, in order to achieve the most beneficial effect of Ritalin, the treatment regimen should also include EE, physical exercise, and cognitive training. The Nrgn-KO mouse model will be useful for the development of new treatment strategy for certain behavioral deficits related to ADHD, schizophrenia, and bipolar disorder.

Enhancement of hippocampal synaptic plasticity in Nrgn-knockout mice by phorbol ester

In neurons, stimulation of PKC is known to enhance transmitter release and facilitate postsynaptic responses by insertion of AMPA receptors. Short-term treatment of hippocampal slices from Nrgn-KO mice with PKC–activating phorbol ester caused synaptic facilitation at the hippocampal CA1 region that lasted for several hours. The treatment also promoted the redistribution of PKCs from soma to dendrites. The phorbol ester–mediated effects were most prominent among those tissue slices from dorsal hippocampus that exhibited positive responses in the field excitatory postsynaptic potential (fEPSP) and amplitude of population spike (POPS). In contrast, for tissue slices from the ventral hippocampus, phorbol ester only enhanced the amplitude of POPS, without significantly affecting fEPSP. For the dorsal hippocampal slices, the phorbol ester–induced stimulation of fEPSP was inhibited by the PKC inhibitor chelerythrine but not by the CaMKII inhibitor KN93, the MEK inhibitor U0126, the protein-synthesis inhibitor anisomycin, or the NMDA–receptor antagonist APV. Following maximal stimulation by phorbol ester, application of theta-burst stimulation (TBS) caused no additional response. However, TBS followed by phorbol ester caused additional potentiation of fEPSP, suggesting that the phorbol ester–mediated responses also overlap with those by TBS. It is intriguing that, for the tissue slices from ventral hippocampus, application of phorbol ester following TBS induced depotentiation.

To further investigate the mechanism of action of phorbol ester, we generated a specific antibody against the PKC phosphorylation sites of Ng and GAP-43/neuromodulin, which share sequence homology and are the specific targets of PKC in the brain. The antibody recognized both the PKC–phosphorylated Ng and PKC-phosphorylated GAP-43 on Western blot but only PKC-phosphorylated GAP-43 in Nrgn-KO mice. Treatment of Nrgn-KO dorsal hippocampal slices with active phorbol ester caused sustained potentiation in the CA1 region and extensive phosphorylation of GAP-43, which is known to promote presynaptic neurotransmitter release. The highest level of GAP-43 phosphorylation was in the stratum lacunosum-moleculare (SLM), where perforant path input from the entorhinal cortex (EC) innervates the CA1 distal apical dendrites. Given that the CA1 neurons from the dorsal and ventral hippocampus receive different input from EC and that activation of PKC among different populations of interneurons in the SLM can cause either activation or inhibition of synaptic response, these complex interactions among different neurons could contribute to the observed different responses to phorbol ester of the dorsal and ventral hippocampus. The positive response of the dorsal hippocampus of Nrgn-KO mice to PKC–mediated long-term facilitation suggests that treatment of these animals with an activator of PKC may improve the synaptic efficacy of this area, which is thought to be associated with cognitive functions.

Identification of neurogranin in cerebral spinal fluid as a biomarker of Alzheimer’s and other neurodegenerative diseases

Ng is one of the most abundant postsynaptic neuronal proteins in the brain and enhances synaptic plasticity by controlling the availability of CaM and elevation of neurotransmitter-evoked calcium transients. In rodents, the hippocampal levels of Ng correlate positively with their cognitive performances and behavioral well-being. In several human diseases associated with cognitive deficits and behavioral abnormalities, such as Alzheimer’s disease and schizophrenia, the levels of Ng in the brain are reduced. Synaptic degeneration occurs early in Alzheimer’s disease and may cause the reduction of Ng in the brain and a rise in its level in the cerebral spinal fluid (CSF). Indeed, CSF Ng levels among Alzheimer’s disease patients are higher than in healthy controls; however, the current assay method is not sensitive enough to detect minor increase during early stage of disease, when therapeutic intervention is most effective. For sensitive detection of the native as well as the proteolyzed Ng in CSF, we generated three Ng–specific antibodies encompassing the entire molecule. In collaboration with Kaj Blennow, we are using these antibodies to characterize the CSF Ng level in patients with cognitive deficits. A successful development of Ng as a biomarker for Alzheimer’s and neurodegenerative diseases will aid the diagnosis, in assessing disease progression, in developing treatment strategies to prevent/retard synaptic degeneration, and in studying the mechanisms of these diseases.

Publications

Huang K-P, Huang FL, Shetty PK. Stimulation-mediated translocation of calmodulin and neurogranin from soma to dendrites of mouse hippocampal CA1 pyramidal neurons. Neuroscience 2011;178:1-12.
Huang K-P, Huang FL. Calcium-sensitive translocation of calmodulin and neurogranin between soma and dendrites of mouse hippocampal CA1 neurons. ACS Chem Neurosci 2011;2:223-230.
Huang FL, Huang K-P. Methylphenidate improves the behavioral and cognitive deficits of neurogranin knockout mice. Genes Brain Behav 2012;11:794-805.

Collaborators

Kaj Blennow, MD, PhD, Göteborgs Universitet, Göteborg, Sweden

Contact

For more information, email kphuang@helix.nih.gov or visit neuroscience.nih.gov/Faculty/Profile/kuo-ping-huang.aspx

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