This laboratory investigates the signal transduction in synaptic transmission and plasticity by biochemical, behavioral, and electrophysiological approaches using a strain of genetically modified mice. The mutant mouse was generated by deleting a gene coding for neural-specific protein, neurogranin (Ng). This protein is expressed at high levels in hippocampus, cerebral cortex, and amygdala, and has been implicated in the modulation of synaptic plasticity. Ng is a small molecular weight protein, which binds calmodulin (CaM) under basal physiological conditions when cellular calcium level is low. The binding of Ng and CaM is weakened upon neuronal stimulation that leads to calcium influx. When dissociated from CaM, Ng is phosphorylated by PKC and/or oxidized by nitric oxide and other oxidants. The phosphorylated and oxidized Ngs are poor binding partners of CaM. These multifaceted regulations of Ng provide a fine tune mechanism to control the calcium transients and availability of CaM depending on the strength of stimulation to the neurons and, thus, gate the output response. Deletion of Ng gene in mice causes deficits in learning the hippocampus- and amygdala-dependent behavioral tasks, and the high-frequency stimulation (HFS)-induced long-term potentiation (LTP). In human, mutation of Ng gene has been linked to cognitive deficits, schizophrenia, and bipolar disorder. Deletion of Ng in mice (NgKO) caused deficits in calcium-mediated signaling in hippocampus, impairment in learning and memory, and high frequency stimulation (HFS)-induced long-term potentiation (LTP). These animals also exhibited other behavioral abnormalities, including hyperactivity and social withdrawal. These behavioral deficits could not be improved by environmental enrichment (EE). We have treated these NgKO mice with Ritalin while they were also kept under EE conditions. Ritalin is a psychostimulant drug known to increase the extracellular neurotransmitters. Experimental animals were injected with Ritalin or saline for three weeks and, afterward, subjected to a battery of behavioral tests for two weeks. Treatment of NgKO with Ritalin improved their cognition 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 NgKO in the open field and an increase in the immobility time in the forced-swim chamber. The drug-treated mutant mice also exhibited improvement of their social interaction. The drug treatment, however, only had a marginal effect on the performances of wild type mice. At the cellular level, treatment of NgKO with Ritalin increased glial fibrillary acidic protein (GFAP)-positive astrocytes in the hilus of dentate gyrus (DG) and stratum radiatum of the CA1 region. Also, the drug-treated NgKO exhibited an increase in the number of proliferative Ki-67-positive cells and doublecortin-positive neuronal precursor cells. A separate set of experiment was done with the same drug treatment regimen for five weeks without behavioral testing to assess cell proliferation by injection of BrdU for immunohistochemical localization of newly generated cells. For both wild type and NgKO mice, EE was beneficial for increasing the number of new born cells and treatment of Ritalin further elevated those of the wild type mice. Surprisingly, the drug-treated NgKO exhibited a reduction of both the Ki-67- and BrdU-positive cells in the DG subgranular zone. Thus, drug treatment plus intensive behavioral testing are essential to promote neurogenesis of NgKO. Under these conditions, structural remodeling may underlie drug-mediated neurobehavioral responses. These results indicate that Ritalin, a drug commonly used for the treatment of attention-deficit hyperactivity disorder (ADHD), may exert different effects on individual of different genetic background. These 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 NgKO mice model will be useful for the development of new treatment strategy for certain behavioral deficits related to ADHD. Treatment of hippocampal slices from NgKO mice with PKC-activating phorbol ester caused synaptic facilitation at the hippocampal CA1 region. The phorbol ester-mediated effects were most prominent among those tissue slices from dorsal hippocampus, which exhibited elevation in the field excitatory postsynaptic potential (fEPSP) and amplitude of population spike (POPS). In contrast, for tissue slices from ventral hippocampus, phorbol ester only enhanced POPS without significantly affecting fEPSP. For the dorsal hippocampal slices, the phorbol ester-induced stimulation in fEPSP was inhibited by PKC inhibitor chelerythrine but not by inhibitors of CaMKII, MEK, and protein synthesis, nor by NMDA receptor antagonist, APV. Following 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 study the action of phorbol ester, we generated an antibody against PKC phosphorylation sites of Ng and GAP-43, which share sequence homology. This antibody recognized both the PKC-phosphorylated Ng and GAP-43 and only GAP-43 in NgKO. Treatment of NgKO dorsal hippocampal slices with phorbol ester caused potentiation in the CA1 region and phosphorylation of GAP-43, which is known to promote neurotransmitter release. The highest level of GAP-43 phosphorylation was in stratum lacunosum-moleculare (SLM), where perforant path input from the entorhinal cortex (EC) innervates the CA1 distal apical dendrites. Since the CA1 neurons from dorsal and ventral hippocampus receive different input from EC and that activation of PKC among different population of interneurons in SLM can cause either activation or inhibition of synaptic response, these complex interactions among different neurons could contribute to the observed differential responses to phorbol ester between the dorsal versus ventral hippocampus. The positive response of dorsal hippocampus of NgKO to PKC-mediated facilitation suggests that treatment of these animals with activator of PKC may improve the synaptic efficacy of this area, which is thought to associate with cognitive functions. Ng is one of the most abundant neuronal proteins in the brain and, in rodents, the hippocampal levels of Ng correlate positively with their cognitive performances. 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 were reduced. Synaptic degeneration occurs early in Alzheimer's disease development that may cause the reduction of Ng in the brain and increase its level in the cerebral spinal fluid (CSF). Indeed, the CSF Ng levels among Alzheimer's disease patients are elevated compared to healthy control;however, the current assay method is not sensitive enough to detect minor increase during early stage of disease development when therapeutic intervention is most effective. We have generated three Ng-specific antibodies encompassing the entire molecule for detection of the native and the proteolyzed Ng in CSF. In collaboration with Kaj Blennow these antibodies are being used 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 and assessing the disease progression.
|Shetty, Pavan K; Huang, Freesia L; Huang, Kuo-Ping (2008) Ischemia-elicited oxidative modulation of Ca2+/calmodulin-dependent protein kinase II. J Biol Chem 283:5389-401|