A) NEUREGULIN/ERB-B SIGNALING REGULATES NEURONAL PLASTICITY: POSSIBLE
TO SCHIZOPHRENIA 1. Molecular and Cellular Characterization of Neuregulin-1 type IV: Polymorphisms in genes encoding Neuregulin-1 (NRG1) and its receptor ErbB4 are associated with a risk for developing schizophrenia. A single nucleotide polymorphism (SNP) in NRG1, designated SNP8NRG243177 (T/T), is associated with reduced prefrontal cortical function, working memory, myelination and premorbid IQ. Because SNP8NRG243177 (T/T) has characteristics of a functional polymorphism and maps close to DNA sequences encoding NRG1 type IV, 1 of 7 NRG1 mRNA isoforms, our goal was to precisely map the type IV transcription initiation site and to investigate the properties and subcellular distribution of NRG1 type IV protein. We mapped a novel type IV transcription initiation site and isolated two full-length mRNAs encoding type IV proteins. Using our type IV-specific antiserum, we found that pro-NRG1 type IV is targeted to the plasma membrane where it is proteolitically released through a PKC-dependent mechanism, similar to other NRG1 isoforms. However, unlike NRG1 type III, which is expressed in the somato-dendritic and axonal compartments of neurons, NRG1 type IV and its close homolog NRG1 type I are excluded selectively from axons. These results constitute an important step for understanding how alterations in NRG1 type IV expression levels associated with SNP8NRG243177 (T/T) could selectively modify signaling from NRG1 released from somato-dendritic compartments. 2. Conservation of ErbB4 expression in GABAergic interneurons in the hippocampus and cortex, from rodents to primates: The NRG1-ErbB4 pathway is genetically associated with schizophrenia, and because knowledge of the cellular and subcellular localization of the receptor is important for understanding how NRG1 regulates neuronal network activity and behavior, it is critical to resolve where ErbB4 receptors are expressed in the primate cortex using reagents with demonstrated specificity. Using a combination of electrophysiological profiling of pyramidal and GABAergic cells and single-cell RT-PCR in mice, we demonstrated in collaboration with Dr. McBain's group that ErbB4 transcripts are expressed selectively in interneurons in the frontal cortex. Using highly specific monoclonal antibodies we developed and carefully characterized, we showed that ErbB4 protein is undetectable in pyramidal cells of rodent, rhesus monkey, and human frontal cortex. In collaboration with Dr. Lewis's group, we found that almost all interneurons positive for parvalbumin, calretinin or cholecystokinin, but only a minority of calbindin-positive cells, coexpress ErbB4. Of note, no presynaptic ErbB4 expression was detected in any species. Our data validate the use of rodents to analyze cellular and neural circuit effects of abnormal ErbB4 function as a means to model endophenotypes pertinent to psychiatric disorders. 3. Analysis of ErbB4 protein expression throughout the primate cerebrum: Using the highly specific monoclonal antibodies described above, we found that ErbB4 levels are high in association cortices, intermediate in sensory cortices, and relatively low in motor cortices. The overall immunoreactivity in the hippocampal formation is intermediate, but is high in a subset of interneurons. We detected the highest overall immunoreactivity in distinct locations of the ventral hypothalamus, medial habenula, intercalated nuclei of the amygdala and structures of the ventral forebrain, such as the islands of Calleja, olfactory tubercle and ventral pallidum. While this pattern is generally consistent with ErbB4 mRNA expression data, further investigations are needed to identify the exact cellular and subcellular sources of mRNA and protein expression in these areas. We detected only low levels of ErbB4-immunoreactivity in dopaminergic nuclei of the mesencephalon but additional medium-to-high expression in the dorsal striatum and high expression in the ventral forebrain, suggesting that most ErbB4 protein in dopaminergic neurons could be transported to axons. Based on these findings, we conclude that the NRG-ErbB4 signaling pathway can potentially influence many functional systems throughout the brain of primates, and suggest that major sites of action are areas of the corticolimbic network. This interpretation is functionally consistent with the genetic association of NRG1 and ERBB4 with schizophrenia. 4. NRG1-signaling influences AMPA receptor mediated responses in primary cerebellar granule neurons: Previous studies demonstrated that NRG1 affects plasticity at glutamatergic synapses in principal glutamatergic neurons in the hippocampus and frontal cortex;however, these effects are indirect because expression of ErbB4 is confined to GABAergic interneurons in these brain regions. In collaboration with Dr. Catherine Fenster we analyzed the effects of NRG1 on developing cultured cerebellar granule cells (CGCs). These cultures have the advantage that they are relatively homogenous and consist primarily of granule neurons that express ErbB4. We found that NRG1 does not affect whole-cell AMPAR or NMDAR mediated currents, nor the frequency or amplitude of spontaneous NMDAR- or AMPAR-mediated miniature excitatory post-synaptic currents, in baseline conditions of CGCs grown for 10-12 days in vitro. However, we found that high-glycine induces a form of chemical potentiation (chemLTP) in CGCs characterized by an increase in AMPAR-mEPSC frequency that is decrease by NRG1 treatment. Because in our culture conditions CGCs express very low levels of the AMPA receptor GluR1 subunit, our data suggest that the NRG1 effect could be mediated via GluR4 subunits that are highly expressed. This study provides first evidence that (1) high-glycine can induce plasticity at glutamatergic synapses in CGCs, and that acute NRG1/ErbB-signaling can regulate glutamatergic plasticity in CGCs. Taken together with previous reports, our results suggest that, similar to Schaeffer collateral to CA1 synapses, NRG1 effects are activity dependent and mediated via modulation of synaptic AMPARs. B. ACTIVITY-DEPENDENT REGULATION OF MUSCLE TYPES 1. Activity-Dependent Regulation of Fast Muscle Genes by ets-family Factors: Most studies on the activity-dependent regulation of muscle genes have focused on genes encoding slow contractile proteins in response to slow-patterned motoneuron depolarization (approx. 10 -20 Hz). However, as we and others have shown, fast-patterned (>50 Hz) activity regulates the expression of genes encoding muscle proteins specific to fast-twitch muscles. In collaboration with Dr. Kristian Gundersen, we have used cDNA microarrays to analyze the effects of skeletal muscle denervation, and stimulation of denervated muscles with either slow or fast patterns of activity, on myofiber-type-specific gene expression. Expression of mRNA was analyzed at multiple time points following the stimulation of muscles to analyze temporal expression profiles of transcription factors and myofibril proteins. Interestingly, we identified that members of the ets family of transcription regulators, which are sensitive to calcium levels, are selectively regulated by fast-patterned activity shortly after commencing stimulation. Utilizing the Troponin I fast (TnIf) intronic regulatory element (FIRE), we identified sequences that are necessary to regulate transcription in response to fast patterned activity and correspond to potential ets consensus binding sites. siRNA-mediated knockdown experiments confirm the specificity and importance of these factors to regulate TnIf in response to fast depolarization patterns.
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