A) NEUREGULIN/ERB-B SIGNALING REGULATES NEURONAL PLASTICITY: POSSIBLE
TO SCHIZOPHRENIA 1. Neuregulin-1 Modulates Hippocampal Gamma Oscillations: Implications for Schizophrenia: Alterations in gamma-frequency oscillations are implicated in psychiatric disorders, and their amplitude (power) have been reported to increase selectively during psychotic episodes. In collaboration with Dr. Andre Fisahn at the Karolinska Institute, we found that NRG-1 dramatically increases the power of kainate-induced gamma oscillations in acute hippocampal slices. NRG-1 effects are blocked by PD158780, a pan-specific antagonist of ErbB receptors, and are absent in slices prepared from ErbB4 null mice. Moreover, we demonstrate that 50% of GABAergic parvalbumin-positive interneurons, which heavily contribute to the generation of gamma oscillations, express ErbB4 receptors. Importantly, both the number of parvalbumin-immunoreactive interneurons and the power of kainate-induced gamma oscillations are reduced in ErbB4 knockout mice. This study provides the first plausible link between NRG-1/ErbB4 signaling and rhythmic network activity that may be altered in persons with schizophrenia. 2. Cellular and Subcellular Expression of the Neuregulin Receptor ErbB4: In order to understand the cellular mechanisms that mediate the above-mentioned effects of NRG-1 on synaptic plasticity and network activity we identified the ErbB4 expressing neurons. We generated novel monoclonal antibodies, and demonstrated they are highly specific for ErbB4. Using these antibodies we analyzed the expression pattern of ErbB4 in four functionally distinct classes of interneurons that represent the majority of all inhibitory neurons in the adult hippocampus of mice. We found high expression levels in three of the four cell classes, indicating that NRG can modulate several inhibitory pathways in the hippocampus. ErbB4 has also been implicated in the generation and maturation of interneurons during development, and consistent with this we found significant reductions of two classes of interneurons in mice that lack ErbB4. We next investigated the subcellular expression of ErbB4 in interneurons, because the exact location is again crucial for understanding the physiological effects of NRG-ErbB4 signaling. Ultrastructural analysis in CA1 interneurons using immunoelectron microscopy revealed abundant ErbB4 expression in the somatodendritic compartment where it accumulates at, and adjacent to, glutamatergic postsynaptic sites. By contrast, we found no evidence for presynaptic expression in cultured GAD67-positive hippocampal interneurons and in CA1 basket cell terminals. Our findings identify ErbB4-expressing interneurons, but not pyramidal neurons, as a primary target of NRG signaling in the hippocampus, and furthermore implicate ErbB4 as a selective marker for glutamatergic synapses on inhibitory interneurons. 3. Conservation of ErbB4 expression in GABAergic interneurons in the hippocampus and cortex, from rodents to primates: Before extrapolating from rodent work how the NRG/ErbB signaling may be affected in psychiatric disorders, it is critical to know that this signaling pathway functions similarly in rodents and primates. To this end, we analyzed the distribution of ErbB4 protein using tissue sections obtained from the hippocampus and cortices of mice and monkeys. We found that, similarly to the hippocampus, ErbB4 receptors accumulate on the somatodendritic compartment of GABAergic interneurons. In the cortex, expression was high in fast-spiking parvalbumin-positive interneurons. This cellular pattern of expression was conserved in rodents and primates, supporting the use of mice as a model system. 4. Neuregulin-1 Regulates LTP at CA1 Hippocampal Synapses Through Activation of Dopamine D4 Receptors: Neuregulin-1 (NRG-1) and ErbB4 are genetically associated with schizophrenia, a neurodevelopmental cognitive disorder characterized by imbalances in glutamatergic and dopaminergic function. Previously we reported NRG-1 suppresses or reverses long-term potentiation (LTP) at hippocampal glutamatergic synapses. Now we demonstrate that NRG-1 stimulates dopamine release in the hippocampus, and reverses early-phase LTP via activation of D4 dopamine receptors (D4R). NRG-1 fails to depotentiate LTP in hippocampal slices treated with the antipsychotic clozapine and other more selective D4R antagonists. Moreover, LTP is not depotentiated in D4R null mice by either NRG-1 or theta-pulse stimuli. Conversely, direct D4R activation mimics NRG-1 and acts by reducing AMPAR currents and increasing receptor internalization. This novel functional link between NRG-1, dopamine and glutamate has important implications for understanding how imbalances in Neuregulin-ErbB signaling can impinge on dopaminergic and glutamatergic function, neurotransmitter pathways associated with schizophrenia. 5. Molecular and Cellular Characterization of NRG-1 (type IV): NRG-1 encodes a family of growth and differentiation factors transcribed from distinct promoters designated type I through type VII, and it has been reproducibly identified as an """"""""at risk gene"""""""" for schizophrenia. Interestingly one of the four single nucleotide polymorphisms comprising HAPice designated as SNP8NRG243177 T/T, is associated with endophenotypes related to schizophrenia, such as reduced prefrontal cortical function, working memory, myelination and premorbid IQ. Since SNP8NRG243177 T/T has characteristics of a functional polymorphism and maps close to DNA sequences encoding NRG-1 type IV transcripts, our goal was to precisely map the type IV transcription initiation site and to investigate the properties and subcellular distribution of NRG-1 type IV protein. We mapped a novel type IV transcription initiation site and isolated two full-length mRNAs encoding type IV proteins. Using an antiserum we raised against the unique type IV N-terminal end of the protein, we found that NRG-1 type IV is targeted to the cell surface and proteolytic cleavage and release of the extracellular domain is promoted by PKC activation. Also we demonstrated that NRG1 type IV is possessed biological activity similar to other releasable NRG-1 isoforms. However, the subcellular distributions of distinct NRG-1 isoforms differ. Unlike NRG-1 type III which are expressed in the somato-dendritic and axonal compartments of neurons, NRG-1 type IV and its close homolog NRG-1 type I are excluded selecctively from axons. These results constitute an important step for understanding how alterations in NRG-1 type IV expression levels associated with SNP8NRG243177 T/T could selectively modify signaling from NRG-1 released from somato-dendritic compartments. B. ACTIVITY-DEPENDENT REGULATION OF MUSCLE TYPES 1. Activity-Dependent Repression of Fast Muscle Genes by NFAT: The NFAT family of calcium-dependent transcription factors has been implicated in the upregulation of genes encoding slow contractile proteins in response to slow-patterned motoneuron depolarization. In collaboration with Dr. Kristian Gundersen, we demonstrated a novel, and unexpected, function of NFATc1 in slow-twitch muscles. Utilizing the Troponin I fast (TnIf) intronic regulatory element (FIRE), we identified sequences that downregulate its function selectively in response to patterns of electrical activity that mimic slow motoneuron firing. A bona fide NFAT binding site in the TnIf FIRE was identified by site directed mutations and EMSAs, and shown to mediated the activity-dependent transcriptional repression of FIRE. siRNA-mediated knockdown of NFATc1 in adult muscles resulted in ectopic activation of the FIRE in the slow soleus, without affecting enhancer activity in the fast EDL muscle. These findings demonstrate a novel function of NFAT as a repressor of transcription of fast contractile genes in slow muscle
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