A) NEUREGULIN/ERB-B SIGNALING REGULATES NEURONAL PLASTICITY: POSSIBLE
TO SCHIZOPHRENIA? ? 1. 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.? ? 2. 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.? ? 3. Regulation of ErbB-4 Endocytosis by Neuregulin-1 in GABAergic Hippocampal Interneurons: We investigated the developmental expression and trafficking the receptor. Interactions between ErbB4 and PSD95 are of special interest because they are altered in postmortem brain from schizophrenia patients. Using immunofluorescence and dissociated neurons in culture, we found that ErbB4 receptors are expressed predominantly at glutamatergic synapses in GABAergic interneurons. ErbB4 trafficking studies, using biotinylation and antibody live labeling, indicate that surface receptors precedes PSD-95 expression. ErbB-4clusters initially form in the absence of, but later associate with, PSD-95-positive puncta. The surface fraction of dendritic ErbB-4 increases with age, and NRG-1 triggers its internalization in young and mature neurons. These findings enhance our understanding of the role of ErbB-4/PSD-95 protein interaction for NRG-mediated signaling at glutamatergic synapses.? ? 4. Novel Regional and Developmental NMDA Receptor Expression Patterns Uncovered in NR2C Subunit-beta-Galactosidase Knock-in Mice: We previously reported that expression of the NR2C subunit of the NMDAR is regulated by NRG-1 in cultured organotypic slices from cerebellum. To study NR2C expression and function, NMDA receptor """"""""knock-in"""""""" mice were generated by inserting the nuclear beta-galactosidase reporter at the translation initiation site using homologous recombination. Novel cell-types and dynamic patterns of NR2C expression were identified using these mice, which went previously unnoticed because reagents that specifically recognize NR2C-containing receptors were non-existent. We found that NR2C is expressed in transient caudalrostral gradients and parasagittal bands in subsets of cerebellar granule cells residing in the internal granular layer. We identified a transition zone from NR2C-expressing neurons to astrocytes in an area connecting the retrosplenial cortex and hippocampus. We demonstrated that NR2C is expressed in a subset of S100beta-positve/GFAP-negative glial cells in the striatum, olfactory bulb and cerebral cortex. We also found novel areas of neuronal expression such as pontine and vestibular nuclei, thalamus and retrosplenial cortex. In collaboration with Drs. Logan and Vicini, we found that magnesium-insensitive NMDAR currents in medium spiny neurons of the striatum are not mediated by NR2C-containing receptors, consistent with our observation that striatal NR2C is restricted to other cells. ? ? 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 muscles.? ? 2. PPARdelta Expression is Influenced by Muscle Activity and Induces Slow Muscle Properties: Metabolites like free fatty acids (FFAs), generated during exercise, could activate transcriptional pathways that regulate fiber types. In collaboration with Dr. Gundersen, we found that mRNA levels for peroxisome proliferator-activated receptor (PPAR)-delta were 3-fold higher in the slow/oxidative soleus compared to the fast/glycolytic EDL muscle. PPARdelta mRNA levels were reduced by > 50% in solei stimulated with fast patterned activity, while levels increased by 3-fold in EDLs stimulated with slow-patterned activity. Over-expression of a constitutively active form of PPARdelta in normally active adult fibers tripled the number of I/IIa hybrids in the fast EDL muscle and increased the activity of the oxidative enzyme succinate dehydrogenase. Therefore, PPARdelta may provide an important link coupling activity with transcriptional changes in slow muscles.? ? 3. Transcription Factor TEAD4 Specifies the Trophectoderm Lineage at the Beginning of Mammalian Development, but Is Not Necessary for Skeletal Muscle Formation: A targeting vector was generated to conditionally mutate the Tead4 gene by homologous recombination, since this factor was found to bind the TnI slow enhancer. Initial analysis using Tead4 null mice, in collaboration with Dr. DePamphilis's lab, indicated Tead4-/- embryos do not to express trophectoderm specific genes, and the morulae do not produce trophoblast stem cells, trophectoderm or blastocoel cavities, and therefore fail to implant. In contrast, progeny from Tead4floxed X Meox2-Cre mice used to circumvent early lethality (after E5.5), did not manifest noticeable abnormalities. Future experiments using Teadflox mice will be used to assess possible role in fiber type properties in the adult.
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