A) NEUREGULIN/ERB-B SIGNALING REGULATES NEURONAL PLASTICITY: POSSIBLE
TO SCHIZOPRENIA? ? 1. Neuregulin Regulates LTP: Neuregulin-1 (NRG-1) is a trophic/differentiation factor that signals via receptor tyrosine kinases (ErbB 2-4). Understanding NRG/ErbB-4 signaling in the brain is important because this pathway is altered in postmortem brains of persons with schizophrenia and polymorphisms in both genes have been associated with the disorder. We found that NRG-1 reverses ('depotentiates') long-term potentiation (LTP) at hippocampal CA1 glutamatergic synapses in an activity-dependent fashion. Inhibitors that selectively target ErbB tyrosine kinases block NRG-1-dependent depotentiation, and increase LTP levels at synapses already potentiated. Using patch clamp and cell biological techniques, we demonstrated that NRG-1 depotentiates LTP by selectively reducing AMPA, but not NMDA, receptor currents. Live imaging of hippocampal neurons transfected with AMPA receptors fused to superecliptic green fluorescent protein (seGFP), a form of GFP that only fluoresces strongly when expressed on the cell surface, indicates that NRG-1 stimulates the internalization of surface seGluR1-containing AMPA receptors. Consistent with these and our earlier findings, others have shown that NRG-1 and ErbB receptor hypomorphic mice develop normally, but have a reduction in glutamate receptor levels and manifest behavioral deficits. This novel regulation of LTP by NRG-1 has important implications for the modulation of synaptic homeostasis at glutamatergic synapses, which can affect cognition, learning and memory, and for understanding molecular mechanisms that underlie complex disorders like schizophrenia. ? ? 2. ErbB-4 Surface Clustering by PSD-95 at Inhibitory Hippocampal Neurons: To extend on our earlier work, showing that ErbB-4 directly interacts with the postsynaptic density protein PSD-95 at glutamatergic synapses, we investigated the developmental expression and trafficking the receptor. This interaction is of special interest because it may be altered in schizophrenia. Using immunofluorescence analysis in hippocampal slices and dissociated neurons in culture, we found that ErbB-4 receptors are expressed predominantly at glutamatergic synapses in GABAergic interneurons. The trafficking of ErbB-4 in cultured hippocampal neurons was investigated by surface protein biotinylation and antibody labeling of receptors in live cells. We found that ErbB-4 immunoreactivity in developing neurons precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating 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.? ? 3. Function of NMDA NR2C Receptor tested in cells from knockout 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 the function of the NR2C subunit, DNA sequences corresponding to the first 11 exons of the gene protein were removed by homologous recombination. In collaboration with the groups of Drs. Vicini and Wolfe, the NMDAR excitatory postsynaptic currents (EPSCs) were studied in solitary cerebellar neurons cultured in microislands from wild-type (WT) and NR2C-Bgal knock-in mice, as well as NR2A subunit knockout mice. Compared to WT cells, NR2C null granule neurons have larger NMDA-EPSCs, fast decaying currents and increased quantal content. The most striking result is a significant increase in the NMDA-EPSC peak amplitude and charge transfer in NR2C mutant cells which is mostly due to an increase in quantal size, as estimated from miniature NMDA-EPSCs. Interestingly, the protein levels of NR1, NR2A and NR2B in cerebella from 21-day-old NR2C mutant mice are decreased, as compared to wild-type, suggesting a possible compensatory response to the increased NMDA-EPSCs.? ? B. ACTIVITY-DEPENDENT REGULATION OF MUSCLE TYPES? ? The long-term goals of this project are to identify and characterize transcription factors that modify the contractile properties of skeletal muscles during development, and in response to different types of activity in the adult (i.e., exercise). There is emerging evidence that genetic background determines or influences the contractile properties of slow- and fast-twitch skeletal muscles during early development, but that these properties are plastic and can be modified by activity.? ? 1. GTF3 Regulates Skeletal Muscle Properties During Development: Transcription is one of the major mechanisms that determines the fiber-type-specific properties of muscles. We have used the TnIs and TnIf genes as our experimental model system because their expression in mature muscle is restricted to distinct fiber types and is controlled by neuronal activity. We identified a slow (SURE) and a fast (FIRE) TnI enhancer that regulate their fiber-type-specific transcription. A short DNA sequence in the TnIs SURE, necessary to confer slow-specific transcription, interacts with General Transcription Factor 3 (GTF3). GTF3 is expressed and regulates TnIs transcription during fetal and neonatal development. Using a method to select transcription factor DNA binding sites from pools of random oligonucleotides, we delineated the GTF3 consensus site (G/A)GATT(A/G) and confirmed that binding sites in the TnIs SURE and other slow muscle genes conform to this motif. Our studies using ectopically transfected GTF3 constructs in adult muscles and GTF3 knock-out mice support a possible role for this factor in regulating muscle contractile properties. Interestingly, GTF3 is lost in a ~2.0 Mb micro-deletion of chromosome 7q11.2 in individuals with Williams Syndrome (WS), who in addition to having the more commonly reported impairment in spatial cognitive skills, also present myopathies. ? ? 2. TnI SURE and FIRE Transcription is Regulated Differentially by Patterned Activity: Distinct patterns of electrical impulses elicited by motor neurons during exercise differentially regulate the metabolic and contractile properties of adult skeletal muscles, as well as muscle mass. To map the precise DNA regulatory sites that convey the effects of activity, we hooked up the green fluorescent protein (GFP) reporter to the SURE and FIRE TnI enhancers to measure transcription levels in vivo in electrically stimulated adult muscles. We found that slow, tonic depolarization up-regulates transcription from the SURE, while fast, phasic stimulates the FIRE. These results indicate that the TnI slow and fast enhancers can sense, and respond to, distinct patterns of neuronal activity. Experiments are in progress to identify the DNA regulatory elements that mediate the specific responses to patterned activity.
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