The general interest of the laboratory is to understand the molecular mechanisms that regulate neuronal and muscle plasticity during development and in response to experience. We are investigating how Neuregulins NRG 1-3), a family of neural factors related to EGF, regulate synaptic transmission at interneuronal synapses. These factors and their receptors have recently been implicated in the neurological disorder, schizophrenia. In a second project, we are studying how different muscle types emergence during development, and how they are differentially regulated by distinct frequencies of muscle depolarization. We have identified a transcription factor, known as General Transcription Factor-3 (GTF3), that contributes the slow mucle phenotype. Interestingly, the gene encoding GTF3 is deleted in individuals with Williams Syndrome. A. NEUREGULIN EXPRESSION IN THE NERVOUS SYSTEM: POSSIBLE ROLE IN SCHIZOPHRENIA. Earlier work by our group and by others, showed that NRG-1 regulates the expression of neurotransmitter receptors for glutamate (NMDA subtype), GABA and acetylcholine. The subsequent demonstration that ErbB4 and NMDA receptors co-localize at glutamatergic synapses with PSD-95, a PDZ protein that couples postsynaptic receptors to signaling complexes, lead us to hypothesize that the NRG/ErbB signaling pathway may modify synaptic properties (Garcia et al. [2000], PNAS, 97:3596-3601). Recent research has supported the view that NRG-1 alters synaptic transmission. The implications of these findings for clinical and basic research are important because genetic studies have associate NRG-1 mutations with schizophrenia, and because heterozygote mice with targeted mutations in the NRG-1 and ErbB receptor genes have reduced NMDA receptor levels and manifest behavioral deficits reported to be consistent with schizophrenia. Future studies on the cellular and molecular targets of the NRG/ErbB signaling pathway should contribute to understanding distinct mechanisms that regulate synaptic plasticity and that may be associated with neurological disorders. As a first step to understanding how the NRG/ErbB signaling pathway regulates synaptic plasticity and its relation to neurological disorders, we characterized the regional and subcellular expression patterns of NRGs 1-3 in developing brain. NRG-1 expression is highest at birth, while NRG-2 mRNA levels increase with development; expression of both genes is restricted to distinct brain regions. In contrast, NRG-3 transcripts are abundant in most brain regions throughout development. The marked differences in the regional and temporal expression patterns of NRGs are consistent with the idea that these factors serve different functions during development. Next, NRG-2 antibodies were generated to analyze the processing, expression and subcellular distribution of this factor in central neurons. Like NRG-1, the transmembrane NRG-2 pro-protein is proteolytically processed in transfected cells and in neural tissues, and its active extracellular domain accumulates on the neuron surface. Despite the structural similarities between NRG-1 and NRG-2, we found that both factors are targeted to distinct subcellular compartments. The distinct temporal, regional and subcellular expression of NRGs suggests their unique and non-redundant roles in neural function. To test this hypothesis, our laboratory has begun to analyze the phenotypes of different NRG knock-out mice. By utilizing multidisciplinary approaches, that include electrophysiology, genomics, and cellular and molecular biology, we aim to elucidate how this signaling pathway regulates synaptic plasticity. This work should lead to a better understanding of the role of NRGs in neurological disorders. B. DEVELOPMENTAL AND ACTIVITY-DEPENDEN REGULATION OF MUSCLE TYPES. The developmental and neuronal regulation of skeletal muscle fiber types provides an excellent model to study how patterned activity regulates plasticity of postsynaptic targets. Our long-term objectives are to identify the DNA regulatory elements and transcription factors that regulate the emergence of slow- and fast-twitch fibers during development, and that are necessary to intraconvert the contractile properties muscles in response to specific patterns of motoneuron activity. The Troponin I slow (TnIs) and fast (TnIf) genes have served as our experimental model because expression of both genes is fiber-type-specific and regulated by selective patterns of electrical impulses that mimic motoneuron activity. Our earlier work showed that General Transcription Factor 3 (GTF3) is highly expressed in most tissues during early fetal development (when muscle types emerge), binds to a DNA element in the TnIs enhancer required for fiber-type specificity, and regulates the slow muscle program in regenerating adult muscles (Calvo et al., [2001] Mol. Cell. Biol. 21: 8490-8503). Utilizing GTF3-mutant mice, we are presently analyzing the role of this factor during early muscle development. Biochemical characterization of GTF3 indicates that its reiterated HLH domains, regulated in part by differential splicing, contribute to the complexity of this nuclear factor. GTF3, like its paralog TFII-I, contains multiple HLH domains and an N-terminal leucine zipper motif. We found that HLH domain 4 is necessary and sufficient for binding the BLM, and that removal of GTF3 N-terminal sequences may unmask the DNA binding domain. Six GTF3 splice variants were found in mice. The gamma-isoform lacking exon 23 and exons 26-28 (encoding the sixth HLH domain) interacted most avidly with the TnIs DNA regulatory element, while the alpha- and beta- isoforms that include these exons fail to bind. The GTF3 leucine zipper promotes homo-dimerization, but not heterodimerization with its close relative TFII-I. We speculate that a large number of GTF3 proteins with different DNA binding properties can be generated in each cell by alternative splicing and combinatorial association of GTF3 polypeptides. The genes encoding GTF3 and TFII-I are lost in a ~2.0 Mb micro-deletion of chromosome 7q11.2 in individuals with Williams Syndrome (WS). Persons with WS have distinctive physical, cognitive and behavior abnormalities that include impaired spatial cognitive skills and myopathies. Although approximately 20 genes are associated with the micro-deletion, recent studies strongly implicate GTF3 and TFII-I with the cognitive deficiencies observed in WS patients. 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, which could be related to myopathies observed in WS. The observation that GTF3 and TFII-I are highly expressed in developing musculature and neurons, raises the possibility that reduction of these nuclear factors during embryogenesis may affect the expression of target genes later in development.
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