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developmental regulation of

neuronal and muscle plasticity

Andres Buonanno, PhD, Head, Section on Molecular Neurobiology Irina Karavanov, PhD, Senior Staff Fellow Detlef Vullhorst, PhD, Staff Fellow Oh-Bin Kwon, PhD, Visiting Fellow Marines Longart, PhD, Visiting Fellow Lequin Yun, PhD, Visiting Fellow Zaheer Rana, MS, Predoctoral Visiting Fellow^a

A central question in neurobiology is how nature and nurture contribute to remodeling of the nervous system during development and in response to experience. The general goal of our laboratory is to investigate molecular mechanisms that regulate neuronal and muscle plasticity during development. We are studying how neuregulins, a family of neural factors related to epidermal growth factor (EGF), regulate synaptic transmission at interneuronal synapses. These factors and their receptors are important for regulating behavior in rodents. Recently, numerous genetic studies have linked single nucleotide polymorphisms in the neuregulin gene with schizophrenia in different populations. In a second project, we are studying the emergence of muscle types during prenatal development and how, later in the adult, muscles are regulated by distinct patterns of motor neuron activity that mimic various types of exercise. The developmental and neuronal regulation of skeletal muscle fiber types provides an excellent model for studying how patterned activity regulates plasticity of postsynaptic targets. Our long-term objectives are to identify the signaling pathways and transcription factors that regulate the development of slow- and fast-twitch muscles and their properties in response to specific patterns of motor neuron activity. The slow and fast troponin I genes serve as our experimental model; the expression of both genes is muscle-type–specific and regulated by distinct patterns of electrical impulses that mimic motoneuron activity. Regulation of neuronal plasticity by neuregulin: possible relevance to schizophrenia Longart, Kwon, Buonanno The neuregulin (NRG) family of growth/differentiation factors comprises three genes that give rise to numerous transcripts generated by differential gene splicing. The active domains of NRGs 1-3 are homologous to EGF; the factors all bind and signal via the family of receptor tyrosine kinases known as ErbB 2-4. Earlier work by our group and others showed that NRG-1 elicits changes in the composition of neurotransmitter receptors for glutamate (NMDA subtype), GABA, and acetylcholine. Initially, we found that the co-activation of ErbB receptors by NRG-1 and of NMDA receptors by glutamate is necessary to modify the expression of an NMDA receptor subunit gene, suggesting a cross-talk between these two types of signaling pathways (Ozaki et al., Nature 1997;390:691). 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, led us to hypothesize that the NRG/ErbB signaling pathway may acutely modify synaptic properties (Garcia et al., Proc Natl Acad Sci USA 2000;97:3596). To understand how NRGs contribute to distinct aspects of neural development and function, we initially characterized their regional and subcellular expression patterns in the developing brain. In general, we found that 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. We generated NRG-2 antibodies in order 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 neural tissues, and its active extracellular domain accumulates on the neuron surface. However, despite the structural similarities between NRG-1 and NRG-2, we found that each factor is targeted to distinct subcellular compartments. NRG-2 accumulates in proximal primary dendrites of hippocampal neurons in culture and in vivo but is not detectable in axons or synaptic terminals. We observed a similar dendritic distribution in cortical neurons and cerebellar Purkinje cells. In contrast, NRG-1 is highly expressed in axons of dissociated hippocampal neurons as well as in somas and dendrites. The distinct temporal, regional, and subcellular expression of NRGs suggests their unique and nonredundant roles in neural function. Recent research performed by our group and others supports the view that NRG-1 alters synaptic transmission. The implications of these findings for basic and clinical science may be extremely important in light of recent evidence that associates single nucleotide polymorphisms in the NRG-1 gene with a hightened risk for schizophrenia. To date, studies performed in seven cohorts, including populations in Iceland, Scotland, Ireland, and China, have shown that NRG-1 is a susceptibility gene for schizophrenia. Mutant mice with decreased levels of NRG-1 and ErbB receptors develop normally but exhibit a reduction in NMDA receptors and manifest behavioral deficits. The mice are hyperactive when exposed to new environments and have an altered pre-pulse inhibition startle response, consistent with behaviors seen in schizophrenia. Interestingly, treatment of mice with clozapine, a pharmacological agent used to treat schizophrenia, ameliorated hyperactivity. Future studies on the cellular and molecular targets of the NRG/ErbB signaling pathway should contribute to an understanding of the distinct mechanisms that regulate synaptic plasticity and that may be associated with neurological disorders. Longart M, Liu Y, Karavanov I, Buonanno A. Neuregulin-2 is developmentally regulated and targeted to dendrites of central neurons. J Comp Neurol 2004;472:156-172. GTF3 and development of the slow muscle program Vullhorst, Karavanov, Buonanno General Transcription Factor 3 (GTF3) is highly expressed in most tissues during early fetal development when muscle types emerge. The factor binds to the bicoid-like motif (BLM) in the TnIs enhancer known as SURE (for slow upstream regulatory element), which is required for specific transcription in slow-twitch muscles. We found that differential splicing of the six helix-loop-helix (HLH) motifs of GTF3 contributes to the factor’s complexity. The use of SELEX, a method that selects specific DNA binding sites from random pools of sequences, indicated that several of the HLH motifs exhibited different preferences for DNA sequence. We found that the HLH motif 4 in GTF3 has the highest avidity for DNA and is necessary and sufficient for binding to the BLM site in the TnI SURE. We also found that a leucine zipper domain located at the N-terminus promotes GTF3 homodimerization but not heterodimerization with GTF2i, a protein closely related to GTF3. 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. We are using proteomic approaches to identify proteins that may form larger transcriptional complexes with GTF3. The genes encoding GTF3 and GTF2i are lost in an approximately 2.0 Mb microdeletion of chromosome 7q11.2 in individuals with Williams syndrome (WS). WS patients display distinctive physical, cognitive, and behavioral abnormalities, including impaired spatial cognitive skills and myopathies. Although approximately 20 genes are associated with the microdeletion, recent studies strongly implicate GTF3 and GTF2i in the cognitive deficiencies observed in WS patients. Our studies using ectopically transfected GTF3 constructs in adult muscles and GTF3 knockout mice support a possible role for these factors in regulating muscle contractile properties, which could be related to myopathies observed in WS. The observation that GTF3 and GTF2i are highly expressed in developing musculature and neurons raises the possibility that reduction of these nuclear factors during embryogenesis affects the expression of target genes later in development. Imaging transcription in vivo: regulation of TnI genes by different activity patterns Vullhorst, Rana, Buonanno; in collaboration with Gundersen Firing patterns typical of slow and fast motor units activate genes for, respectively, slow and fast isoforms of contractile proteins. The mechanisms responsible for sensing and decoding distinct patterns of action potentials and converting them into specific changes in gene expression remain unknown. We have used a combination of in vivo muscle transfection, live imaging, and fluorescence quantification to investigate the transcriptional control of the TnIs and TnIf genes in muscles stimulated with activity patterns that mimic either slow or fast motor neurons. We measured transcription in adult muscles by following the fluorescence of the green fluorescent protein (GFP) expressed under the control of the TnIs- and TnIf-regulatory sequences. We found that transcription from the TnIs and TnIf enhancers was increased only when matched with the corresponding slow or fast pattern, respectively. Removal of nerve-evoked activity by denervation, or stimulation with a mismatching pattern, reduced transcriptional activity of both enhancers. The results indicate that the TnI slow and fast enhancers, which we have isolated, can sense and respond to distinct patterns of neuronal activity. Future experiments will focus on identifying signal transduction pathways and transcription factors that mediate these responses. Vullhorst D, Buonanno A. Structure and function of the nuclear factor GTF3. J Biol Chem 2003;278:8370-8379. ^aUniversity of Oslo, Norway COLLABORATOR Kristian Gundersen, PhD, University of Oslo, Norway For further information, contact buonanno@helix.nih.gov