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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 |
|
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 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 COLLABORATOR Kristian
Gundersen, PhD, For further
information, contact buonanno@helix.nih.gov |