The general interest of the laboratory lies in understanding the molecular
mechanisms that regulate neuronal and muscle plasticity during development.
Until recently, a commonly held view in neurobiology was that neural inputs
(particularly neuronal activity) are instructive to the functional properties
of postsynaptic targets. Recently, a different picture has begun to emerge.
Early in development and independently of innervation, lineage plays a
fundamental role in the pre-patterning of targets. Later, during the maturation
of neural connections, activity can modify the plastic properties of postsynaptic
targets. Moreover, the view that electrical activity exerts its effects
predominantly by regulating the levels of intracellular calcium is undergoing
revision because of the realization that the action of growth/differentiation
factors on synaptic function is activity-dependent and can occur in the
time scale of minutes. We are using two experimental models to investigate
how neural factors and activity regulate neuronal and muscle plasticity
during development. In the first project, we are investigating how neuregulins
acutely regulate synaptic transmission at interneuronal synapses. In the
second, we are studying how lineage and motoneuron activity contribute
to the emergence and plasticity of different muscle types.
Neuregulin Effects on Synaptic Plasticity: Possible
Role in Schizophrenia
Buonanno
Neuregulins (NRG 1-3) are growth/differentiation factors that signal by
means of a family of receptor tyrosine kinases known as ErbB 1-4. Present
knowledge of NRGs mostly originates from studies on NRG-1. The pro-NRG-1
is synthesized as a transmembrane precursor in the soma, then transported
down axons, and proteolytically released in its active form in an activity-dependent
fashion. Earlier work by our group and others showed that long-term exposure
(more than two days) of neurons to NRG-1 elicits changes in the composition
of neurotransmitter receptors for glutamate (NMDA subtype), GABA, and
acetylcholine by selectively regulating the expression of distinct receptor
subunits. We found that co-activation of both glutamate and ErbB receptors
is necessary for NRG-1 to induce NMDA receptor expression, suggesting
a cross-talk between these signaling pathways. The subsequent demonstration
that ErbB4 and NMDA receptors co-localize at glutamatergic synapses with
PSD-95, a PDZ protein coupling postsynaptic receptors to signaling complexes,
led us to hypothesize that the NRG/ErbB signaling pathway may acutely
modify synaptic properties (Garcia et al.,
2000).
FIGURE 20
Expression of NRG-1, NRG-2, and NRG-3 mRNAs in the
developing central nervous system. Sections from E15, P0, P7.5, and
adult mice were hybridized with specific P33-labeled
cRNA probes for NRGs 13. The regional and developmental profiles
of each NRG differ, suggesting that they may have different biological
functions. |
NRG and ErbB Receptor Expression in the Developing
Central Nervous System
Longart, Liu, Vasudevan, Karavanova, Buonanno;
in collaboration with Carroll
We investigated the regional and temporal expression of NRGs and their
receptors because such information is essential for understanding how
the NRG/ErbB pathway contributes to neuronal function. We used multiple
approaches in our studies, including in situ
hybridization, Western blots, and immunofluorescence histochemistry. The
patterns of NRG 1-3 mRNA expression differ markedly during development
(Fig. 20). In general, NRG-1 expression is highest early in development
and becomes restricted postnatally. NRG-2 mRNA levels in embryonic and
newborn mouse brains are low compared with those of NRG-3. Seven days
after birth, the highest levels of NRG-2 are found in the dentate gyrus,
olfactory bulb, and cerebellum. In contrast, NRG-3 is the most highly
expressed NRG in the brain and shows the least regional or developmental
regulation. To study the cellular and subcellular distribution of these
proteins in brain, we generated and characterized antibodies against NRG-2
and NRG-3. Preliminary studies indicate that NRG-2 and NRG-3 accumulate
in neurons, although expression in glia cannot be ruled out. Interestingly,
the subcellular distributions of NRG 1-3 differ, suggesting that NRGs
may perform distinct functions during neuronal development and maturation.
Although ErbB receptors are critical for neural development, their regional
distributions during development and in the adult were unknown. ErbB receptors
are differentially expressed in neurons and glia. ErbB2 is expressed in
most cells, ErbB3 is predominantly found in glia and subpopulations of
neurons, and ErbB4 is mostly restricted to neurons and oligodendrocytes.
The punctate labeling with ErbB2 and ErbB4 antibodies is highest in synapse-rich
regions, which is consistent with the previous finding that ErbB receptors
accumulate at postsynaptic densities (Garcia et
al., 2000). ErbB4 receptors are mostly restricted to the dendrites
and cell bodies of GABAergic neurons, where they co-localize at excitatory
synapses with NMDA receptors and PSD-95. The existence of complexes of
these proteins at postsynaptic densities suggests that the NRG/ErbB pathway
may function to regulate synaptic function acutely.
NRG-1 Acutely Modifies Synaptic Transmission
in Cultured Hippocampal Neurons
Liu, Longart, Buonanno; in collaboration with
Vicini
To test the aforementioned hypothesis, we analyzed the acute affects of
NRG on synaptic transmission. A brief application of 5nM NRG1b1
(about two minutes), which causes the phosphorylation of ErbB2 and ErbB4,
induces a persistent and significant increase in the firing patterns of
cultured hippocampal neurons. These effects are specific because they
are not observed with the NRG-a2 splice variant
that differs slightly with NRG1-b1 and is
a weaker activator of ErbB receptors. The findings suggest that NRGs may
play important roles in synchronizing synaptic or network activity of
hippocampal circuitry. Experiments are in progress to understand the underlying
mechanisms regulating synaptic plasticity in response to NRG in dissociated
hippocampal fresh slices and cultures.
The implications of these findings for basic and clinical science may
be extremely important in light of a recent study associating NRG-1 mutations
with schizophrenia in Icelandic families. Moreover, mutant mice with decreased
levels of NRG-1 and ErbB receptors have fewer NMDA receptors and manifest
behavioral deficits reported to be consistent with schizophrenia. Pharmacological
agents used to treat schizophrenia reverse these behavioral changes in
mutant mice.
Contribution of Developmental History and Neural
Activity to the Fiber-Type Specificity of Troponin I Genes
Buonanno
The developmental and neuronal regulation of skeletal muscle fiber types
provides an excellent model in which to study how patterned activity regulates
plasticity of postsynaptic targets. Our long-term objectives are to identify
transcription factors that both regulate the emergence of slow- and fast-twitch
fibers during development and intraconvert their contractile properties
in response to specific patterns of motoneuron activity. The troponin
I slow (TnIs) and fast (TnIf) genes serve as our experimental paradigm
because their expression is fiber-typespecific and regulated by
selective patterns of electrical impulses that mimic motoneuron activity.
We have determined that the General Transcription Factor 3 (GTF3), expressed
in numerous tissues including muscle and brain, contributes to the establishment
of fiber types during perinatal development (Calvo et
al., 2001).
Transcription Factor GTF3 Contributes to the
Slow Muscle Program
Vullhorst, Karavanova, Buonanno
The TnIs is activated during terminal myogenic differentiation in all
skeletal muscles regardless of their future fiber type and is later confined
to prospective slow fibers during fetal development. The SURE element
(for slow upstream regulatory enhancer), which
confers slow fiber specificity to TnIs expression, requires interactions
between multiple transcription factors. The bicoid-like
motif (BLM) in SURE is bound by GTF3 and is required for slow fiber-specific
expression. GTF3 is expressed at its highest levels during fetal development
in numerous tissues, and its expression in muscles is repressed after
birth. Experiments in adult regenerating muscles (which re-express GTF3)
transfected with GTF3 by using electroporation, as well as analysis of
GTF3 mutant mice, support the role of GTF3 in regulating the slow-twitch
muscle program.
FIGURE 21
GTF3 splice variants bind to the SURE BLM with
different avidities. Left: Partial 3' exon-intron structure of mouse
GTF3 splice variants isolated from skeletal muscle. Dark gray boxes
represent alternatively spliced exons, dark portions of 3' exons
30a and 30b are noncoding. Right: Electrophoretic mobility shift
assays of full-length (FL) mouse GTF3g
and aminoterminally truncated (D1-3)
mutant versions of GTF3a, b,
and g isoforms. Arrowhead indicates
specific shift obtained with full-length GTF3g. |
Biochemical Characterization of
GTF3 and Its Novel Splice Variants
Vullhorst, Buonanno
To map the GTF3 DNA binding domain, we generated a series of human GTF3
constructs harboring different truncations. In TFII-I, the paralog of
GTF3, a basic motif that maps between the reiterated helix-loop-helix
(HLH) domains R1 and R2 is necessary for DNA binding. In contrast, analysis
of numerous GTF3 deletion constructs identified the HLH domain 4 (R4)
as necessary and sufficient to mediate DNA binding and showed that sequences
in the N-terminal region interfere with binding. The mouse GTF3 gene
gives rise to a, b,
and g splice variants that differ in sequences
carboxyterminal to the DNA-binding domain R4. To determine which GTF3
splice variants are expressed in skeletal muscle, we employed RT-PCR.
We identified three novel splice variants (called GTF3 a2,
a3, and g2 for
their exonic structure), in addition to the known GTF3 a1
and g1 isoforms, in all muscle and non-muscle
tissues examined. Using EMSAs, we compared the interactions between
the different GTF3 splice variants with the BLM. As was the case for
the human GTF3, the full-length mouse proteins interacted poorly with
the BLM, but N-terminally truncated versions of GTF3 a3,
g1, and g2 avidly
bound to the probe. These experiments suggest that muscle gene expression
may be differently regulated by distinct GTF3 isotypes.
Next, we investigated if an aminoterminal leucine zipperlike
(LZ) motif in GTF3 is required for homomeric or heteromeric interactions
with TFII-I. We followed the interactions of GTF3 with itself or with
TFII-I by using full-length and truncated GTF3 proteins on co-immunoprecipitation
assays (co-IP) and Western blots. The experiments demonstrated that
GTF3 polypeptides interact with each other, but not with TFII-I, via
the LZ domain. We confirmed the significance of the interactions in
myoblasts by using immunofluorescence cytochemistry. A GTF3 mutant protein
(GTF3.D3-6) lacking a bona fide nuclear
localization signal located near the carboxyterminus remains cytosolic
unless co-expressed with full-length GTF3. In agreement with the co-IP
experiments, GTF3.D3-6 fails to localize to the nucleus when co-expressed
with a full-length GTF3 lacking only the LZ zipper (GTF3. DLZ). In conclusion,
GTF3 can form complexes via the LZ motif with itself, but not with TFII-I,
indicating that both transcription factors act independently and possess
distinct DNA binding properties.
Possible Implications of GTF3
and GTF2i in Williams Syndrome
Vullhorst, Karavanova, Buonanno
The genes encoding GTF3 and its related homolog GTF2i are deleted in
individuals with Williams syndrome (WS). The biochemical characterization
of GTF3 (and GTF2i) and the study of its function in
vivo could provide important information about the molecular
basis for WS. Patients suffering from WS have distinctive physical,
cognitive, and behavioral abnormalities that include impaired spatial
cognitive skills and myopathies. WS is a rare, sporadic disorder resulting
from the loss (haplotype) of approximately 20 genes (including the gtf3
and gtf2i) located in about 2.0 Mb of
chromosome 7q11.23. Recent studies strongly implicate GTF3 and TFII-I
as candidate proteins that contribute to the deficiencies observed in
WS patients. Our studies using ectopically transfected GTF3 constructs
in adult muscles and GTF3 knock-out mice strongly support a role for
this factor in regulating muscle contractile properties, which could
be related to myopathies observed in WS. The observation that GTF2I
and GTF3 are mostly expressed in developing musculature and neurons
raises the possibility that reduction of these factors during embryogenesis
could affect the expression of target genes later in development.
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SELECTED PUBLICATIONS
- Buonanno A, Fischbach G. Neuregulin and ErbB receptor signalling
in the nervous system. Curr Opin Neurobiol. 2001;11:287-296.
- Calvo S, Vullhorst D, Venapally P, Karavanova I, Cheng J, Buonanno
A. Molecular dissection of DNA sequences and factors involved in slow
muscle-specific transcription. Mol Cell Biol. 2001;21:8490-8503.
- Desai A, Turetsky D, Vasudevan K, Buonanno A. Analysis of the NMDA
receptor 2A subtype gene in transgenic mice and in transfected cortical
neurons. J. Biol. Chem. 2002; 277:46374-46384.
- Garcia R, Vasudevan K, Buonanno A. The neuregulin receptor ErbB-4
interacts with the PDZ domain protein at neuronal synapses. Proc Natl
Acad Sci USA. 2000;97:3596-3601.
- Gerecke KM, Wyss JM, Karavanova I, Buonanno A, Carroll SL. ErbB transmembrane
tyrosine kinase receptors are differentially expressed in adult rodent
central nervous system. J Comp Neurol. 2001;433:86-100.
- Longart M, Buonanno A. Neuregulins: a family of factors with critical
functions during nervous system development and in the cellular transformation
and differentiation. Rev Neurol. 2002;34:91-97.
- Villegas R, Villegas G, Hernandez M, Longart M, Maqueira B, Buonanno
A, Garcia R, Castillo C. Neuregulin found in cultured sciatic nerve
conditioned medium causes neuronal differentiation of PC12 cells. Brain
Res. 2001;852:305-318.
COLLABORATORS
Steven Carroll, M.D., Ph.D., University of Alabama,
Birmingham, AL
Stefano Vicini, Ph.D., Georgetown University,
Washington, DC
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