DEVELOPMENTAL REGULATION OF NEURONAL AND MUSCLE PLASTICITY
, Head, Section on Molecular Neurobiology
Detlef Vullhorst, PhD, Staff Scientist
Irina Karavanov, PhD, Biologist
Carmen Gonzalez, PhD, Visiting Fellow
Oh-Bin Kwon, PhD, Visiting Fellow
Joerg Neddens, PhD, Visiting Fellow
Zaheer Rana, MS, Visiting Fellow1
Alon Shamir, PhD, Visiting Fellow
Leqin Yan, PhD, Visiting Fellow
How experience and genes interact to remodel the nervous system during development is a central question in biology with major implications for disease. Our laboratory investigates the molecular mechanisms that regulate neuronal and muscle plasticity during development in response to experience. We are studying how the trophic differentiation factor known as neuregulin-1 (NRG-1) and its cognate receptor tyrosine kinases (ErbB 2-4) regulate synaptic plasticity in the brain and modulate behavior. Importantly, polymorphisms in the NRG-1 and ErbB-4 genes are associated with higher risk for developing schizophrenia, and the NRG/ErbB-4 signaling pathway is altered in post mortem brains of persons diagnosed with schizophrenia. Our laboratory also works to identify and characterize transcription factors that modify the contractile properties of skeletal muscles both during development and in response to different types of activity in the adult such as exercise.
Isolation and characterization of the neuregulin-1 type IV isoform
Accumulating evidence supports the involvement of NRG-1 and ErbB-4 receptors in the etiology of schizophrenia. The NRG-1 gene generates numerous transcripts by using different transcriptional promoters and alternative splicing. Interestingly, a single nucleotide polymorphism (SNP8NRG243177) is located close to a region proposed to function as a promoter for the novel NRG-1 isoform denoted as Type IV. The SNP8NRG243177 [T/T] polymorphism is associated with higher levels of Type IV transcripts in post mortem tissue of schizophrenia patients. Moreover, SNP8NRG243177 [T/T], which maps within the previously identified schizophrenia at-risk haplotype, is associated with lower prefrontal activation and the development of psychotic symptoms.
NRG-1 Type IV transcripts were originally identified by RT-PCR (reverse transcriptase-polymerase chain reaction) as partial RNA fragments. Therefore, it is not yet known whether these partial transcripts originated from full-length NRG-1 mRNAs and whether these mRNAs encode pro-NRG-1 proteins that are post-translationally processed to produce a biologically active form of NRG-1. Toward defining a possible role of Type IV NRG-1 in the human brain, we isolated two full-length mRNAs encoding Type IV proteins. We found that the transcripts are translated to generate pro–NRG-1 Type IV protein that is post-translationally processed, released from cells, and capable of activating ErbB receptors and their downstream signaling pathways. Our research provides the first evidence for the existence of NRG-1 Type IV protein. Experiments are in progress to determine if and how expression of NRG-1 Type IV protein is altered in schizophrenia.
Neuregulin and synaptic plasticity: possible relevance in schizophrenia
Although NRG-1 function has been extensively studied in the developing peripheral nervous system, its role in the developing and adult brain remained largely unknown until recently. The fact that ErbB-4 and NMDA receptors (NMDARs) colocalize at glutamatergic postsynaptic densities, where they interact directly with scaffolding proteins that integrate synaptic signaling, led to the hypothesis that the NRG/ErbB signaling pathway regulates activity-dependent synaptic plasticity. Our recent work supports that hypothesis.
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 that are 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 fluoresces strongly only when expressed on the cell surface, indicates that NRG-1 stimulates the internalization of surface seGluR1-containing AMPA receptors.
Consistent with our findings, others have shown that NRG-1 and ErbB receptor–hypomorphic mice develop normally but exhibit reduced glutamate receptor levels and behavioral deficits. This novel regulation of LTP by NRG-1 has important implications for modulating synaptic homeostasis at glutamatergic synapses, which can affect cognition, learning, and memory, and for understanding molecular mechanisms that underlie complex disorders such as schizophrenia.
Kwon O, Longart M, Vullhorst D, Hoffman D, Buonanno A. Neuregulin depotentiates long term potentiation at hippocampal CA1 synapses. J Neurosci 2005;25:9378-83.
ErbB-4 surface clustering by PSD-95 at inhibitory hippocampal neurons
To extend our earlier work, which showed that ErbB-4 directly interacts with the postsynaptic density protein PSD-95 at glutamatergic synapses, we investigated the receptor’s developmental expression and trafficking. The interactions may be of particular interest because they appear altered in postmortem brain tissue isolated from persons diagnosed with 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. We investigated the trafficking of ErbB-4 in cultured hippocampal neurons by using 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 clusters initially forming in the absence of, but later associating with, PSD-95–positive puncta. The surface fraction of dendritic ErbB-4 increases from 30 percent at 6 days in culture to 65 percent by day 16 (DIV 16). Interestingly, receptor activation by NRG-1 triggers significant internalization in young and mature neurons despite increased association of ErbB-4 with PSD-95. PSD-95 seems primarily to control receptor clustering. These findings enhance our understanding of the role of ErbB-4/PSD-95 protein interaction for NRG-mediated signaling at glutamatergic synapses.
Longart M, Chatani-Hinze M, Gonzalez C, Vullhorst D, Buonanno A. Regulation of ErbB-4 endocytosis by neuregulin in GABAergic hippocampal interneurons. Brain Res Bull 2007;73:210-9.
Expression and function of NMDA NR2C receptor in beta-galactosidase knockin mice
We previously reported that expression of the NR2C subunit of the NMDAR is regulated by NRG-1 in cultured organotypic slices from cerebellum. Generally, NR2C has been considered a cerebellar subunit because its expression is strikingly higher in the cerebellum than in other brain areas. To study the precise expression and function of the NR2C subunit in the developing brain, we developed knockin mice. Using homologous recombination, we replaced DNA sequences encoding most of the NR2C protein with the E. coli nlacZ gene that encodes the beta-galactosidase (B-gal) reporter. To make a histological identification of cells that express the B-gal reporter under control of NR2C transcriptional regulatory elements, we stained whole brains and brain sections of the knockin mice with X-gal and found that NR2C is more dynamically and broadly expressed in brain than previously reported. In the cerebellum, NR2C is expressed in a caudal-rostral gradient and in a series of parasagittal bands in subsets of cerebellar granule cells. We also found NR2C expression in previously unappreciated areas, such as the retrosplenial and cerebral cortex, hippocampus, and basal ganglia. Using cell type–specific antibodies, we unexpectedly found that NR2C is expressed by glial cells, not neurons, dispersed in the hippocampus, striatum, olfactory bulb, and cerebral cortex. We confirmed all novel sites of expression, identified in NR2Ctg-nlacZ knockin mice by in situ hybridization using 33P-labeled NR2C cRNA probes. The expression of NR2C in discrete brain areas outside the cerebellum and in glia suggests that NMDARs with different subunit compositions may serve distinct functions.
In collaboration with Stefano Vicini and Barry Wolfe and their colleagues, we studied the NMDAR excitatory postsynaptic currents (EPSCs) in solitary cerebellar neurons cultured in microislands from wild-type (WT) and NR2Ctg-nlacZ knockin mice and from NR2A subunit knockout mice. Compared with WT cells, NR2Ctg-nlacZ granule neurons have larger NMDA-EPSCs. The decay times of the currents were all unexpectedly fast, and the quantal content was elevated in the mutant mice. The most striking result was a significant increase in the NMDA-EPSC peak amplitude and charge transfer in the NR2Ctg-nlacZ knockin mice; the increase was mostly attributable to an increase in quantal size as estimated from miniature NMDA-EPSCs.
Karavanov I, Vasudevan K, Cheng J, Buonanno A. Novel regional and developmental NMDA receptor expression patterns uncovered in NR2C subunit-beta-galactosidase knock-in mice. Mol Cell Neurosci 2007;34:468-80.
Logan S, Partridge J, Matta J, Buonanno A, Vicini S. Long-lasting NMDA receptor-mediated EPSCs in mouse striatal medium spiny neurons. J Neurophysiol 2007;98:2693-704.
Lu C, Fu Z, Karavanov I, Buonanno A, Vicini S. NMDA receptor subtypes at autaptic synapses of cerebellar granule neurons. J Neurophysiol 2006;96:2282-94.
Transcription factors that differentially regulate transcription of muscle genes
Genetic background and energetic demands from the environment determine the contractile properties of adult slow- and fast-twitch skeletal muscles. During early development, lineage is important in determining whether muscles become slow- or fast-twitch, but the properties of myofibers remain plastic and are later modified by activity (i.e., exercise). Transcription is the major mechanism determining the fiber type–specific properties of muscles; it regulates the expression of genes encoding contractile proteins and metabolic enzymes characteristic of slow and fast muscles. The troponin I slow (TnIs) and fast (TnIf) genes are selectively expressed in slow and fast muscles during development and are later regulated by distinct patterns of electrical impulses elicited by motor neurons. Using the TnI genes as a model system, we identified (SURE) and fast (FIRE) enhancers that regulate fiber type–specific transcription. We used reporter constructs driving the expression of either luciferase or GFP to map the transcription regulatory elements in SURE and FIRE.
Using a yeast-1-hybrid approach, we found that the General Transcription Factor 3 (GTF3) binds to a region of the TnIs SURE necessary for slow-specific transcription during early development. 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 knockout mice support a role for GTF3 in regulating the contractile properties of muscle.
We measured the effects of motor neuron electrical activity on TnI transcription in vivo by imaging individual myofibers. We determined the levels of transcription in adult muscles transfected with the SURE and FIRE GFP reporter constructs before and after electrical stimulation. We found that slow, tonic depolarization upregulated SURE transcription, whereas fast, phasic stimuli enhanced FIRE transcription. The results indicated that the TnI slow and fast enhancers sense and respond to distinct patterns of neuronal activity.
Next, we set out to identify the DNA elements and transcription factors that respond differentially to activity. Numerous lines of evidence indicate that the effects of activity are mediated by calcium, which is released from the sarcoplasmic reticulum after depolarization of the sarcolemma. Sustained low-frequency muscle depolarization causes a sustained elevation of calcium in the cell, whereas short depolarization bursts give rise to transient spikes of elevated calcium. NFAT and NFkB are two transcription factors that differentially respond to calcium transients in T cells in order to regulate genes differentially. Unexpectedly, we found that both transcription factors are also involved in the regulation of the TnI FIRE. Whereas NFAT repressed transcription from FIRE in response to slow-patterned activity, NFkB increased FIRE transcription in response to fast-patterned stimuli. These experiments exemplify how muscles can modify their adult contractile properties in response to distinct types of exercise.
Lunde JG, Ekmark M, Rana ZA, Buonanno A, Gundersen K. PPARd expression is influenced by muscle activity and induces slow muscle properties in adult rat muscles after somatic gene transfer. J Physiol 2007;582:1277-87.
Rana Z, Gundersen K, Buonanno A, Vullhorst D. Imaging transcription in vivo: distinct regulatory effects of fast and slow activity patterns on promoter elements from troponin I isoform genes. J Physiol 2005;562:815-28.
Vullhorst D, Buonanno A. Multiple GTF2i-like repeats of general transcription factor 3 exhibit DNA binding properties: evidence for a common origin as a sequence-specific DNA interaction module. J Biol Chem 2005;280:31722-31.
TEF3/Tead4 knockout mice
Based on the analysis of TnI SURE regulatory elements, we analyzed the transcription factor Tead4 (also known as TEF3) for its potential role in regulating early muscle development and regeneration in the adult. To this end, we generated Tead4-floxed mice by homologous recombination. We could not, however, study a possible role of Tead4 in muscle development because null mice were not born. Developmental studies indicated that ablation of the gene resulted in a pre-implantation failure. Tead4−/− embryos do not express trophectoderm-specific genes, and the morulae do not produce trophoblast stem cells, trophectoderm, or blastocoel cavities and therefore fail to implant. Tead4−/− embryos can produce embryonic stem cells, and the embryos can complete development if the gene is deleted after the implantation period, as shown by crossing the Tead4-floxed mice with Meox2-Cre recombinase mice. Consequently, to determine whether Tead4 plays any role in regulating fiber type properties during development, additional experiments using cre-recombinase to delete Tead4 later in development will be necessary.
Yagi R, Kohn MJ, Karavanova I, Kaneko KJ, Vullhorst D, DePamphilis ML, Buonanno A. Transcription factor TEAD4 specifies the trophectoderm lineage at the beginning of mammalian development. Development 2007;134:3827-36.
1 University of Oslo, Norway
2 Marines Longart, PhD, former Guest Worker, Instituto de Estudios Avanzados, Venezuela
3 Mayumi Chatani-Hintze, PhD, on leave
COLLABORATOR
Melvin DePamphilis, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Kristian Gundersen, PhD, University of Oslo, Oslo, Norway
Dax Hoffman, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
Kotaro Kaneko, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Matthew Kohn, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Congyi Lu, MS, Georgetown University, Washington, DC
Stefano Vicini, PhD, Georgetown University, Washington, DC
Barry Wolfe, PhD, Georgetown University, Washington, DC
Rieko Yagi, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
For further information, contact buonanno@helix.nih.gov.
