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Developmental Regulation of Neuronal and Muscle Plasticity

Andres Buonanno, PhD, Head, Section on Molecular Neurobiology
Detlef Vullhorst, PhD, Staff Scientist
Irina Karavanova, PhD, Biologist
Joerg Neddens, PhD, Visiting Fellow
Daniel Paredes, PhD, Fellow
Alon Shamir, PhD, Visiting Fellow
Oh-Bin Kwon, PhD, Visiting Fellow

Genetic (nature) and epigenetic (nurture) interactions influence the functional properties of neurons and skeletal muscles during development. Our lab is investigating how Neuregulin-1 (NRG-1), a growth and differentiation factor, regulates synaptic formation and plasticity during development. We found that NRG-1, signaling via its receptor ErbB4, functions to repress or reverse hippocampal long-term potentiation (LTP)—a cellular mechanism implicated in learning, memory, and cognition. We identified a novel and intriguing interrelationship between the NRG-1, glutamate, and dopamine signaling pathways that regulate LTP at central synapses, and found that NRG-1 regulates gamma rhythm network activity in the brain. These findings could have important implications for biological mechanisms affected in psychiatric disorders because these three signaling pathways, as well as alterations in gamma oscillations, are implicated genetically and pharmacologically in schizophrenia. To understand the mechanisms that underlie the physiological effects of NRG/ErbB4 signaling, we analyzed the cellular expression of the ErbB4 receptor. We found that ErbB4 is not detectable in excitatory pyramidal cells but that three classes of inhibitory GABAergic interneurons express high levels of the receptor. Moreover, we found that, within these interneurons, ErbB4 is selectively targeted to glutamatergic postsynaptic sites, constituting a novel specific marker for glutamatergic synapses on inhibitory neurons. Interestingly, the number of fast-spiking parvalbumin-containing GABAergic interneurons, which express ErbB4, are reduced in persons with schizophrenia. These findings provide an important and novel framework to test how these different signaling systems, all implicated either genetically or pharmacologically in schizophrenia, may contribute to psychiatric disorders.

In another series of studies, we investigated how developmental factors and motoneuron activity differentially regulate the contractile properties of slow-twitch (red) and fast-twitch (white) muscles. We identified DNA-regulatory elements and transcription factors that confine contractile protein expression to either slow- or fast-twitch fibers in response to "slow" and "fast" patterned motoneuron electrical stimulation. These studies emphasize the importance of both nature and nurture in modulating the plastic properties of excitable tissues.

Neuregulin-1 regulates LTP at CA1 hippocampal synapses through activation of dopamine D4 receptors: possible implications for schizophrenia

Although NRG-1 function has been extensively studied in the developing peripheral nervous system, until recently, its role in the developing and adult brain remained largely unknown. 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 this hypothesis.

We previously reported that NRG-1 reverses ("depotentiates") long-term potentiation (LTP) at hippocampal CA1 glutamatergic synapses, in an activity-dependent fashion, by selectively reducing AMPA-type glutamate receptor currents. We have now demonstrated that NRG-1 stimulates dopamine release in the hippocampus, and reverses early-phase LTP via activation of D4 dopamine receptors (D4R). NRG-1 fails to depotentiate LTP in hippocampal slices treated with the antipsychotic clozapine and other more selective D4R antagonists. Moreover, LTP is not depotentiated in D4R knockout mice by either NRG-1 or theta-pulse stimuli. Conversely, direct D4R activation mimics NRG-1 and acts by reducing AMPAR currents and increasing receptor internalization. This novel functional link between NRG-1, dopamine, and glutamate is consistent with animal studies reporting behavior deficits in NRG-1– and ErbB–receptor hypomorphic mice—deficits that are ameliorated by treatment with the antipsychotic clozapine. This functional link also has important implications for understanding how imbalances in Neuregulin-ErbB signaling can impact dopaminergic and glutamatergic function—neurotransmitter pathways associated with schizophrenia.

Neuregulin-1 modulates hippocampal gamma oscillations

Alterations in gamma-frequency oscillations are implicated in psychiatric disorders, and their amplitudes (power) have been reported to increase selectively during psychotic episodes. In collaboration with Andre Fisahn, we found that NRG-1 dramatically increases the power of kainate-induced gamma oscillations in acute hippocampal slices. NRG-1 effects are blocked by PD158780, a pan-specific antagonist of ErbB receptors, and are absent from slices prepared from ErbB4 null mice. Moreover, we demonstrate that 50% of GABAergic parvalbumin-positive interneurons, which heavily contribute to the generation of gamma oscillations, express ErbB4 receptors. Importantly, both the number of parvalbumin-immunoreactive interneurons and the power of kainate-induced gamma oscillations are reduced in ErbB4 knockout mice. This study provides the first plausible link between NRG-1/ErbB4 signaling and rhythmic network activity that may be altered in persons with schizophrenia.

Cellular and subcellular expression of the neuregulin receptor ErbB4

To understand the cellular mechanisms that mediate the above-mentioned effects of NRG-1 on synaptic plasticity and network activity, we have to identify the neurons that express the ErbB4 receptor. Previous studies by several laboratories have pointed to ErbB4 expression in both excitatory pyramidal cells and inhibitory GABAergic interneurons of the cortex; however, many of these studies relied on unspecific reagents. To resolve this issue, we generated novel rabbit monoclonal antibodies that were stringently characterized and proven to be highly specific for ErbB4. Using these antibodies, we analyzed the expression pattern of ErbB4 in four functionally distinct classes of GABAergic interneurons that represent the majority of all inhibitory neurons in the adult hippocampus of mice. We found high expression levels in three of the four cell classes, indicating that NRG can modulate several inhibitory pathways in the hippocampus. ErbB4 has also been implicated in the generation and maturation of interneurons during development. Consistent with this idea, we found significant reductions of two classes of GABAergic interneurons in ErbB4 knockout mice.

We next investigated the subcellular expression of ErbB4 in interneurons, because, again, the exact location is crucial for understanding the physiological effects of NRG-ErbB4 signaling. Ultrastructural analysis in CA1 interneurons using immunoelectron microscopy revealed abundant ErbB4 expression in the somatodendritic compartment, where it accumulates at, and adjacent to, glutamatergic postsynaptic sites. By contrast, we found no evidence for presynaptic expression in cultured GAD67–positive hippocampal interneurons and in CA1 basket cell terminals. Our findings identify ErbB4–expressing interneurons, but not pyramidal neurons, as a primary target of NRG signaling in the hippocampus and furthermore implicate ErbB4 as a selective marker for glutamatergic synapses on inhibitory interneurons.

ErbB-4 surface clustering by PSD-95 at inhibitory hippocampal neurons

To extend on our earlier work, showing that ErbB-4 directly interacts with the postsynaptic density protein PSD-95 at glutamatergic synapses, we investigated the developmental expression and trafficking the receptor. These interactions may be of special 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. The trafficking of ErbB-4 in cultured hippocampal neurons was investigated by surface protein biotinylation and antibody labeling of receptors in live cells. We found that, in developing neurons, ErbB-4 immunoreactivity precedes PSD-95 expression, with ErbB-4 cluster initially forming in the absence of, but later associating with, PSD-95–positive puncta. The surface fraction of dendritic ErbB-4 increases from 30% at 6 days in culture to 65% by day 16 (DIV 16). Interestingly, receptor activation by NRG-1 triggers significant internalization in young and mature neurons, despite of the 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.

Isolation and characterization of the neuregulin-1 type IV isoform

NRG-1 encodes a family of growth and differentiation factors transcribed from distinct promoters designated type I through type VII. NRG-1 has been reproducibly identified as an "at risk gene" for schizophrenia (HAP_ICE—at risk haplotype). Interestingly, one of the four single-nucleotide polymorphisms comprising HAP_ICE, designated SNP8NRG243177 [T/T], is associated with endophenotypes related to schizophrenia, such as reduction in prefrontal cortical function, working memory, and myelination and a premorbid IQ. Given that SNP8NRG243177 [T/T] has characteristics of a functional polymorphism and maps close to DNA sequences encoding NRG-1 type IV transcripts, our goal was to precisely map the type IV transcription initiation site and to investigate the properties and subcellular distribution of NRG-1 type IV protein. We mapped a novel type IV transcription initiation site and isolated two full-length mRNAs encoding type IV proteins. Using an antiserum we raised against the unique type IV N-terminal end of the protein, we found that NRG-1 type IV is targeted to the cell surface and proteolytic cleavage and that release of the extracellular domain is promoted by PKC activation. Also we demonstrated that NRG-1 type IV possesses biological activity similar to other releasable NRG-1 isoforms. However, the subcellular distributions of distinct NRG-1 isoforms differ. Unlike type III, which associates with axons, NRG-1 type IV and the type I family member are excluded selectively from axons. These results constitute an important step toward understanding how alterations in NRG-1 type IV expression levels associated with SNP8NRG243177 [T/T] can selectively modify signaling from NRG-1 released from somato-dendritic compartments.

Expression and function of NMDA NR2C receptor in beta-galactosidase knockin mice

We previously reported that, in cultured organotypic slices from cerebellum, expression of the NR2C subunit of the NMDAR is regulated by NRG-1. NR2C has generally 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, DNA sequences encoding most of the NR2C protein were replaced by the E. coli nlacZ gene, which encodes the beta-galactosidase (B-gal) reporter. Cells that express the B-gal reporter, under control of NR2C transcriptional regulatory elements, were identified histologically by staining whole brains and sections with X-gal. Using these knockin mice, we found that NR2C is more dynamically and broadly expressed in brain than previously reported. In the cerebellum, NR2C is expressed in a caudalrostral 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. All novel sites of expression, identified in NR2Ctg-nlacZ knock-in mice, were confirmed 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 differing subunit compositions may serve distinct functions.

In collaboration with the groups of Stefano Vicini and Barry Wolfe, we studied the NMDAR excitatory postsynaptic currents (EPSCs) in solitary cerebellar neurons cultured in microislands from wild-type (WT) and NR2Ctg-nlacZ knock-in mice as well as NR2A subunit knockout mice. NR2Ctg-nlacZ granule neurons have larger NMDA-EPSCs than WT cells. The decay times of these currents were all unexpectedly fast, and the quantal content was higher in the mutant mice. The most striking result is a significant increase in the NMDA-EPSC peak amplitude and charge transfer in NR2Ctg-nlacZ knock-in mice, which is mostly due to an increase in quantal size, as estimated from miniature NMDA-EPSCs.

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 for determining the fate of muscles as either slow and fast twitch, but the properties of myofibers remain plastic and are later modified by activity (i.e., exercise). Transcription is the major mechanisms determining the fiber type–specific properties of muscles, by regulating 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 a slow (SURE) and a fast (FIRE) enhancer that regulate their fiber type–specific transcription. Reporter constructs driving the expression of either luciferase or the green fluorescent protein (GFP) were used to map the transcription-regulatory elements in SURE and FIRE. Our initial studies showed that General Transcription Factor 3 (GTF3) binds to a region of the TnIs SURE necessary for slow-specific transcription during early development. The next question was to determine how distinct patterns of motoneuron activity in the adult are coupled to different transcriptional programs of slow- and fast-twitch myofibril expression.

a) Activity-dependent repression of fast muscle genes by NFAT: Activity is presumably coupled to distinct programs of transcription by modulating the levels of intracellular calcium and metabolites. The NFAT family of calcium-dependent transcription factors has been implicated in the upregulation of genes encoding slow contractile proteins in response to slow-patterned motoneuron depolarization. We uncovered a novel, and unexpected, function of NFATc1 in slow-twitch muscles. Utilizing the TnIf intronic regulatory element FIRE, we identified sequences that downregulate FIRE's function selectively in response to patterns of electrical activity that mimic slow motoneuron firing. A bona fide NFAT binding site in the TnIf FIRE was identified by site-directed mutations and EMSAs (electrophoretic mobility shift assays), and shown to mediated the activity-dependent transcriptional repression of FIRE. When we knocked down NFATc1 in adult muscles with siRNA, the result was ectopic activation of the FIRE in the slow soleus without an effect on enhancer activity in the fast EDL (extensor digitorum longus) muscle. These findings demonstrate a novel function of NFAT as a repressor of transcription of fast contractile genes in slow muscles and underscore how muscles can modify their adult contractile properties in response to distinct types of exercise.

b) PPARdelta expression is influenced by muscle activity and induces slow muscle properties: Metabolites such as free fatty acids (FFAs), generated during exercise, could activate transcriptional pathways that regulate fiber types. In collaboration with Kristian Gundersen, we found that mRNA levels for peroxisome proliferator–activated receptor- (PPAR) delta were three-fold higher in the slow/oxidative soleus than in the fast/glycolytic EDL muscle. PPARdelta mRNA levels were reduced by more than 50% in solei stimulated with fast patterned activity, while levels increased three-fold in EDL muscle stimulated with slow-patterned activity. Overexpression of a constitutively active form of PPARdelta in normally active adult fibers tripled the number of I/IIa hybrids in the fast EDL muscle and increased the activity of the oxidative enzyme succinate dehydrogenase. Therefore, PPARdelta provides an additional important link coupling motoneuron activity with transcriptional changes in slow muscles.

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, Tead4-floxed mice were generated by homologous recombination. A possible role of Tead4 in muscle development could not be studied, however, because no null mice were born. Developmental studies indicated that ablation of the gene resulted in a preimplantation failure. Tead4^−/− embryos do not to 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 has any role in regulating fiber-type properties during development, additional experiments using cre-recombinase to delete Tead4 later in development would be necessary.

Publications

Kwon O, Paredes D, Gonzalez C, Neddens J, Hernandez L, Vullhorst D, Buonanno A. Neuregulin-1 regulates LTP at CA1 hippocampal synapses through activation of dopamine D4 receptors. Proc Natl Acad Sci USA 2008 105:15587-15592.
Fisahn A, Neddens J, Yan L, Buonanno A. Neuregulin-1 modulates hippocampal gamma oscillations: implications for schizophrenia. Cereb Cortex 2009 19:612-618.
Neddens J, Buonanno A. Selective populations of hippocampal interneurons express ErbB4 and their number and distribution is altered in ErbB4 knockout mice. Hippocampus 2009 [E-pub ahead of print].
Vullhorst D, Neddens J, Karavanova I, Tricoire L, Petralia R, McBain CJ, Buonanno A. Selective expression of ErbB4 in interneurons, but not pyramidal cells, of the rodent hippocampus. J Neurosci 2009 29:12255-12264.
Rana ZA, Gundersen K, Buonanno A. Activity-dependent repression of muscle genes by NFAT. Proc Natl Acad Sci USA 2008 105:5921-5926.

Collaborators

Andre Fisahn, PhD, Karolinska Institute, Stockholm, Sweden
Kristian Gundersen, PhD, University of Oslo, Oslo, Norway
Luis Hernández, MD, Universidad de los Andes, Mérida, Venezuela
Stephen Logan, PhD, Georgetown University School of Medicine, Washington, DC
Melvin DePamphilis, PhD, Program in Genomics of Differentiation, 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
Christopher McBain, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
Ronald Petralia, PhD, Laboratory of Neurochemistry, NIDCD, Bethesda, MD
Ludovic Tricore, PhD, Program in Developmental Neuroscience, NICHD, Bethesda, MD
Stefano Vicini, PhD, Georgetown University School of Medicine, Washington, DC
Barry B. Wolfe, PhD, Georgetown University School of Medicine, Washington, DC
Rieko Yagi, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD

Contact

For more information, email buonanno@mail.nih.gov or visit smn.nichd.nih.gov.

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