Developmental Regulation of Neuronal and Muscle Plasticity

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

Genetic (nature) and epigenetic (nurture) interactions influence the functional properties of neurons and skeletal muscles during development. We are 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 interrelationship among 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. Given that these three signaling pathways and alterations in gamma oscillations are implicated both genetically and pharmacologically in schizophrenia, our findings could have important implications for understanding biological mechanisms affected by psychiatric disorders. In another series of studies, we investigated how developmental factors and motoneuron activity work to 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. The studies emphasize the importance of both nature and nurture in modulating the plastic properties of excitable tissues.

Isolation and characterization of the neuregulin-1 type IV isoform

Shamir, Buonanno

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, which maps within the previously identified “schizophrenia at-risk haplotype,” is associated with higher levels of Type IV transcripts in postmortem tissue from persons diagnosed with schizophrenia and with lower prefrontal activation and the development of psychotic symptoms.

NRG-1 Type IV transcripts were originally identified by RT-PCR as partial RNA fragments. Therefore, it currently is not known whether these partial transcripts originated from full-length NRG-1 mRNAs and whether the mRNAs encode pro-NRG-1 proteins that are post-translationally processed to produce a biologically active form of NRG-1. Toward understanding 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 the pro–NRG-1 Type IV protein, which is post-translationally processed, released from cells, and capable of activating the ErbB receptor and its downstream signaling pathways. This study provides the first evidence for the existence of the NRG-1 Type IV protein. Experiments are in progress to determine whether (and how) expression of the NRG-1 Type IV protein is altered in schizophrenia.

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

Kwon, Paredes, Gonzalez, Neddens, Vullhorst, Buonanno; in collaboration with Hernández

Although NRG-1 function has undergone extensive study in the developing peripheral nervous system, its role in the developing and adult brain remained, until recently, largely unknown. Our hypothesis that the NRG/ErbB signaling pathway regulates activity-dependent synaptic plasticity was based on the fact that ErbB-4 and NMDA receptors (NMDAR) co-localize at glutamatergic postsynaptic densities, where they interact directly with scaffolding proteins that integrate synaptic signaling. Our recent work supports this hypothesis.

We previously reported that, by selectively reducing AMPA-type glutamate receptor currents, NRG-1 reverses (depotentiates) LTP at hippocampal CA1 glutamatergic synapses in an activity-dependent fashion. 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 among NRG-1, dopamine, and glutamate is consistent with animal studies reporting behavior deficits in NRG-1 and ErbB receptor hypomorphic mice that are ameliorated by treatment with the antipsychotic clozapine. The link also has important implications for understanding how imbalances in Neuregulin-ErbB signaling can impinge on dopaminergic and glutamatergic function—neurotransmitter pathways associated with schizophrenia synapses.

Buonanno A, Kwon OB, Yan L, Gonzalez C, Longart M, Hoffman D, Vullhorst D. Neuregulins and neuronal plasticity: possible relevance in schizophrenia. Novartis Found Symp 2008;289:165-177.
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.

Neuregulin-1 modulates hippocampal gamma oscillations

Neddens, Yan,¹ Buonanno; in collaboration with Fisahn

Alterations in gamma-frequency oscillations are implicated in psychiatric disorders, and their amplitude (power) has 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 demonstrated that 50 percent 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. Our investigation provides the first plausible link between NRG-1/ErbB4 signaling and rhythmic network activity that may be altered in persons with schizophrenia.

Fisahn A, Neddens J, Yan L, Buonanno A. Neuregulin-1 modulates hippocampal gamma oscillations: implications for schizophrenia. Cereb Cortex 2008; [E-pub ahead of print].

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

Chatani-Hinze,² Longart,³ Gonzalez, Vullhorst, Yan,¹ Buonanno

To extend 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 of 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. We investigated the trafficking of ErbB-4 in cultured hippocampal neurons by using surface protein biotinylation and antibody labeling of receptors in live cells and found that ErbB-4 immunoreactivity in developing neurons 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 percent at 6 days in culture to 65 percent by day 16. Interestingly, despite the increased association of ErbB-4 with PSD-95, receptor activation by NRG-1 triggers significant internalization in young and mature neurons. PSD-95 primarily seems 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;73:210-219.

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

Karavanov, Buonanno; in collaboration with Lu, Logan, Vicini, Wolfe

We previously reported that, in cultured organotypic slices from cerebellum, expression of the NR2C subunit of 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, we replaced DNA sequences encoding most of the NR2C protein with the E. coli nlacZ gene that encodes the beta-galactosidase (B-gal) reporter. We histologically identified cells that express the B-gal reporter, under control of NR2C transcriptional regulatory elements, 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 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 the 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 the laboratories of Stefano Vicini and Barry Wolfe, we studied the NMDAR excitatory postsynaptic currents (EPSC) in solitary cerebellar neurons cultured in microislands from wild-type (WT) and NR2Ctg-nlacZ knockin mice and in 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 elevated in the mutant mice. The most striking result was a significant increase in the NMDA-EPSC peak amplitude and charge transfer in NR2Ctg-nlacZ knockin mice, which is 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;34:468-480.
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;98:2693-2704.
Lu CC, Fu Z, Karavanov I, Buonanno A, Vicini S. NMDA receptor subtypes at autaptic synapses of cerebellar granule neurons. J Neurophysiol 2006;96:2282-2294.

Transcription factors that differentially regulate transcription of muscle genes

Rana,⁴ Vullhorst, Buonanno; in collaboration with Gundersen

Genetic background and energetic demands from the environment determine the contractile properties of adult slow- and fast-twitch skeletal muscles. Lineage is important during early development for determining the fate of muscles as either 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 a slow (SURE) and fast (FIRE) enhancer that regulate the genes’ fiber type–specific transcription. We used reporter constructs driving the expression of either luciferase or the green fluorescent protein (GFP) 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. Our studies using ectopically transfected GTF3 constructs in adult muscles and GTF3 knockout mice support a role for GTF3 in regulating muscle contractile properties during development.

Activity-dependent repression of fast muscle genes by NFAT. Activity presumably is 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. Using 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. We identified a bona fide NFAT-binding site in the TnIf FIRE by site-directed mutations and EMSAs and showed that NFAT mediated the activity-dependent transcriptional repression of FIRE. Knockdown, mediated by siRNA, of NFATc1 in adult muscles resulted in ectopic activation of FIRE in the slow soleus without affecting enhancer activity in the fast EDL (extensor digitorum longus) muscle. Our 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.

PPARδ expression is influenced by muscle activity and induces slow muscle properties. Metabolites such as free fatty acids (FFA) that are 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)-δ were three times higher in the slow/oxidative soleus than in fast/glycolytic EDL muscle. PPARδ mRNA levels were more than 50 percent lower in solei stimulated with fast-patterned activity while levels increased three-fold in EDLs stimulated with slow-patterned activity. Overexpression of a constitutively active form of PPARδ in normally active adult fibers tripled the number of I/IIa hybrids in fast EDL muscle and increased the activity of the oxidative enzyme succinate dehydrogenase. Therefore, PPARδ provides an additional important link between motoneuron activity and transcriptional changes in slow muscles.

Lunde IG, Ekmark M, Rana ZA, Buonanno A, Gundersen K. PPARδ expression is influenced by muscle activity and induces slow muscle properties in adult rat muscles after somatic gene transfer. J Physiol 2007;582:1277-1287.
Rana ZA, Gundersen K, Buonanno A. Activity-dependent repression of muscle genes by NFAT. Proc Natl Acad Sci USA 2008;105:5921-5926.

TEF3/Tead4 knockout mice

Karavanov, Vullhorst, Buonanno; in collaboration with Yagi, Kohn, Kaneko, DePamphilis

Based on the analysis of TnI SURE regulatory elements, we analyzed another 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 if Tead4 plays any role in regulating fiber type properties during development, we would need to undertake additional experiments using cre-recombinase to delete Tead4 later in development.

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;134:3827-3836.

¹ Leqin Yan, PhD, MD, Anderson Center, former laboratory member
² Mayumi Chatani-Hinze, PhD, currently on leave, former laboratory member
³ Marines Longart, PhD, Instituto de Estudios Avanzados, Venezuela, former laboratory member
⁴ Zaheer Rana, PhD, University of Oslo, Norway, former laboratory member

Collaborators

Melvin DePamphilis, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
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
Congyi Lu, MS, Georgetown University School of Medicine, Washington, DC
Kotaro Kaneko, PhD, Program in Genomics of Differentiation, NICHD, Bethesda, MD
Matthew Kohn, PhD, Program in Genomics of Differentiation, 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

For further information, contact buonanno@helix.nih.gov or visit http://smn.nichd.nih.gov.