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Developmental Regulation of Neuronal and Muscle Plasticity

Andres Buonanno, PhD
  • Andres Buonanno, PhD, Head, Section on Molecular Neurobiology
  • Detlef Vullhorst, PhD, Staff Scientist
  • Irina Karavanova, PhD, Biologist
  • Alon Shamir, PhD, Visiting Fellow
  • Joerg Neddens, PhD, Visiting Fellow
  • Elias Leiva-Salcedo, PhD, Visiting Fellow
  • Ryoichi Kimura, PhD, Visiting Fellow
  • Claudia Colina, PhD, Visiting Fellow

Genetic and epigenetic factors determine the functional properties of the brain and skeletal muscles during development and can be altered in disease. Our laboratory has focused on elucidating the functions of a factor known as Neuregulin-1 (NRG-1) and its cognate receptor ErbB4 in the developing brain. NRG-1 and ErbB4 have been genetically identified as schizophrenia "at risk" genes. We recently demonstrated that NRG-1 and its receptor ErbB4 function to regulate synaptic plasticity in the hippocampus by modulating long-term potentiation, a cellular mechanism implicated in learning, memory, and cognition. Our newer studies demonstrated that NRG-1 regulates gamma rhythm activity in the brain and uncovered an important relationship between the NRG-1, glutamate, and dopamine-signaling pathways. We also found that ErbB4 is not detectable in excitatory pyramidal cells; instead, the receptor is expressed in three classes of inhibitory interneurons in the hippocampus. Moreover, we found that within these interneurons ErbB4 is targeted to glutamatergic postsynaptic sites. In our most recent studies, we found that in the frontal cortex of mice, as well as in the cortex of primates, ErbB4 expression is also confined to GABAergic interneurons. The conserved regional and subcellular expression of ErbB4 from rodents to primates validates the use of mice to investigate the cellular mechanisms that may be altered in psychiatric disorders. Our findings provide an important and novel framework within which to test how these signaling systems, which include the NRG/ErbB network and various neurotransmitters, all implicated either genetically or pharmacologically in schizophrenia, may contribute to psychiatric disorders.

The contractile properties of slow-twitch (red) and fast-twitch (white) muscles are also determined by genetic and epigenetic factors. Genetic cues are important during early development to differentiate muscle types; later in development, activity, in the form of exercise, can modify muscle types. Our group identified DNA regulatory sequences and transcription factors that modify the contractile properties of skeletal muscles during development and, in an activity-dependent fashion, in the adult. Interestingly, these factors regulate different contractile genes in response to distinct frequencies of muscle depolarization. Our goal is to uncover how these transcription factors can "sense" slow and fast patterns of motor neuron depolarization and consequently intraconvert the contractile properties of slow and fast muscles.

NRG-1 modulates hippocampal gamma oscillations: implications for schizophrenia.

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 André 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 not found in slices prepared from ErbB4 null mice. Moreover, we demonstrated 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. The study provides the first plausible link between NRG-1/ErbB4 signaling and rhythmic network activity that may be altered in individuals 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 identified the ErbB4-expressing neurons. We generated novel monoclonal antibodies, and demonstrated they are highly specific for ErbB4. Using these antibodies, we analyzed the expression pattern of ErbB4 in four functionally distinct classes of 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 finding, we observed significant reductions in the numbers of two classes of interneurons in mice that lack ErbB4. We next investigated the subcellular expression of ErbB4 in interneurons, because the exact location is again 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 ErbB4 accumulates at, and adjacent to, glutamatergic postsynaptic sites. By contrast, we found no evidence for presynaptic expression in cultured GAD67-positive hippocampal interneurons or 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.

Conservation of ErbB4 expression in GABAergic interneurons in the hippocampus and cortex, from rodents to primates

Before extrapolating from rodent work as to how the NRG/ErbB signaling may be affected in psychiatric disorders, it is critical to know that this signaling pathway functions in similar fashion in rodents and primates. To this end, we analyzed the distribution of ErbB4 protein using tissue sections obtained from the hippocampus and cortices of both mice and monkeys. We found that, similarly to the hippocampus, ErbB4 receptors accumulate on the somatodendritic compartment of GABAergic interneurons. In the cortex, expression was high in fast-spiking parvalbumin-positive interneurons. This cellular pattern of expression was conserved in both rodents and primates, supporting the use of mice as a model system.

NRG-1 regulates LTP at CA1 hippocampal synapses through activation of dopamine D4 receptors.

NRG-1 and ErbB4 are genetically associated with schizophrenia, a neurodevelopmental cognitive disorder characterized by imbalances in glutamatergic and dopaminergic function. Previously, we reported that NRG-1 suppresses or reverses long-term potentiation (LTP) at hippocampal glutamatergic synapses. More recently, we 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 null 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 has important implications for understanding how imbalances in NRG-1–ErbB signaling can impinge on dopaminergic and glutamatergic function—neurotransmitter pathways associated with schizophrenia.

Molecular and cellular characterization of NRG-1 (type IV)

NRG-1 encodes a family of growth and differentiation factors transcribed from distinct promoters designated type I through type VII and has been reproducibly identified as an "at risk gene" for schizophrenia. Interestingly, SNP8NRG243177 T/T, one of the four single nucleotide polymorphisms comprising the NRG-1 haplotype HAPice, is associated with endophenotypes related to schizophrenia, such as reduced prefrontal cortical function, working memory, myelination, and 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 that 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 that proteolytic cleavage and release of the extracellular domain is promoted by PKC activation. Also we demonstrated that NRG1 type IV possesses biological activity similar to that of other releasable NRG-1 isoforms. However, the subcellular distributions of distinct NRG-1 isoforms differ. Unlike NRG-1 type III, which is expressed in the somato-dendritic and axonal compartments of neurons, NRG-1 type IV and its close homolog NRG-1 type I are excluded selectively from axons. These results constitute an important step in our understanding of how alterations in NRG-1 type IV expression levels associated with SNP8NRG243177 T/T could selectively modify signaling from NRG-1 released from somato-dendritic compartments.

Activity-dependent repression of fast-muscle genes by NFAT

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. In collaboration with Kristian Gundersen, we demonstrated a novel and unexpected function of NFATc1 in slow-twitch muscles. Utilizing the Troponin I fast (TnIf) intronic regulatory element (FIRE), we identified sequences that downregulate the element'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 of NFATc1 in adult muscles mediated by siRNA resulted in ectopic activation of the FIRE in the slow soleus, without affecting enhancer activity in the fast EDL muscle. These findings demonstrate a novel function of NFAT as a repressor of transcription of fast contractile genes in slow muscle.

Publications

  • Buonanno A. The neuregulin signaling pathway and schizophrenia: From genes to synapses and neural circuits. Brain Res Bull. 2010;83:122-131.
  • 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.
  • Sharmir A, Buonanno A. Molecular and cellular characterization of Neuregulin-1 type IV isoforms. J Neurochem. 2010;113:1163-1176.

Collaborators

  • Jacqueline N. Crawley, PhD, Laboratory of Behavioral Neuroscience, NIMH, Bethesda, MD
  • André Fisahn, PhD, Karolinska Institute, Stockholm, Sweden
  • Kristian Gundersen, PhD, University of Oslo, Oslo, Norway
  • Luis Hernández, MD, Universidad de los Andes, Mérida, Venezuela
  • Sanford P. Markey, PhD, Laboratory of Neurotoxicology, NIMH, Bethesda, MD
  • Christopher McBain, PhD, Program on Developmental Neuroscience, NICHD, Bethesda, MD
  • Daniel Paredes, PhD, Laboratory of Molecular Biology, NINDS, Bethesda, MD
  • Ronald Petralia, PhD, Laboratory of Neurochemistry, NIDCD, Bethesda, MD
  • Zaheer Rana, PhD, University of Oslo, Oslo, Norway
  • Ludovic Tricore, PhD, Program on Developmental Neuroscience, NICHD, Bethesda, MD

Contact

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

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