1. Functional analysis of the extrasynaptic ErbB4 receptor proteome using proteomics approach - identifying an interaction with GABA-A receptors: Most studies on Neuregulin-ErbB4 signaling have focused on the receptor that accumulates at glutamatergic post-synaptic densities (PSDs);however, little research has been performed on the pool of extrasynaptic ErbB4 receptors that may constitute up to approximately 80% of the total receptor population in cortical and hippocampal interneurons. Our laboratory, in collaboration with Sanford Markey's group at NIMH, has used ErbB4 immunoprecipitation from the soluble fraction of metabolically active synaptosomes followed by LC/MS/MS to characterize the ErbB4 proteome. Using this approach we identified the GABA-A receptor α1 subunit (GABAR α1) as an ErbB4 interacting protein. Remarkably, we found that this novel ErbB4 signaling pathway, which decreases postsynaptic GABAR currents on GABAergic interneurons, acts independently of ErbB4s canonical receptor tyrosine kinase (RTK) activity. While the effects of NRG on GABAR α1 internalization do not require RTK activity, the ErbB4 protein is necessary for clathrin-mediated endocytosis and reduction of GABA receptor IPSCs (Mitchell et al., PNAS 2013). 2. Importance of Neuregulin/ErbB4 and dopamine (DA) signaling in parvalbumin-positive fast-spiking interneurons: Using reverse-microdialysis neurochemistry and electrophysiology, we have reported that NRG1 potently triggers DA release in the hippocampus, reverses LTP at Schaeffer collateral-to-CA1 (SC-CA1) synapses and regulates the power of kainate-induced (KI) gamma oscillations in acute hippocampal slices through activation of dopamine D4 receptors (D4Rs). Importantly, we found that ErbB4 is not detectable in excitatory pyramidal neurons but is abundantly expressed in GABAergic interneurons, in particular Pv+ fast-spiking basket cells. These observations strongly suggested that effects of NRG/ErbB4 on SC-CA1 synaptic plasticity and KI gamma oscillations results from an interplay between the glutamatergic and DAergic neurotransmitter systems First, we addressed the role of ErbB4 receptors specifically in Pv+ GABAergic interneurons by comparing the effects of full vs. interneuron-specific Erbb4 ablation in genetically engineered mice. Unexpectedly, ErbB4 ablation in Pv+ interneurons was sufficient to reproduce the effects of the full ErbB4 knockout on LTP. Because our earlier studies found that D4Rs are necessary to mediate the effects of ErbB4 on LTP, experiments are in progress to determine the role of D4Rs in Pv+ interneurons. This is a particularly interesting question because a subset of Pv+ fast-spiking basket cells co-express D4 and ErbB4 receptors that modulate KI gamma oscillation power in acute hippocampal slices (Andersson et al. PNAS 2012). This novel cross-talk between D4 and ErbB4 receptor signaling to augment gamma oscillation power suggests Pv+ interneurons as a critical nexus of NRG/ErbB4 and DA co-signaling, and provide further evidence in support of a possible contribution of this pathway to the pathophysiology underlying psychiatric disorders (Furth et al., Front Cell Neurosci 2013;Penzes et al., J Neurochem 2013). 3. Mesocortical and nigrostriatal DA function in mice with targeted mutations of ErbB4 in Pv+ and tyrosine hydroxylase-positive (TH+) neurons: Dysfunctional Neuregulin-ErbB4 signaling in the hippocampus, pre-frontal cortex (PFC) and striatum may contribute to alterations in DA function associated with several schizophrenia symptoms. Because we have shown that NRG1 acutely increases extracellular DA levels to regulate LTP and gamma oscillations, and that ErbB4 expression is confined to GABAergic interneurons (cortex) and TH+ mesocortical DA, we are investigating the relative role NRG/ErbB4 signaling in these two different neuronal populations. To this end, we are measuring basal extracellular DA levels in the PFC, hippocampus and striatum in mice harboring targeted mutations of the receptor in either Pv+ or TH+ neurons. We also are comparing their behaviors to begin unraveling the role of Neregulin-ErbB4 signaling in Pv+ interneurons vs. TH+ terminals in regulating schizophrenia-associated behaviors (Skirzewski et al., in preparation). 4. Dopamine regulation of prefrontal cortical gamma oscillations in freely moving rats: Mounting evidence suggests that gamma oscillations are unnaturally high at baseline in disorders that affect attention, such as schizophrenia and ADHD, and that these patients often display problems increasing oscillatory activity relative to healthy controls.These abnormal oscillatory patterns can be used as an endophenotype in schizophrenia. Based on these observations, we hypothesized that drugs targeting either the D4 or ErbB4 receptors may have therapeutic potential in treating cognitive deficits in patients with schizophrenia. To examine abnormal oscillatory patterns and connect them to the firing patterns of single neurons, in collaboration with Dr. Judith Walters at NINDS, we are using multielectrode recordings from the medial prefrontal cortex, dorsomedial thalamus, and hippocampus of rats to analyze the effects of D4- and ErbB4-targeting drugs to regulate the state-dependent regulation of gamma oscillations. 5. A new paradigm for Neuregulin signaling in the CNS: Little is known about the cellular and molecular processes that promote the conversion of Neuregulin ligands from inactive pro-forms to signaling-competent ligands that can engage ErbB4 receptors to mediate their aforementioned biological effects in the developing and maturing brain. To address this major unresolved question, we investigated the role of Neuregulin 2 (NRG2), a Neuregulin isotype that is prominently expressed in the developing postnatal and adult CNS. Traditionally, cortical ErbB4 signaling has been viewed as being mediated primarily by axonally derived Neuregulins from glutamatergic and cholinergic inputs. Consistent with this notion, NRG2 mRNA has previously been reported in dentate gyrus granule cells. However, using double-labeling in situ hybridization and newly generated monoclonal antibodies, we found that in the rodent hippocampus NRG2 mRNA and protein are also expressed in ErbB4-positive GABAergic interneurons, suggesting that NRG2 engages in both autocrine and paracrine ErbB4 signaling. Interestingly, we found no evidence of NRG2 protein in axons or axon terminals. Instead, NRG2 accumulates on the soma and proximal dendrites where it forms large puncta on the plasma membrane that typically do not colocalize with pre- or post-synaptic markers. Rather, immunogold electron microscopy of NRG2 in dissociated hippocampal neurons, performed in collaboration with Dr. Susan Cheng from NINDS, revealed that these puncta are frequently found atop subsurface cisterns - sites of close apposition between the plasma membrane and the endoplasmic reticulum. Double-immunofluorescence labeling of NRG2 with antibodies against the extra- and intracellular domains indicates that these clusters are comprised of the unprocessed pro-form. Taken together, these data suggest that NRG2 subserves distinct and non-overlapping roles. We hypothesize that clustering of pro-NRG2 serves to protect the protein from constitutive processing by extracellular sheddases, and to render the process of ectodomain shedding subject to some form of regulation by neural signals (Vullhorst et al., submitted).
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