1. Subcellular Distribution of Neuregulin Isoforms in Central Neurons: Numerous Neuregulins (NRGs) are generated through the use of four different genes (NRG1-NRG4), promoters (NRG1: types -I, -II and -II)) and alternative splicing, but the functional significance of this evolutionary conserved diversity remains poorly understood. Our recent studies reveal that NRGs can be categorized by their distinct membrane topologies that impart fundamentally different subcellular trafficking properties. As discussed below, NRGs whose pro-forms contain a single transmembrane domain, like NRG1 (types I and II) and NRG2, target to the cell body plasma membrane where they accumulate at specialized contact sites with the underlying endoplasmic reticulum. Their ectodomains are cleaved by sheddases in an activity-dependent manner to signal in paracrine fashion (see #2 and #3). By contrast, we found that NRGs translated as dual transmembrane domain proteins, such as CRD type III-NRG1 and NRG3 (our recent studies uncovered it also is a dual transmembrane protein, are instead targeted to axons where they signal in juxtacrine mode. These findings reveal a previously unknown functional relationship between membrane topology and subcellular targeting, and suggest that single- and dual-pass NRGs regulate neuronal functions in fundamentally different ways. This work was supported by a Directors Investigator Award and has recently been submitted for publication (Ahmed, Vullhorst et al., Journal of Neuroscience, in review). 2. Unprocessed NRG2 accumulates at endoplamic reticulum-plasma membrane (ER-PM) junctions where it is protected from alpha-secretase processing: The cellular and molecular processes that promote the conversion of NRG 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 remain mostly unknown. 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. Using a novel double-labeling in situ hybridization technique (RNAScope) and newly generated monoclonal antibodies, we found that in the rodent hippocampus NRG2 mRNA and protein are highly expressed in ErbB4-positive GABAergic interneurons, suggesting that NRG2 can engage in autocrine ErbB4 signaling. Interestingly, we found no evidence of NRG2 protein in axons; instead, we found that unprocessed proNRG2 accumulates at large somato-dendritic puncta on the plasma membrane of GABAergic interneurons. Immunogold electron microscopy of NRG2 in dissociated hippocampal neurons, performed in collaboration with Dr. Susan Cheng, revealed that these puncta are found atop of subsurface cisterns (SSCs) - sites of close ER-PM apposition that are proposed to function as signaling platforms (Vullhorst et al., Nat Commun 1;6:7222, 2015). 3. NMDA receptor function on cortical interneurons promotes proNRG2 processing and, in turn, NRG2 signaling via ErbB4 downregulates NMDAR function on interneurons: We found that the proNRG2 puncta atop SSCs are localized to the empty center of doughnut-shaped Kv2.1 clusters at the edges of SSCs. Interestingly, treatment of neuronal cultures with glutamate or NMDA rapidly causes the dispersal of both proteins from the SSC and processing of proNRG2 by shedasses to release the signaling-competent NRG2. What is the function of released NRG2 from GAGAergic interneurons, which are known to express the ErbB4 receptor? In collaboration with Sanford Markey's group at NIMH, we 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 NMDAR GluN2B subunit as an ErbB4 interacting protein. This interaction was confirmed in cultured hippocampal neurons, where NRG2 treatment was shown to enhance the internalization of GluN2B-containing, but not GluN2A-containing, NMDARs. Consistent with this observation, we found that NRG2 also caused a dramatic reduction of whole cells NMDAR currents in dissociated hippocampal ErbB4-positive interneurons, but not in ErbB4-negative glutamatergic neurons. Lastly, using whole-cell voltage-clamp recordings in acute medial prefrontal cortical slices, we found that NRG2 selectively reduced NMDAR synaptic currents (EPSCs), and not AMPAR EPSCs, at glutamatergic synapses onto GABAergic interneurons. These results are consistent with the idea that the bidirectlional signaling between NRG2/ErbB4 and NMDAR activity can play a major role in modulating the activity of GABAergic neurons and cortical E/I balance (Vullhorst et al., Nat Commun 1;6:7222, 2015). 5. Analysis of ErbB4 function in mice harboring targeted mutations in GABAergic and dopaminergic neurons: Dysfunctional Neuregulin-ErbB4 signaling in the hippocampus, pre-frontal cortex (PFC) and striatum may contribute to alterations in dopamine (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 receptor expression is confined to GABAergic interneurons (cortex) and TH+ mesocortical DAergic neurons, we have used genetic, biochemical and behavioral approaches to measure DA function in the hippocampus, PFC and striatum in mice harboring targeted mutations of ErbB4 in either PV+ or TH+ neurons. Interestingly, we have found that in contrast to GABAergic neurons, ErbB4 is highly expressed on the axons of DA neurons, suggesting that NRG/ErbB4 signaling may directly regulate the presynaptic function of these neurons. We found NRG regulates the increase in extracellular DA levels, at least in part, by regulating DAT function. In contrast to mice harboring CNS-wide or GABAergic-restricted mutation of ErbB4, which show sensory-motor gating deficits and increases in motor activity, mice with ablation of ErbB4 in TH+ neurons only manifest behavioral deficits in cognitive-related tasks, such as: performance on the T-maze, Y-maze and Barnes maze. Our findings suggest that direct effects of NRG/ErbB4 signaling in GABAergic and DAergic neurons in combination regulate cortical circuits and DA homeostasis to affect numerous behaviors relevant to schizophrenia (Skirzewski et al., in preparation). 6. Effects of ketamine on cortical gamma oscillations and role of dopamine receptors. Mounting evidence suggests that gamma oscillations are atypically high at baseline in disorders that affect attention such as schizophrenia and ADHD. Ketamine, an antagonist of the NMDAR that elicits psychosis and affects cognitive functions in healthy individuals that phenocopy schizophrenia. In collaboration with Dr. Judith Walters lab, we are using multi-electrode recordings from the medial prefrontal cortex and dorsomedial thalamus of rats acutely treated with ketamine to analyze the effects of D4 and ErbB4targeting drugs on gamma oscillations in this rodent model with face validity for schizophrenia (Furth et al. Psychopharmacology, in review).

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U.S. National Inst/Child Hlth/Human Dev
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Yan, L; Shamir, A; Skirzewski, M et al. (2017) Neuregulin-2 ablation results in dopamine dysregulation and severe behavioral phenotypes relevant to psychiatric disorders. Mol Psychiatry :
Skirzewski, M; Karavanova, I; Shamir, A et al. (2017) ErbB4 signaling in dopaminergic axonal projections increases extracellular dopamine levels and regulates spatial/working memory behaviors. Mol Psychiatry :
Vullhorst, Detlef; Ahmad, Tanveer; Karavanova, Irina et al. (2017) Structural Similarities between Neuregulin 1-3 Isoforms Determine Their Subcellular Distribution and Signaling Mode in Central Neurons. J Neurosci 37:5232-5249
Canfield, Kaleigh; Li, Jiaqi; Wilkins, Owen M et al. (2015) Receptor tyrosine kinase ERBB4 mediates acquired resistance to ERBB2 inhibitors in breast cancer cells. Cell Cycle 14:648-55
Vullhorst, Detlef; Mitchell, Robert M; Keating, Carolyn et al. (2015) A negative feedback loop controls NMDA receptor function in cortical interneurons via neuregulin 2/ErbB4 signalling. Nat Commun 6:7222
Padmakumar, Vc; Masiuk, Katelyn E; Luger, Dror et al. (2014) Detection of differential fetal and adult expression of chloride intracellular channel 4 (CLIC4) protein by analysis of a green fluorescent protein knock-in mouse line. BMC Dev Biol 14:24
Golani, Idit; Tadmor, Hagar; Buonanno, Andres et al. (2014) Disruption of the ErbB signaling in adolescence increases striatal dopamine levels and affects learning and hedonic-like behavior in the adult mouse. Eur Neuropsychopharmacol 24:1808-18
Penzes, Peter; Buonanno, Andres; Passafaro, Maria et al. (2013) Developmental vulnerability of synapses and circuits associated with neuropsychiatric disorders. J Neurochem 126:165-82
Mitchell, Robert M; Janssen, Megan J; Karavanova, Irina et al. (2013) ErbB4 reduces synaptic GABAA currents independent of its receptor tyrosine kinase activity. Proc Natl Acad Sci U S A 110:19603-8
Furth, Katrina E; Mastwal, Surjeet; Wang, Kuan H et al. (2013) Dopamine, cognitive function, and gamma oscillations: role of D4 receptors. Front Cell Neurosci 7:102

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