1. Subcellular Distribution and Functions of Single Transmembrane (TM) Neuregulins 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. 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. As discussed here and below, our recent studies reveal that NRGs can be categorized by their distinct membrane topologies that impart fundamentally different subcellular trafficking properties. 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. First we analyzed NRG2, an 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 (Vullhorst et al., Nat Commun 1;6:7222, 2015). Our more recent studies on the other single TM NRGs (NRG1 types I and II) demonstrate a similar subcellular distribution. Moreover, we found that the ectodomains of single TM are cleaved by sheddases in an activity-dependent manner to signal in paracrine and autocrine fashion, as NMDA receptor activation on cortical interneurons promotes proNRG2 shedding that in turn activates ErbB4 receptor signaling. The activation of ErbB4 promotes its association with NMDARs and their internalization. In this fashion, there is a bidirectional signaling pathway between NRG/ErbB4 and NMDAR that can function as a homeostatic mechanism to regulate interneuron excitability. 2. Subcellular Distribution of Dual Transmembrane (TM) Neuregulins in Central Neurons: By contrast single TM NRGs, we found that dual TM NRGs, such as CRD type III-NRG1 and NRG3 (our recent studies uncovered it also is a dual transmembrane protein) are 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 (Vullhorst et al., Journal of Neuroscience 37(21):5232-5249). 3. Neuregulin-2 Knockout Mice Exhibit Dopamine Dysregulation and Severe Behavioral Phenotypes with Relevance to Psychiatric Disorders: We found that NRG2 expression in the adult rodent brain does not overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC), and therefore generated NRG2 knockout mice (KO) to study its function. NRG2 KOs have higher extracellular dopamine levels in the dorsal striatum but lower levels in the mPFC; a pattern with similarities to dopamine dysbalance in schizophrenia. Like ErbB4 KO mice, NRG2 KOs performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. NRG2 KOs exhibit hyperactivity in a novelty-induced open field, deficits in prepulse inhibition, hypersensitivity to amphetamine, antisocial behaviors, reduced anxiety-like behavior in the elevated plus maze and deficits in the T-maze alteration reward test-a task dependent on hippocampal and mPFC function. Acute administration of clozapine rapidly increased extracellular dopamine levels in the mPFC and improved alternation T-maze performance. Similar to mice treated chronically with N-methyl-d-aspartate receptor (NMDAR) antagonists, we demonstrate that NMDAR synaptic currents in NRG2 KOs are augmented at hippocampal glutamatergic synapses and are more sensitive to ifenprodil, indicating an increased contribution of GluN2B-containing NMDARs. Our findings reveal a novel role for NRG2 in the modulation of behaviors with relevance to psychiatric disorders (Yan et al. Molecular Psychiatry 2017). 4. Analysis of ErbB4 function in mice harboring targeted mutations in GABAergic and dopaminergic neurons: Dysfunctional NRG-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., Molecular Psychiatry 2017). 5. 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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