1. Subcellular Distribution and Functions of Single- vs. Dual-Transmembrane (TM) Neuregulins in Central Neurons: Numerous Neuregulins (NRGs) are generated through the use of four different genes (NRG1-NRG4) and promoters (NRG1: types -I, -II and -II)), but the functional significance of this evolutionary conserved diversity remains poorly understood. We showed that NRGs can be categorized by their distinct transmembrane (TM) topologies that impart different subcellular trafficking properties. Initially, we studied the single-TM NRG2 that is prominently expressed in the developing postnatal and adult CNS. Using RNAScope and newly generated monoclonal antibodies, we found that 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 in GABAergic interneurons unprocessed proNRG2 accumulates at somato-dendritic junctions between the endoplasmic reticulum and plasma membrane (ER-PM junctions). 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 all single-TM NRGs are cleaved by alpha-sheddases in an activity-dependent manner and shed from the cell surface to signal in paracrine/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 and internalization with NMDARs and, therefore in this fashion, establishes a bidirectional signaling pathway between NRG/ErbB4 and NMDAR that can function as a homeostatic mechanism to regulate interneuron excitability (Vullhorst et al., Nat Commun 2015). In contrast to single-TM NRGs, we found that dual-TM CRD-NRG1 (type III) and NRG3 (our recent studies uncovered it also is a dual-TM NRG) 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-TM NRGs regulate neuronal functions in fundamentally different ways (Vullhorst, Ahmad et al., J Neurosci 2017). This work was supported by a Directors Investigator Award. 2. NMDA Receptors Regulate NRG2 Binding to ER-PM Junctions and Ectodomain Release by ADAM10: We investigated the temporal and downstream targets of NMDAR activity that mediate proNRG2 processing in hippocampal neurons. Using pharmacological and genetic approaches, we found that activation of NMDARs causes the dissociation of single-TM proNRGs from SSCs/ER-PM junctions and subsequent processing by ADAM10 - not ADAM17 as initially reported in studies using non-neuronal cell lines. Concomitantly, we observed the dephosphorylation in a conserved Ser/Thr-rich region in the intracellular domain of proNRG2. Most of the biologically active NRG1 (type-I or -II) or NRG2 ectodomains are shed into the extracellular space within minutes of NMDAR activation, which can then promote signaling via ErbB4 receptors (Vullhorst & Buonanno, Mol Neurobiol 2019). 3. A Novel Ultrasensitive In Situ Hybridization Approach to Detect Short Sequences and Splice Variants with Cellular Resolution: Detection of short isoform-specific sequences requires RNA isolation for PCR analysis-an approach that loses the regional and cell-type-specific distribution of isoforms. Having the capability to distinguish the differential expression of RNA variants in tissue is critical because alterations in mRNA splicing and editing, as well as coding single nucleotide polymorphisms, have been associated with numerous cancers, neurological and psychiatric disorders. We reported on a novel highly specific and sensitive single-probe colorimetric/fluorescent ISH approach, called BaseScope, that targets short exon/exon RNA splice junctions using single-pair oligonucleotide probes (50 bp). We used this approach to investigate, with single-cell resolution, the expression of four ErbB4 encoding transcripts that differ by alternative splicing of exons encoding two juxtamembrane (JMa/JMb) and two cytoplasmic (CYT-1/CYT-2) domains. First, by comparing ErbB4 hybridization on sections from wild-type and ErbB4 knockout mice (missing exon 2), we demonstrated that single-pair probes have the specificity and sensitivity to visualize and quantify the differential expression of ErbB4 isoforms. Next, we demonstrated that expression of ErbB4 isoforms differs between neurons and oligodendrocytes. Basescope could serve as an invaluable diagnostic tool to detect alternative spliced isoforms, and potentially single base polymorphisms, associated with disease (Erben et al. Mol Neurobiol 2018; Erben & Buonanno Curr Protoc Neurosci 2019). 4. 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, Shamir, Skirzewski et al. Mol Psych 2018). 5. 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 NRG1 acutely increases extracellular DA levels and regulates LTP and gamma oscillations, and ErbB4 is expressed in GABAergic (Pv+) and mesocortical DAergic (TH+) 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. Unexpectedly we have found that, in contrast to GABAergic neurons, ErbB4 is expressed DA neuron axons, and that NRG regulates extracellular DA levels by modulating DAT function. In contrast to mice harboring mutations in GABAergic neurons, which show sensory-motor gating deficits and increases in motor activity, ErbB4 TH KO mice exhibit deficits in cognitive-related tasks (in the T-, Y- and Barnes- mazes). Therefore, direct effects of NRG/ErbB4 signaling in GABAergic vs. DAergic neurons differentially affect cortical circuits and DA homeostasis and behaviors relevant to schizophrenia (Skirzewski et al., Mol Psych 2018).
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