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. We showed that NRGs can be categorized by their distinct trans-membrane (TM) topologies that impart different subcellular trafficking properties. Single TM NRGs, like NRG1 (types I and II) and NRG2, target to the cell body plasma membrane and 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 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 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, and therefore in this fashion 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. 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). 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 et al. Mol Psych 2017). 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 2017). 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. PLoS One 2017).
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