Neuregulin-ErbB Signaling in Neuronal Development and Psychiatric Disorders

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

1. Subcellular Distribution, Processing and Function of Single-Transmembrane (TM) NRGs in Central Neurons: We recently found that single-TM NRGs, such as NRG1 type II and NRG2, traffic as unprocessed pro-forms to the neuronal cell surface where they accumulate at ER-PM junctions on neuronal soma and proximal dendrites. Shedding of the signaling-competent ectodomain of single-TM NRGs is triggered by calcium entry through excitatory glutamatergic NMDA receptors, and mediated by membrane-bound ADAM-type metalloproteinases (Vullhorst, Ahmad et al., 2017). NMDA receptor activity promotes dissociation of unprocessed proNRGs from ER-PM contact sites through dephosphorylation of multiple conserved Ser/Thr residues located in their intracellular region and ectodomain shedding by ADAM10. Together, these two processes promote rapid regulated release of biologically active single-TM NRGs within minutes of NMDA receptor activation to promote ErbB4 signaling (Vullhorst & Buonanno, Mol Neurobiol 2019). In turn, the processed NRG is released and binds ErbB4 receptors expressed at excitatory post-synaptic densities of GABAergic interneurons, where it selectively down-regulates NMDA, but not AMPA, receptors. We hypothesized (Vullhorst et al., Nat Comm 2015) that this bidirectional autocrine NRG/ErbB4 signaling mode could serve as a homeostatic mechanism to regulate calcium entry in GABAergic interneurons. 2. Polarized Axonal Expression of Dual-TM NRGs in Central Neurons by Trans-Synaptic Retention: In stark contrast to single-TM NRGs, dual-TM NRG1 type III and NRG3 are targeted to axons and accumulate at glutamatergic presynaptic terminals onto GABAergic interneurons where they signal in juxtacrine mode via postsynaptic ErbB4 receptors expressed at postsynaptic densities on GABAergic interneurons. Our published (Vullhorst, Ahmad et al., J Neurosci 2017) and ongoing (Ahmad et al., in progress) studies demonstrate that cleavage of proNRG3 in the trans-Golgi network (TGN) is required for its release from this organelle. Once cleaved near the second TM domain by BACE-1, the membrane bound fragment containing the first cytoplasmic sequence, TM and EGF-like domain (necessary for ErbB binding) is sorted and trafficked to axons by transcytosis. Through a novel mechanism that we denote trans-synaptic retention, the polarized accumulation of NRG3 on axonal presynaptic terminals is maintained through its trans-synaptic interaction with postsynaptic ErbB4 receptors expressed on dendrites of GABAergic interneurons. 3. Neuregulin-2 and ErbB4 Knockout Mice Exhibit Dopamine Dysregulation and Severe Behavioral Phenotypes with Relevance to Psychiatric Disorders: We found that NRG2 expression is more extensive than originally reported, extending to striatal and medial prefrontal cortical (mPFC) neurons, and its receptor ErbB4 are expressed on presynaptic process from mesencephalic dopamine (DA) neurons that innervate these structures. Therefore, to investigate the function of NRG2-ErbB4 signaling, we generated NRG2 and ErbB4 knockout mice (KO). We found NRG2 andd ErbB4 KOs have higher extracellular DA levels in the dorsal striatum but lower levels in the mPFC and hippocampus; a pattern with similarities to DA dysbalance in schizophrenia. NRG2 and ErbB4 KO mice performed abnormally in a battery of behavioral tasks relevant to psychiatric disorders. They 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, a potent atypical antipsychotic, to NRG2 KO mice rapidly increased extracellular DA levels in the mPFC and improved alternation T-maze performance. This work emphasizes the importance of the NRG2-ErbB4 signaling pathway on the nigrostriatal, mesocortical and mesolimbic DA systems (Yan, Shamir, Skirzewski et al. Mol Psych 2018; Skirzewski et al., ENeuro 2020). 4. Analysis of ErbB4 function in mice harboring targeted mutations in either GABAergic or DAergic neurons: Dysfunctional NRG-ErbB4 signaling in the hippocampus, pre-frontal cortex (PFC) and striatum may contribute to alterations in 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). 5. 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 targetsshort 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).