Neuregulin-ErbB Signaling in Neuronal Development and Psychiatric Disorders

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1. Subcellular Distribution and Functions of Single- vs. Dual-Transmembrane Neuregulins in Central Neurons: We recently found that single-pass transmembrane NRGs (sp-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 sp-NRGs is triggered by glutamatergic (i.e., excitatory) activity acting on 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 sp-NRGs within minutes of NMDA receptor activation to promote ErbB4 signaling (Vullhorst & Buonanno, Mol Neurobiol 2019). In stark contrast to sp-NRGs, dual-pass transmembrane (dp) 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. Published and ongoing studies, primarily on NRG3, demonstrate that their unprocessed proforms are cleaved near the second TM domain by BACE-1, which is required for NRG3 to be released from the Golgi compartment and transport into axons (Vullhorst, Ahmad et al., J Neurosci 2017). 2. 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). 3. 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). 4. 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). 5. Transduction of mesencephalic DAergic neurons via AAV_Cre microinjection can lead to off-target toxicity: Stereotaxic microinjection of adeno-associated virus (AAV) expressing Cre recombinase (AAV_Cre) into specific brain nuclei has become a popular approach to ablate genes by loxP/Cre recombination in a regional- and temporal-specific fashion. In the process of investigating the functional role of the ErbB4 Cyt-1 isoform in DAergic neurons by microinjecting AAV_Cre into the ventral tegmental area (VTA) of mice harboring a floxed Cyt-1 allele of ErbB4, using AAV_Cre titers commonly reported in the literature (1013 GC/mL), we found that it is toxic to mesencephalic DA neurons. The toxicity observed is independent of ErbB4 deletion, as AAV_Cre microinjection in both ErbB4 Cyt-1fl/fl and wildtype C57Bl/6J mice resulted in the loss of the DA transporter (DAT) and tyrosine hydroxylase (TH), proteins critical for DA function, and behavioral changes previously associated with loss of this neurotransmitter. These phenotypes occur regardless of mouse genotype, AAV serotype, or vector packaging (Cre/GFP mixture or Cre-GFP fusion protein). Interestingly, we observed that the AAV1 serotype affects DAT and TH expression, whereas AAV9 is toxic to DAergic neurons when used at 1013 GC/mL. Importantly, we found that diluting the viral titer by 1000-fold to 1010 GC/mL effectively prevents these undesired effects while still efficiently recombining floxed targets. This work highlights the critical need for thorough and appropriate controls, such as including WT mice in experimental design, to account for potential off-target effects when working with AAV_Cre recombinase (Erben et al., under review in Journal of Neurochemistry).