Neuregulin-ErbB and NMDA Receptor Signaling in Neuronal Development and Psychiatric Disorders

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1. Presynaptic accumulation of NRG3 in Central Neurons is Achieved by Trans-Synaptic Retention - a novel mechanism for polarized axonal expression of proteins: How stable axonal polarity is maintained remains a central question in neuroscience. We previously demonstrated that dual-TM proNRGs, comprised by CRD-NRG1 type III and NRG3, are targeted to axons and accumulate at glutamatergic presynaptic terminals where they signal in juxtacrine mode via postsynaptic ErbB4 receptors expressed at postsynaptic densities on GABAergic interneurons (Vullhorst, Ahmad et al., J Neurosci 2017). Our new study aimed to understand how and where proNRG3 is cleaved by BACE1, and how the resulting biologically active NRG3 peptide is sorted and selectively retained in axons. For this study an LOV-based optogenetic proNRG3 reporter (LA143-NRG3) was designed, which in response to blue light, it undergoes a conformational change that allows BACE1 to cleave proNRG3. Using LA143-NRG3, we found proNRG3 is retained in the TGN until cleaved by BACE1, then mature NRG3 emerges on the somatodendritic plasma membrane, and by transcytosis is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons. Importantly, NRG3 accumulation at axonal presynaptic terminals is maintained by its continued trans-synaptic interaction with ErbB4 receptors expressed at postsynaptic glutamatergic synapses on GABAergic interneurons. We denote this novel mechanism "transsynaptic retention" and propose it can accounts for polarized axonal expression of other transmembrane ligands and receptors (Ref. 1). 2. Single-pass TM NRG2 in Central Neurons: Single-pass 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. Activation of excitatory glutamatergic NMDARs located on neuronal soma/dendrites promote calcium entry and activate phosphatases that dephosphorylate Ser/Thr residues in the proNRG2 intracellular region, resulting in the dissociation of proNRG2 from ER-PM junctions and ectodomain cleavage by the metalloproteinase ADAM10 (Vullhorst & Buonanno, Mol Neurobiol 2019). The released biologically active NRG2 binds and activates ErbB4 receptors expressed at postsynaptic excitatory glutamatergic synapses onto GABAergic interneurons, which in turn selectively downregulates NMDA excitatory currents (Vullhorst et al., Nat Comm 2015). We hypothesize that this bidirectional NMDAR-NRG2 (up)/ErbB4-NMDAR (down) signaling mode serves as a homeostatic mechanism that regulates the activity of GABAergic interneurons. A remaining challenge was to understand how proNRG2 clusters at ER-PM junctions, a site where Kv2.1 potassium channels also accumulate via their interaction with VAP, and how NMDAR activation causes their dissociation from the junction. Using a combination of cell biological and protein biochemical approaches, we identified two conserved protein sequences (denoted C- and D-boxes) in the cytoplasmic tails of proNRG1 and proNRG2 that interact with the ER-resident protein VAP to promote the formation of ER-PM contact sites on the proximal somatodendritic region of GABAergic interneurons. Although the sequences of C/D-boxes do not conform to canonical FFAT VAP-binding sites, the proNRG2 D-box contains acidic residues required for VAP binding and the C-box harbors a cryptic, phosphorylation-dependent VAP binding site. Importantly, NMDAR activation stimulates C-box Ser/Thr dephosphorylation, dissociation of proNRG2 from VAP and dispersion of NRG2 from ER-PM junctions. Based on these findings, we hypothesize that Kv2.1 channels and bidirectional NRG2/ErbB4 autocrine signaling function synergistically as a homeostatic mechanism both to protect fast-spiking parvalbumin-positive interneurons from excitotoxic damage and to regulate the activity of GABAergic interneurons, which affect E-I balance and neuronal network activity associated with psychiatric-relevant behaviors disrupted in NRG2 and ErbB4 knockout mice (Ref. 2). 3. Developmental, Neurochemical and Behavioral Analyses of ErbB4 Cyt-1 Knockout Mice: ErbB4 receptor transcripts are alternatively spliced to generate isoforms that either include (Cyt-1) or exclude (Cyt-2) exon 26; an exon that encodes a cytoplasmic domain that imparts on ErbB4 receptors the ability to signal via the PI3K/Akt pathway rather than the MAPK pathway. To investigate the effects of germline (constitutive) and conditional (acute) deletions of the Cyt-1 exon, we generated and studied ErbB4-floxed (ErbB4-Cyt1fl/fl ) mice because ErbB4 Cyt-1/2 isoforms had been only studied in cultured cells, and clinical studies implicated ErbB4 Cyt-1 variants as schizophrenia risk-factors. We found Cyt-1 knockouts only encode ErbB4 Cyt- 2 variants, but in contrast to ErbB4 null mice, GABAergic interneuron migration and number are unaltered in Cyt-1 KOs. Interestingly, basal extracellular dopamine (DA) levels are augmented in the medial prefrontal cortex of Cyt-1 mice but they don't manifest the behavioral abnormalities we observed in mice lacking ErbB4 in DA neurons. To address the possibility that Cyt-2 variants compensate for the lack of Cyt-1 during development, we microinjected AAV-Cre into the DA-rich VTA of adult ErbB4-Cyt1fl/fl mice to acutely target exon 26. These conditional Cyt-1 KOs were found to exhibit behavioral abnormalities in the elevated plus maze and startle response, consistent with the idea that late exon 26 ablations may circumvent compensation by Cyt-2 variants. Our findings suggest that ErbB4 Cyt-1 function in vivo is important for modulating DA levels and for regulating behaviors in adult mice (Refs. 3,4). 4. Pathway-specific contribution of parvalbumin interneuron NMDARs to synaptic currents and thalamocortical feedforward inhibition: The prefrontal cortex (PFC) is a site of convergence of long-range excitatory glutamatergic inputs that integrates multiple modalities of information to produce goal-directed behaviors, and drugs selectively targeting NMDA-type glutamate receptors alter PFC function and elicit numerous deficits associated with psychiatric disorders. Inhibitory GABAergic fast-spiking parvalbumin-expressing (PV+) interneurons are uniquely suited to coordinate the firing of pyramidal neurons in response to these converging excitatory inputs, and to induce gamma oscillations in cortical networks that modulate behaviors and that may be disrupted in several psychiatric disorders. Despite the importance of understanding how glutamatergic inputs onto PV+ interneurons affect network activity, behavior and disease, there continues to be controversy if both AMPA- and NMDA-type glutamate receptors or only AMPARs contribute to excitatory drive. We have used a combination of molecular, electrophysiological and optogenetic approaches, in combination with selective gene targeting techniques in PV+ interneurons, to resolve this long-standing controversy. We found that nearly 100% of PV+ interneurons in adult medial PFC express transcripts encoding GluN1 and GluN2B that assemble to form functional NMDARs. Importantly, by using selective optogenetic stimulation of corticocortical vs. thalamocortical inputs onto PV+ interneurons, we found that the relative synaptic NMDAR contribution to excitatory post-synaptic currents is pathway-specific. NMDARs contribute more at thalamocortical, rather than corticocortical, synapses. We then went on to demonstrate that PV+ interneuron NMDAR currents contribute to thalamus-mediated feedforward inhibition in PFC circuits. These findings demonstrating a molecular and circuit-based mechanisms for cognitive impairment under conditions of reduced NMDAR function, represent an important conceptual advance with implications for understanding the pathogenesis of psychiatric disorders (Re