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

Eunice Kennedy Shriver National Institute Of Child Health & Human Development

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Abstract

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 recently 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). In a new series of studies, we aimed to understand how and where proNRG3 is cleaved, and its biologically active NRG3 peptide is sorted to and then retained in axons. To this end, we investigate the spatial-temporal dynamics of NRG3 processing and sorting in neurons using an optogenetic proNRG3 cleavage reporter (LA143-NRG3) that we developed. In dark conditions, unprocessed LA143-NRG3 is retained in the trans-Golgi network (TGN) but, following blue-light photoactivation, it is cleaved by BACE1 and released from the TGN. We found that mature NRG3 initially emerges on the somatodendritic plasma membrane from where it is re-endocytosed and anterogradely transported on Rab4+ vesicles into axons via a process known as transcytosis. Interestingly, our work shows that NRG3 accumulation at axonal presynaptic terminals is mediated by interactions with ErbB4 receptors expressed by postsynaptic GABAergic interneurons. We then go on to show that the continuous interaction between the NRG3 EGF-like domain and its receptor are necessary for NRG3 retention at presynaptic sites, as either addition of a competing peptide or knock-down of ErbB4 from GABAergic neurons, prevents its accumulation. We denote this mechanism "transsynaptic retention" and propose that it may account for polarized expression of other neuronal transmembrane ligands and receptors (Ahmad et al., J Cell Biol 2022). 2. Single-pass TM NRG2 in Central Neurons: We recently found that 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 (Vullhorst et al., Nat Comm 2015). Activation of glutamatergic (excitatory) 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. Together, these two processes promote rapid regulated release of biologically active NRG2 within minutes of NMDA receptor activation to promote ErbB4 signaling (Vullhorst & Buonanno, Mol Neurobiol 2019). In turn, activation of ErbB4 receptors at excitatory post-synaptic densities of GABAergic interneurons selectively down-regulates activity of NMDA, but not AMPA, glutamate receptors (Vullhorst et al., Nat Comm 2015). Based on these findings we hypothesized that this bidirectional NMDAR-NRG2 (up)/ErbB4-NMDAR (down) signaling mode could serve as a homeostatic mechanism to regulate the activity of GABAergic interneurons. Importantly, disruption of such a homeostatic mechanism would alter E-I balance and neuronal network activity, consequently affecting numerous psychiatric-relevant behaviors known to be altered in NRG2 and ErbB4 knockout mice (Yan, Shamir, Skirzewski et al. Mol Psych 2018; Skirzewski et al., Mol Psych 2018; Skirzewski et al., eNeuro 2020). A major unresolved question was to understand mechanistically how proNRG2 clusters at ER-PM contact sites and how it dissociates from them in response to NMDAR activation. Using a combination of cell biological and protein biochemical approaches, we found that proNRG2 promotes the formation of ER-PM contact sites in hippocampal GABAergic interneurons via interactions of its cytoplasmic tail with the ER-resident protein VAP. Interestingly, there are two stretches of amino acids in the intracellular cytoplasmic domain conserved between proNRG1 and proNRG2, denoted as C- and D-boxes, that are required to stabilize proNRG2/VAP complexes during immunoprecipitation. Although the protein sequence of neither box conforms to known FFAT motifs, shown in other proteins to bind VAP, the proNRG2 D-box contains a track of acidic residues required for VAP binding and the C-box harbors a cryptic, phosphorylation-dependent VAP binding site. Importantly, NMDAR activation stimulates dephosphorylation of Ser/Thr residues in the C-box and its dissociation from VAP, which reduces proNRG2 clustering at ER-PM junctions. These observations are interesting because, although both proNRG2 and Kv2.1 are colocalized at ER-PM junctions and clustering at these sites is regulated by NMDA receptor activity, their modes of interaction with VAP differ (Vullhorst et al., submitted). Based on these findings, we hypothesize that autocrine NRG2/ErbB4 signaling and Kv2.1 function synergistically as a homeostatic protective mechanism to downregulate GABAergic interneuron excitability during periods of strong excitatory activity and/or elevated extracellular glutamate levels, which would help to protect these neurons from excitotoxicity. 3. 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 glutamatergic inputs that integrates multiple modalities of information to produce goal-directed behaviors. 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. Altered levels of PV+ interneurons in the PFC and abnormal gamma oscillations have been reported in patients diagnosed with schizophrenia, bipolar depression and autism. Moreover, adolescent disruption of NMDAR function in PV+ interneurons results in altered gamma oscillations and disruption of behaviors associated with 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 glutamate receptors or only AMPARs contribute to excitatory drive. Using a combination of molecular, electrophysiological and optogenetic approaches, in combination with selective gene targeting techniques in PV+ interneurons, we have resolved this long-standing controversy. We found that nearly 100% of PV+ interneurons in adult medial PFC express transcripts encoding GluN1 and GluN2B, and that they have functional NMDARs. With selective optogenetic stimulation of corticocortical or thalamocortical inputs on PV+ interneurons in the PFC, we found that the relative synaptic NMDAR contribution to excitatory post-synaptic currents is pathway-specific, with NMDAR contributing more at thalamocortical synapses a finding that is likely to explain earlier reports of PV+ interneurons without synaptic NMDAR currents. We then went on to ask if NMDAR currents in PV+ interneurons contribute significantly to PFC neuronal network activity. Indeed, we found that PV+ interneuron NMDAR contribute to thalamus-mediated feedforward inhibition in PFC circuits, suggesting molecular and circuit-based mechanisms for cognitive impairment under conditions of reduced NMDAR function (Lewis et al., Mol Psych in review). These findings represent an important conceptual advance that have major implications for potentially understanding the pathogenesis of psychiatric disorders.