1. Unprocessed NRG2 accumulates at endoplamic reticulum-plasma membrane (ER-PM) junctions where it is protected from alpha-secretase processing: The cellular and molecular processes that promote the conversion of NRG ligands from inactive pro-forms to signaling-competent ligands that can engage ErbB4 receptors to mediate their aforementioned biological effects in the developing and maturing brain remain mostly unknown. To address this major unresolved question, we investigated the role of Neuregulin 2 (NRG2), a Neuregulin isotype that is prominently expressed in the developing postnatal and adult CNS. Using a novel double-labeling in situ hybridization technique (RNAScope) and newly generated monoclonal antibodies, we found that in the rodent hippocampus 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. Immunogold electron microscopy of NRG2 in dissociated hippocampal neurons, performed in collaboration with Dr. Susan Cheng, revealed that these puncta are found atop of subsurface cisterns (SSCs) - sites of close ER-PM apposition that are proposed to function as signaling platforms (Vullhorst et al., Nat Commun 1;6:7222, 2015). 2. NMDA receptor function on cortical interneurons promotes proNRG2 processing and, in turn, NRG2 signaling via ErbB4 downregulates NMDAR function on interneurons: We found that the proNRG2 puncta atop SSCs are localized to the empty center of doughnut-shaped Kv2.1 clusters at the edges of SSCs. Interestingly, treatment of neuronal cultures with glutamate or NMDA rapidly causes the dispersal of both proteins from the SSC and processing of proNRG2 by shedasses to release the signaling-competent NRG2. What is the function of released NRG2 from GAGAergic interneurons, which are known to express the ErbB4 receptor? In collaboration with Sanford Markey's group at NIMH, we used ErbB4 immunoprecipitation from the soluble fraction of metabolically active synaptosomes followed by LC/MS/MS to characterize the ErbB4 proteome. Using this approach, we identified the NMDAR GluN2B subunit as an ErbB4 interacting protein. This interaction was confirmed in cultured hippocampal neurons, where NRG2 treatment was shown to enhance the internalization of GluN2B-containing, but not GluN2A-containing, NMDARs. Consistent with this observation, we found that NRG2 also caused a dramatic reduction of whole cells NMDAR currents in dissociated hippocampal ErbB4-positive interneurons, but not in ErbB4-negative glutamatergic neurons. Lastly, using whole-cell voltage-clamp recordings in acute medial prefrontal cortical slices, we found that NRG2 selectively reduced NMDAR synaptic currents (EPSCs), and not AMPAR EPSCs, at glutamatergic synapses onto GABAergic interneurons. These results are consistent with the idea that the bidirectlional signaling between NRG2/ErbB4 and NMDAR activity can play a major role in modulating the activity of GABAergic neurons and cortical E/I balance (Vullhorst et al., Nat Commun 1;6:7222, 2015). 3. Neuregulin-2 Ablation Results in Dopamine Dysregulation and Severe Behavioral Phenotypes Relevant to Psychiatric Disorders. While the neurophysiological and behavioral phenotypes of NRG1 mutant mice have been investigated extensively, little is known about the function of NRG2 (see above), the closest NRG1 homologue. We found that NRG2 expression in the adult rodent brain does not fully overlap with NRG1 and is more extensive than originally reported, including expression in the striatum and medial prefrontal cortex (mPFC). We therefore generated NRG2 knockout mice to study its function. NRG2 KOs perform abnormally in a battery of behavioral tasks relevant to psychiatric disorders. We are studying how dopamine levels and glutamatergic function are affected in these mice, and their response to antipsychotics. Our findings reveal a novel role for NRG2 in the modulation of behaviors relevant to the core symptoms of schizophrenia. 4. Studies on the cellular and subcellular expression of Neuregulin 3 (NRG3) in brain. NRGs are encoded by different genes in the brain (NRG1-3), which are transcribed from different promoters and alternatively spliced to generate a large variety of isoforms. In contrast to NRG1, little is known about the cellular expression and functions of NRG2 and NRG3 in the brain. Our study aimed at determining the cellular and subcellular distribution pattern of NRG3, and its function in neurons. Using a novel triple-fluorescence in situ hybridization technique (RNAScope), we found NRG3 transcripts are widely expressed in the adult mouse brain and are relatively more abundant in V-Glut1 positive excitatory cells than in GAD1-positive inhibitory interneurons. Experiments are in progress to compare how the subcellular distribution of NRG3 in neurons, and how it compares to other NRGs. 5. Mesocortical and nigrostriatal DA function in mice with targeted mutations of ErbB4 in Pv+ and tyrosine hydroxylase-positive (TH+) neurons. Dysfunctional Neuregulin-ErbB4 signaling in the hippocampus, prefrontal cortex (PFC) and striatum may contribute to alterations in DA function associated with several schizophrenia symptoms. Because we have shown that NRG1 acutely increases extracellular DA levels to regulate LTP and gamma oscillations, and that ErbB4 expression is confined to GABAergic interneurons (cortex) and TH+ mesocortical DA, we are investigating the relative role NRG/ErbB4 signaling in these two different neuronal populations. To this end, we are measuring basal extracellular DA levels in the PFC, hippocampus and striatum in mice harboring targeted mutations of the receptor in either Pv+ or TH+ neurons, and comparing their behaviors to unravel the role of Neregulin-ErbB4 signaling in schizophrenia-associated behaviors. 6. ErbB signaling disruption increases striatal dopamine levels and affects learning and hedonic-like behavior in the adult mouse. In collaboration with Drs. Alon Shamir and Idit Golani in Israel, the effects of a chronic pharmacological block of ErbB receptors using the pan-ErbB kinase inhibitor JNJ-28871063 (JNJ) was analyzed in adolescent mice. The mice had an elevation of striatal dopamine levels and reduced preference for sucrose, without affecting locomotor activity and exploratory behavior. The reduced preference for sucrose and poor performance during the discrimination phase of the T-maze reversal-learning task was observed up to adulthood. The results suggest that disruption of ErbB signaling during adolescence can have perduring effects on the dopaminergic systems (Godani et al. Eur Neuropsychpharm 24, 1808, 2014). 7. 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.
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