Elsevier

Neuroscience Research

Volume 70, Issue 2, June 2011, Pages 155-163
Neuroscience Research

The ERBB4 intracellular domain (4ICD) regulates NRG1-induced gene expression in hippocampal neurons

https://doi.org/10.1016/j.neures.2011.02.009Get rights and content

Abstract

The NRG1 growth factor and ERBB4 receptor have been identified as leading schizophrenia risk genes. Although NRG1 and ERBB4 have been shown to modulate neuronal functions involved in schizophrenia, including both GABAergic and glutamatergic synapses, the exact molecular mechanisms remain poorly understood. Here we investigated ERBB4 intracellular domain, 4ICD, transactivator function in rat hippocampal cultures by inhibiting γ-secretase mediated ERBB4 regulated intramembrane proteolysis (RIP). NRG1 stimulation resulted in a dramatic increase in the number of hippocampal cells displaying nuclear 4ICD which was abolished in cultures pretreated with the γ-secretase inhibitor compound E (CE). To identify NRG1-4ICD transactivated genes we compared global gene expression profiles of hippocampal cultures stimulated with NRG1 in the absence or presence of CE. In concordance with the contribution of NRG1-ERBB4 signaling to dendritic spine maturation and schizophrenia, global gene expression analysis followed by Ingenuity Pathway Analysis of the dataset identified NRG1-4ICD regulated genes significantly represented in semaphorin signaling and actin cytoskeletal plasticity and multiple genes with confirmed roles in dendritic spine morphogenesis. Using the power of global gene expression analysis our data provides a proof-of-concept supporting a role for non-canonical NRG1-4ICD signaling in the regulation of gene expression contributing to normal and schizophrenic neuronal function.

Highlights

NRG1 stimulates ERBB4 nuclear accumulation in hippocampal neurons. ► ERBB4 activates expression of multiple genes involved in synapse plasticity. ► Functional pathways associated with schizophrenia are activated by nuclear ERBB4.

Introduction

Schizophrenia is a debilitating mental disorder directly affecting approximately 1% of the global population. Individuals with schizophrenia experience varying degrees of classic symptoms including hallucinations, delusional thoughts, apathy, social withdrawal, and cognitive dysfunction and therefore struggle to function in society (Tandon et al., 2008). Although antipsychotics currently used to treat schizophrenia may relieve positive symptoms such as hallucinations, many of these fail to improve, or even exacerbate, negative and cognitive symptoms (Kim et al., 2009). The lack of effective treatment for these patients stems in part from the complex etiology of schizophrenia which appears to involve environmental, developmental, and genetic factors (Tandon et al., 2008). Accordingly, aberrations in early neural development are believed to play a significant contributing role to the acquisition of schizophrenia later in life. With recent advances in molecular medicine, the genetic and molecular basis of neurological defects and the underlying molecular mechanisms associated with schizophrenia are just emerging.

Recent genetic analyses have identified both the EGF-family member neuregulin 1 (NRG1) and its cognate receptor ERBB4 as leading schizophrenia susceptibility genes (Mei and Xiong, 2008). Accordingly, different experimental systems have implicated NRG1-ERBB4 signaling as critical modulators of multiple neural developmental and functional pathways associated with schizophrenia. For example, both GABAergic and glutamatergic neuronal activities are altered in schizophrenia and modulation of these pathways can either enhance or relieve schizophrenic symptoms (Gaspar et al., 2009, Guidotti et al., 2005, Kim et al., 2009, Konradi and Heckers, 2003). NRG1-ERBB4 signaling regulates the assembly and function of glutamatergic synapses in the hippocampus (Jaaro-Peled et al., 2009, Mei and Xiong, 2008), a region of the brain affected in schizophrenia. In the hippocampus, NRG1 is highly expressed in presynaptic structures of CA3 neurons whereas ERBB4 is predominantly expressed in post-synaptic structures of CA1 neurons (Okada and Corfas, 2004). NRG1-ERBB4 signaling regulates CA3-CA1 circuitry where postsynaptic ERBB4 mediates maturation and plasticity of glutamatergic excitatory synapses (Barros et al., 2009, Krivosheya et al., 2008, Li et al., 2007). In an activity dependent manner ERBB4 stabilizes and maintains post-synaptic dendritic spines (Li et al., 2007). Exogenous NRG1 also increases dendritic spine formation and subsequent stabilization of both excitatory (Barros et al., 2009, Li et al., 2007) and inhibitory synapses (Krivosheya et al., 2008). In fact, ERBB4 is predominantly expressed in inhibitory GABAergic interneurons where NRG1-ERBB4 signaling promotes inhibitory synapse formation (Krivosheya et al., 2008) and GABA release (Woo et al., 2007). Therefore, NRG1-ERBB4 signaling can potentially regulate glutamatergic function through direct modulation of both excitatory and inhibitory synapse circuitry. Consistent with the glutamatergic hypofunction hypothesis in schizophrenia the reduction of dendritic spines and glutamatergic synapses observed in the hippocampus of NRG1 or ERBB4 loss-of-function mouse models can be restored and schizophrenia related behavior ameliorated by treatment with clozapine (Barros et al., 2009, Rimer et al., 2005, Stefansson et al., 2002), an atypical antipsychotic with reported efficacy in treating negative and cognitive symptoms of schizophrenia (McGurk, 1999).

The exact molecular mechanisms underlying ERBB4 regulation of synapse maturation and function remain largely undefined. The vast majority of studies to date have focused on deciphering the influence of canonical NRG1-ERBB4 signaling on neural function. However, NRG1 stimulated ERBB4 undergoes regulated intramembrane proteolysis (RIP) to generate a soluble ERBB4 intracellular domain (4ICD) with independent signaling activities (Lee et al., 2002, Naresh et al., 2006, Ni et al., 2001, Vidal et al., 2005). Through a non-canonical signaling mechanism 4ICD functions in multiple experimental systems as a potent transcriptional coactivator, directly associating with and regulating expression of specific gene promoters (Sardi et al., 2006, Williams et al., 2004, Zhu et al., 2006). High levels of nuclear 4ICD have been reported in hippocampal neurons (Lai and Feng, 2004) and 4ICD is the predominant form of ERBB4 detected in both normal and schizophrenic brains (Hahn et al., 2006). Functionally, RIP of ERBB4 to generate 4ICD mediates maturation of oligodendrocytes (Lai and Feng, 2004) and nuclear 4ICD regulates gene expression required for astrogenesis in the developing brain (Sardi et al., 2006). Here we show for the first time that NRG1 stimulated ERBB4 RIP in hippocampal neurons results in nuclear accumulation of 4ICD and transcriptional activation of multiple genes involved in synapse plasticity and functional pathways associated with schizophrenia.

Section snippets

Animals

All animal protocols used in this study were reviewed and approved by the Tulane University Institutional Animal Care and Use Committee, and were in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. For this study, timed-pregnant Sprague–Dawley rats were obtained from Charles River Laboratories (Wilmington, MA, USA), and housed and maintained on a 12-h light/dark cycle, with unlimited access to food and water. Every effort was made to minimize

NRG1-stimulated 4ICD nuclear accumulation requires γ-secretase activity

To determine if similar to other cellular systems NRG1 promotes ERBB4 RIP and 4ICD nuclear accumulation in neural cell cultures, we mock stimulated or stimulated rat hippocampal cells with 50 ng/ml of recombinant heregulin-β1 EGF-domain (NRG1) for 2 h and examined ERBB4/4ICD localization by immunohistochemistry (IHC) and confocal microscopy. In mock treated cultures ERBB4 was predominantly localized to perinuclear regions (Fig. 1; arrowheads) which most likely represents a combination of

Discussion

Clinical and experimental data implicate both NRG1 and ERBB4 as leading schizophrenia susceptibility genes (Mei and Xiong, 2008). Although a complex disease, recent evidence supports a glutamatergic hypofunction model of schizophrenia. In this model hypoactive glutamate signaling contributes to cognitive deficits through impaired synapse stability and the subsequent decrease in GABAergic inhibitory activity results in glutamate neurotoxicity and psychotic symptoms (Gaspar et al., 2009, Konradi

Conclusions

In summary, we investigated the potential role of NRG1 stimulation of cell surface ERBB4 to activate nuclear 4ICD signaling in rat hippocampal cultures. Although our results require genetic and functional confirmation we have provided important proof-of-concept global expression analyses implicating the 4ICD transactivator as a regulator of multiple functional pathways involved in both normal neuronal function and schizophrenia. Most compelling was the potential role of NRG1-4ICD regulated gene

Acknowledgments

The authors would like to thank Benjamin Hall for critical comments during the preparation of this manuscript. This work is dedicated to Brian Jones who is courageously challenging the symptoms of schizophrenia. These studies were supported in part by funds supplied by the Department of Cell and Molecular Biology at Tulane University.

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