Schizophrenia susceptibility pathway neuregulin 1-ErbB4 suppresses Src upregulation of NMDA receptors

Hypofunction of the N-methyl D-aspartate subtype of glutamate receptor (NMDAR) is hypothesized to be a mechanism underlying cognitive dysfunction in individuals with schizophrenia. For the schizophrenia-linked genes NRG1 and ERBB4, NMDAR hypofunction is thus considered a key detrimental consequence of the excessive NRG1-ErbB4 signaling found in people with schizophrenia. However, we show here that neuregulin 1β-ErbB4 (NRG1β-ErbB4) signaling does not cause general hypofunction of NMDARs. Rather, we find that, in the hippocampus and prefrontal cortex, NRG1β-ErbB4 signaling suppresses the enhancement of synaptic NMDAR currents by the nonreceptor tyrosine kinase Src. NRG1β-ErbB4 signaling prevented induction of long-term potentiation at hippocampal Schaffer collateral-CA1 synapses and suppressed Src-dependent enhancement of NMDAR responses during theta-burst stimulation. Moreover, NRG1β-ErbB4 signaling prevented theta burst-induced phosphorylation of GluN2B by inhibiting Src kinase activity. We propose that NRG1-ErbB4 signaling participates in cognitive dysfunction in schizophrenia by aberrantly suppressing Src-mediated enhancement of synaptic NMDAR function.     

Sub-chronic Antipsychotic Drug Administration Reverses the Expression of Neuregulin 1 and ErbB4 in a Cultured MK801-Induced Mouse Primary Hippocampal Neuron or a Neurodevelopmental Schizophrenia Model

It has been reported that specific environmental influences during the postpartum period might contribute to the development of schizophrenia (SZ). Administration of MK801 during early development led to persistent brain pathology. Glutamate decarboxylase 1 (GAD67) and parvalbumin (PV), and neuregulin 1 (NRG1)/ErbB4 signaling were closely associated with SZ pathology. We postulated therefore that NMDA receptor antagonists exposure during the postpartum period may be associated with expression dysregulation of some of the SZ candidate proteins. To test this, we used mouse primary hippocampal neurons and neonatal male mice treated with the NMDA receptor antagonist, MK801 at postnatal day 4 (P4) or P7, followed by the treatments of antipsychotic drugs (i.e., olanzapine, risperidone, and haloperidol). The expressions of GAD67, PV, NRG1, and ErbB4 in in vitro and in vivo SZ models were detected with Western blot analysis and immunohistochemistry, respectively. Behavioral tests (locomotion activity, social interaction, novel object recognition and prepulse inhibition) were measured. We found MK801 decreased the expression of GAD67, PV, NRG1 and ErbB4, and induced obvious behavioral alterations, while antipsychotics reversed these alterations. These results suggest that exposure to the NMDA receptor antagonist in early development may lead to long-lasting influence on the expression of specific proteins, such as GAD67, PV, NRG1, and ErbB4. Moreover, our results suggest that rescue of the activation of the NRG1/ErbB4 signaling pathway may be one of the mechanisms by which antipsychotic drugs have an antipsychotic effect.     

ErbB4-neuregulin signaling modulates synapse development and dendritic arborization through distinct mechanisms

Perturbations in neuregulin-1 (NRG1)/ErbB4 function have been associated with schizophrenia. Affected patients exhibit altered levels of these proteins and display hypofunction of glutamatergic synapses as well as altered neuronal circuitry. However, the role of NRG1/ErbB4 in regulating synapse maturation and neuronal process formation has not been extensively examined. Here we demonstrate that ErbB4 is expressed in inhibitory interneurons at both excitatory and inhibitory postsynaptic sites. Overexpression of ErbB4 postsynaptically enhances size but not number of presynaptic inputs. Conversely, knockdown of ErbB4 using shRNA decreases the size of presynaptic inputs, demonstrating a specific role for endogenous ErbB4 in synapse maturation. Using ErbB4 mutant constructs, we demonstrate that ErbB4-mediated synapse maturation requires its extracellular domain, whereas its tyrosine kinase activity is dispensable for this process. We also demonstrate that depletion of ErbB4 decreases the number of primary neurites and that stimulation of ErbB4 using a soluble form of NRG1 results in exuberant dendritic arborization through activation of the tyrosine kinase domain of ErbB4 and the phosphoinositide 3-kinase pathway. These findings demonstrate that NRG1/ErbB4 signaling differentially regulates synapse maturation and dendritic morphology via two distinct mechanisms involving trans-synaptic signaling and tyrosine kinase activity, respectively.     

Neuregulin-1 enhances depolarization-induced GABA release

Neuregulin-1 (NRG1), a regulator of neural development, has been shown to regulate neurotransmission at excitatory synapses. Although ErbB4, a key NRG1 receptor, is expressed in glutamic acid decarboxylase (GAD)-positive neurons, little is known about its role in GABAergic transmission. We show that ErbB4 is localized at GABAergic terminals of the prefrontal cortex. Our data indicate a role of NRG1, both endogenous and exogenous, in regulation of GABAergic transmission. This effect was blocked by inhibition or mutation of ErbB4, suggesting the involvement of ErbB4. Together, these results indicate that NRG1 regulates GABAergic transmission via presynaptic ErbB4 receptors, identifying a novel function of NRG1. Because both NRG1 and ErbB4 have emerged as susceptibility genes of schizophrenia, these observations may suggest a mechanism for abnormal GABAergic neurotransmission in this disorder.     

Constitutive loss and acute pharmacological manipulation of ErbB4 signaling do not affect attention and inhibitory control in mice

The receptor tyrosine kinase ErbB4 and its ligand trophic factors of the neuregulin (NRG) family have been associated with schizophrenia and other mental disorders in human genetic studies. In vivo studies in mice have shown how abnormal Nrg–ErbB4 signaling leads to deviant behaviors relevant to distinct aspects of schizophrenia, including hyperactivity, sensory gating deficits, working and spatial memory deficits and impaired social behavior. However, so far little is known on the role of ErbB4 in attention and inhibitory control, two aspects of executive functions that are impaired in schizophrenia. Here we investigated the effects of constitutive loss of ErbB4 in the central nervous system of mice on performance in a 5‐choice serial reaction time task (5CSRTT) assessing attention and inhibitory control. In this task, ErbB4^?/? mice did not show deficits in various parameters of attention, and premature responses as measure of inhibitory control. Nonetheless, ErbB4^?/? mice recapitulated a specific set of behavioral phenotypes associated with schizophrenia, including a deficit in spatial learning and memory in the Barnes Maze and in contextual fear learning, and a trend for a deficit in sensorimotor gating. Furthermore, we investigated the effect of acute pharmacological inhibition of ErbB tyrosine kinase receptor using the pan‐ErbB kinase inhibitor JNJ‐28871063 (JNJ), in an automated version of the 5CSRTT. JNJ did not affect attention and inhibitory control. In conclusion, our data suggest no direct involvement of a classical Nrg‐ErbB4 pathway in attention and inhibitory control in mice, while it confirms the involvement of this pathway in other domains relevant to schizophrenia.     

Neuregulin 1 Controls Glutamate Uptake by Up-regulating Excitatory Amino Acid Carrier 1 (EAAC1)

Neuregulin 1 (NRG1) is a trophic factor that is thought to have important roles in the regulating brain circuitry. Recent studies suggest that NRG1 regulates synaptic transmission, although the precise mechanisms remain unknown. Here we report that NRG1 influences glutamate uptake by increasing the protein level of excitatory amino acid carrier (EAAC1). Our data indicate that NRG1 induced the up-regulation of EAAC1 in primary cortical neurons with an increase in glutamate uptake. These in vitro results were corroborated in the prefrontal cortex (PFC) of mice given NRG1. The stimulatory effect of NRG1 was blocked by inhibition of the NRG1 receptor ErbB4. The suppressed expression of ErbB4 by siRNA led to a decrease in the expression of EAAC1. In addition, the ablation of ErbB4 in parvalbumin (PV)-positive neurons in PV-ErbB4^−/− mice suppressed EAAC1 expression. Taken together, our results show that NRG1 signaling through ErbB4 modulates EAAC1. These findings link proposed effectors in schizophrenia: NRG1/ErbB4 signaling perturbation, EAAC1 deficit, and neurotransmission dysfunction.     

Regulation of neuregulin signaling by PSD-95 interacting with ErbB4 at CNS synapses

Neuregulins (NRGs) and their receptors, the ErbB protein tyrosine kinases, are essential for neuronal development, but their functions in the adult CNS are unknown. We report that ErbB4 is enriched in the postsynaptic density (PSD) and associates with PSD-95. Heterologous expression of PSD-95 enhanced NRG activation of ErbB4 and MAP kinase. Conversely, inhibiting expression of PSD-95 in neurons attenuated NRG-mediated activation of MAP kinase. PSD-95 formed a ternary complex with two molecules of ErbB4, suggesting that PSD-95 facilitates ErbB4 dimerization. Finally, NRG suppressed induction of long-term potentiation in the hippocampal CA1 region without affecting basal synaptic transmission. Thus, NRG signaling may be synaptic and regulated by PSD-95. A role of NRG signaling in the adult CNS may be modulation of synaptic plasticity.     

A reduction in hippocampal GABAA receptor α5 subunits disrupts the memory for location of objects in mice

The memory for location of objects, which binds information about objects to discrete positions or spatial contexts of occurrence, is a form of episodic memory particularly sensitive to hippocampal damage. Its early decline is symptomatic for elderly dementia. Substances that selectively reduce α5‐GABA_A receptor function are currently developed as potential cognition enhancers for Alzheimer's syndrome and other dementia, consistent with genetic studies implicating these receptors that are highly expressed in hippocampus in learning performance. Here we explored the consequences of reduced GABA_Aα5‐subunit contents, as occurring in α5(H105R) knock‐in mice, on the memory for location of objects. This required the behavioral characterization of α5(H105R) and wild‐type animals in various tasks examining learning and memory retrieval strategies for objects, locations, contexts and their combinations. In mutants, decreased amounts of α5‐subunits and retained long‐term potentiation in hippocampus were confirmed. They exhibited hyperactivity with conserved circadian rhythm in familiar actimeters, and normal exploration and emotional reactivity in novel places, allocentric spatial guidance, and motor pattern learning acquisition, inhibition and flexibility in T‐ and eight‐arm mazes. Processing of object, position and context memories and object‐guided response learning were spared. Genotype difference in object‐in‐place memory retrieval and in encoding and response learning strategies for object–location combinations manifested as a bias favoring object‐based recognition and guidance strategies over spatial processing of objects in the mutants. These findings identify in α5(H105R) mice a behavioral–cognitive phenotype affecting basal locomotion and the memory for location of objects indicative of hippocampal dysfunction resulting from moderately decreased α5‐subunit contents.     

The neurosteroid allopregnanolone impairs object memory and contextual fear memory in male C57BL/6J mice

Allopregnanolone (ALLO, or 3α-hydroxy-5α-pregnan-20-one) is a steroid metabolite of progesterone and a potent endogenous positive allosteric modulator of GABA-A receptors. Systemic ALLO has been reported to impair spatial, but not nonspatial learning in the Morris water maze (MWM) and contextual memory in rodents. These cognitive effects suggest an influence of ALLO on hippocampal-dependent memory, although the specific nature of the neurosteroid's effects on learning, memory or performance is unclear. The present studies aimed to determine: (i) the memory process(es) affected by systemic ALLO using a nonspatial object memory task; and (ii) whether ALLO affects object memory via an influence within the dorsal hippocampus. Male C57BL/6J mice received systemic ALLO either before or immediately after the sample session of a novel object recognition (NOR) task. Results demonstrated that systemic ALLO impaired the encoding and consolidation of object memory. A subsequent study revealed that bilateral microinfusion of ALLO into the CA1 region of dorsal hippocampus immediately following the NOR sample session also impaired object memory consolidation. In light of debate over the hippocampal-dependence of object recognition memory, we also tested systemic ALLO-treated mice on a contextual and cued fear-conditioning task. Systemic ALLO impaired the encoding of contextual memory when administered prior to the context pre-exposure session. Together, these results indicate that ALLO exhibits primary effects on memory encoding and consolidation, and extend previous findings by demonstrating a sensitivity of nonspatial memory to ALLO, likely by disrupting dorsal hippocampal function.     

Neuregulin 1 in neural development, synaptic plasticity and schizophrenia

Schizophrenia is a highly debilitating mental disorder that affects approximately 1% of the general population, yet it continues to be poorly understood. Recent studies have identified variations in several genes that are associated with this disorder in diverse populations, including those that encode neuregulin 1 (NRG1) and its receptor ErbB4. The past few years have witnessed exciting progress in our knowledge of NRG1 and ErbB4 functions and the biological basis of the increased risk for schizophrenia that is potentially conferred by polymorphisms in the two genes. An improved understanding of the mechanisms by which altered function of NRG1 and ErbB4 contributes to schizophrenia might eventually lead to the development of more effective therapeutics.     

ErbB3和ErbB4特异性配体筛选系统的建立及neuregulin-1突变体的筛选

Neuregulin-1(NRG1,纽兰格林)通过活化ErbB2/ErbB4二聚体具有治疗心衰的作用,目前已完成临床二期。为避免作为心衰治疗药物时同时激活ErbB3并产生副作用,因此用NRG1变异体的方法寻找能对ErbB4专一性激活的配体。文章构建了带有不同筛选标记的ErbB2、ErbB3、ErbB4细胞表达质粒,将ErbB2/ErbB3、ErbB2/ErbB4质粒共转染至CHO细胞,建立了ErbB2/ErbB3特异性表达和ErbB2/ErbB4特异性表达的细胞株。通过与新生大鼠原代心肌细胞比较,证明ErbB2/ErbB4细胞株信号传导功能与心肌细胞相似,NRG1可以激活下游的AKT信号途径、PI3K信号途径,并表现出良好的剂量效应。因此可以通过检测与心肌功能密切相关的下游信号AKT磷酸化水平快速筛选抗心衰药物,并通过与ErbB2/ErbB3信号激活水平比较鉴定其对心肌细胞的特异性。文章还构建了31个不同的NRG1突变体并在大肠杆菌中成功的表达和纯化。将这些突变体用于刺激两个细胞株,通过检测AKT磷酸化水平,发现这些突变体对ErbB2/ErbB3与ErbB2/ErbB4受体的激活能力不同。进一步检测其中5个ErbB2/ErbB4激活特异性发生改变的突变体与两对受体的亲和力,发现这些突变体和ErbB2/ErbB4与ErbB2/ErbB3受体亲和力的变化有一致性。最终筛选到了4个可以更特异性激活ErbB2/ErbB4受体的突变体作为更有效治疗心衰的候选药物。     

Activation of the neuregulin/ErbB system during physiological ventricular remodeling in pregnancy

The neuregulin-1 (NRG1)/ErbB system has emerged as a paracrine endothelium-controlled system in the heart, which preserves left ventricular (LV) performance in pathophysiological conditions. Here, we analyze the activity and function of this system in pregnancy, which imparts a physiological condition of LV hemodynamic overload. NRG1 expression and ErbB receptor activation were studied by Western blot analyses in rats and mice at different stages of pregnancy. LV performance was evaluated by transthoracic echocardiography, and myocardial performance was assessed from twitches of isolated papillary muscles. NRG1/ErbB signaling was inhibited by oral treatment of animals with the dual ErbB1/ErbB2 tyrosine kinase inhibitor lapatinib. Analyses of LV tissue revealed that protein expression of different NRG1 isoforms and levels of phosphorylated ErbB2 and ErbB4 significantly increased after 1-2 wk of pregnancy. Lapatinib prevented phosphorylation of ErbB2 and ERK1/2, but not of ErbB4 and protein kinase B (Akt), revealing that lapatinib only partially inhibited NRG1/ErbB signaling in the LV. Lapatinib did not prevent pregnancy-induced changes in LV mass and did not cause apoptotic cell death or fibrosis in the LV. Nevertheless, lapatinib led to premature maternal death of ～25% during pregnancy and it accentuated pregnancy-induced LV dilatation, significantly reduced LV fractional shortening, and induced abnormalities of twitch relaxation (but not twitch amplitude) of isolated papillary muscles. This is the first study showing that the NRG1/ErbB system is activated, and plays a modulatory role, during physiological hemodynamic overload associated with pregnancy. Inhibiting this system during physiological overload may cause LV dysfunction in the absence of myocardial cell death.     

Neuregulin-1/ErbB signaling serves distinct functions in myelination of the peripheral and central nervous system

Understanding the control of myelin formation by oligodendrocytes is essential for treating demyelinating diseases. Neuregulin-1 (NRG1) type III, an EGF-like growth factor, is essential for myelination in the PNS. It is thus thought that NRG1/ErbB signaling also regulates CNS myelination, a view suggested by in vitro studies and the overexpression of dominant-negative ErbB receptors. To directly test this hypothesis, we generated a series of conditional null mutants that completely lack NRG1 beginning at different stages of neural development. Unexpectedly, these mice assemble normal amounts of myelin. In addition, double mutants lacking oligodendroglial ErbB3 and ErbB4 become myelinated in the absence of any stimulation by neuregulins. In contrast, a significant hypermyelination is achieved by transgenic overexpression of NRG1 type I or NRG1 type III. Thus, NRG1/ErbB signaling is markedly different between Schwann cells and oligodendrocytes that have evolved an NRG/ErbB-independent mechanism of myelination control.     

Stimulated ErbB4 internalization is necessary for neuregulin signaling in neurons

Neuregulin-1 (NRG1) plays an important role in neural development, synapse formation, and synaptic plasticity by activating ErbB receptor tyrosine kinases. Although ligand-induced endocytosis has been shown to be important for many receptor tyrosine kinases, whether NRG1 signaling depends on ErbB endocytosis remains controversial. Here, we provide evidence that ErbB4, a prominent ErbB protein in the brain, becomes internalized in NRG1-stimulated neurons. The induced ErbB4 endocytosis requires its kinase activity. Remarkably, inhibition of ErbB endocytosis attenuates NRG1-induced activation of Erk and Akt in neurons. These observations indicate a role of ErbB endocytosis in NRG1 signaling in neurons.     

Transient Overexposure of Neuregulin 3 during Early Postnatal Development Impacts Selective Behaviors in Adulthood

Neuregulin 3 (NRG3), a specific ligand for ErbB4 and a neuronal-enriched neurotrophin is implicated in the genetic predisposition to a broad spectrum of neurodevelopmental, neurocognitive and neuropsychiatric disorders, including Alzheimer''s disease, autism and schizophrenia. Genetic studies in schizophrenia demonstrate that risk variants in NRG3 are associated with cognitive and psychotic symptom severity, accompanied by increased expression of prefrontal cortical NRG3. Despite our expanding knowledge of genetic involvement of NRG3 in neurological disorders, little is known about the neurodevelopmental mechanisms of risk. Here we exploited the fact that a paralog of NRG3, NRG1, readily penetrates the murine blood brain barrier (BBB). In this study we synthesized the bioactive epidermal growth factor (EGF) domain of NRG3, and using previously validated in-vivo peripheral injection methodologies in neonatal mice, demonstrate that NRG3 successfully crosses the BBB, where it activates its receptor ErbB4 and downstream Akt signaling at levels of bioactivity comparable to NRG1. To determine the impact of NRG3 overexpression during one critical developmental window, C57BL/6 male mice were subcutaneously injected daily with NRG1-EGF, NRG3-EGF or vehicle from postnatal days 2–10. Mice were tested in adulthood using a comprehensive battery of behavioral tasks relevant to neurocognitive and psychiatric disorders. In agreement with previous studies, developmental overexposure to NRG1 induced multiple non-CNS mediated peripheral effects as well as severely disrupting performance of prepulse inhibition of the startle response. In contrast, NRG3 had no effect on any peripheral measures investigated or sensorimotor gating. Specifically, developmental NRG3 overexposure produced an anxiogenic-like phenotype and deficits in social behavior in adulthood. These results provide primary data to support a role for NRG3 in brain development and function, which appears to be distinct from its paralog NRG1. Furthermore we demonstrate how perturbations in NRG3 expression at distinct developmental stages may contribute to the neurological deficits observed in brain disorders such as schizophrenia and autism.     

Neuregulins: functions,forms, and signaling strategies

The neuregulins (NRGs) are cell-cell signaling proteins that are ligands for receptor tyrosine kinases of the ErbB family. The neuregulin family of genes has four members: NRG1, NRG2, NRG3, and NRG4. Relatively little is known about the biological functions of the NRG2, 3, and 4 proteins, and they are considered in this review only briefly. The NRG1 proteins play essential roles in the nervous system, heart, and breast. There is also evidence for involvement of NRG signaling in the development and function of several other organ systems, and in human disease, including the pathogenesis of schizophrenia and breast cancer. There are many NRG1 isoforms, raising the question "Why so many neuregulins?" Study of mice with targeted mutations ("knockout mice") has demonstrated that isoforms differing in their N-terminal region or in their epidermal growth factor (EGF)-like domain differ in their in vivo functions. These differences in function might arise because of differences in expression pattern or might reflect differences in intrinsic biological characteristics. While differences in expression pattern certainly contribute to the observed differences in in vivo functions, there are also marked differences in intrinsic characteristics that may tailor isoforms for specific signaling requirements, a theme that will be emphasized in this review.     

Neuregulin/ErbB regulate neuromuscular junction development by phosphorylation of α-dystrobrevin

Neuregulin (NRG)/ErbB signaling is involved in numerous developmental processes in the nervous system, including synapse formation and function in the central nervous system. Although intensively investigated, its role at the neuromuscular synapse has remained elusive. Here, we demonstrate that loss of neuromuscular NRG/ErbB signaling destabilized anchoring of acetylcholine receptors (AChRs) in the postsynaptic muscle membrane and that this effect was caused by dephosphorylation of α-dystrobrevin1, a component of the postsynaptic scaffold. Specifically, in mice in which NRG signaling to muscle was genetically or pharmacologically abolished, postsynaptic AChRs moved rapidly from the synaptic to the perisynaptic membrane, and the subsynaptic scaffold that anchors the AChRs was impaired. These defects combined compromised synaptic transmission. We further show that blockade of NRG/ErbB signaling abolished tyrosine phosphorylation of α-dystrobrevin1, which reduced the stability of receptors in agrin-induced AChR clusters in cultured myotubes. Our data indicate that NRG/ErbB signaling maintains high efficacy of synaptic transmission by stabilizing the postsynaptic apparatus via phosphorylation of α-dystrobrevin1.     

ErbB3/HER3 does not homodimerize upon neuregulin binding at the cell surface

To understand signaling by the neuregulin (NRG) receptor ErbB3/HER3, it is important to know whether ErbB3 forms homodimers upon ligand binding. Previous biophysical studies suggest that the ErbB3 extracellular region remains monomeric when bound to NRG. We used a chimeric receptor approach to address this question in living cells, fusing the extracellular region of ErbB3 to the kinase-active intracellular domain of ErbB1. The ErbB3/ErbB1 chimera responded to NRG only if ErbB2 was co-expressed in the same cells, whereas an ErbB4/ErbB1 chimera responded without ErbB2. We, therefore, suggest that ErbB3 is an obligate heterodimerization partner because of its inability to homodimerize.