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.     

Involvement of Protein Kinase D1 in Signal Transduction from the Protein Kinase C Pathway to the Tyrosine Kinase Pathway in Response to Gonadotropin-releasing Hormone

The receptor for gonadotropin-releasing hormone (GnRH) belongs to the G protein-coupled receptors (GPCRs), and its stimulation activates extracellular signal-regulated protein kinase (ERK). We found that the transactivation of ErbB4 was involved in GnRH-induced ERK activation in immortalized GnRH neurons (GT1–7 cells). We found also that GnRH induced the cleavage of ErbB4. In the present study, we examined signal transduction for the activation of ERK and the cleavage of ErbB4 after GnRH treatment. Both ERK activation and ErbB4 cleavage were completely inhibited by YM-254890, an inhibitor of G_q/11 proteins. Down-regulation of protein kinase C (PKC) markedly decreased both ERK activation and ErbB4 cleavage. Experiments with two types of PKC inhibitors, Gö 6976 and bisindolylmaleimide I, indicated that novel PKC isoforms but not conventional PKC isoforms were involved in ERK activation and ErbB4 cleavage. Our experiments indicated that the novel PKC isoforms activated protein kinase D (PKD) after GnRH treatment. Knockdown and inhibitor experiments suggested that PKD1 stimulated the phosphorylation of Pyk2 by constitutively activated Src and Fyn for ERK activation. Taken together, it is highly possible that PKD1 plays a critical role in signal transduction from the PKC pathway to the tyrosine kinase pathway. Activation of the tyrosine kinase pathway may be involved in the progression of cancer.     

Neuregulin-1 only induces trans-phosphorylation between ErbB receptor heterodimer partners

ErbB2, ErbB3 and ErbB4 are members of the Epidermal Growth Factor Receptor (EGFR) sub-family of Receptor Tyrosine Kinases (RTKs). Neuregulin-1 (NRG-1) is a ligand of ErbB3 and ErbB4 receptors. NRG-1-induced ErbB2/ErbB3 or ErbB2/ErbB4 heterodimerization, followed by receptor phosphorylation, plays multiple biological roles. To precisely determine the phosphorylation status of each ErbB receptor in ErbB2/ErbB3 and ErbB2/ErbB4 heterodimers, an immunoprecipitation-recapture of the ErbB receptors was performed to exclude any co-immunoprecipitated heterodimer partners from cells with co-expression of ErbB2/ErbB3, ErbB2/ErbB4, or ErbB2/ErbB4D843N, a kinase-inactive ErbB4 mutant, in which the aspartic acid at 843 (D843) was replaced by an asparagine (N). Here, we provide direct biochemical evidence that ErbB2 was only trans-phosphorylated by ErbB4, but not by ErbB3 or ErbB4D843N. By contrast, ErbB3, ErbB4 and ErbB4D843N were trans-phosphorylated by ErbB2 in the co-transfected cells. Therefore, we conclude that trans-phosphorylation, but not cis-phosphorylation occurred between ErbB2/ErbB3 and ErbB2/ErbB4 heterodimer partners by NRG-1 stimulation.     

Sustained high levels of neuregulin-1 in the longest-lived rodents; a key determinant of rodent longevity

Naked mole-rats (Heterocephalus glaber), the longest-lived rodents, live 7-10 times longer than similarly sized mice and exhibit normal activities for approximately 75% of their lives. Little is known about the mechanisms that allow them to delay the aging process and live so long. Neuregulin-1 (NRG-1) signaling is critical for normal brain function during both development and adulthood. We hypothesized that long-lived species will maintain higher levels of NRG-1 and that this contributes to their sustained brain function and concomitant maintenance of normal activity. We monitored the levels of NRG-1 and its receptor ErbB4 in H. glaber at different ages ranging from 1 day to 26 years and found that levels of NRG-1 and ErbB4 were sustained throughout development and adulthood. In addition, we compared seven rodent species with widely divergent (4-32 year) maximum lifespan potential (MLSP) and found that at a physiologically equivalent age, the longer-lived rodents had higher levels of NRG-1 and ErbB4. Moreover, phylogenetic independent contrast analyses revealed that this significant strong correlation between MLSP and NRG-1 levels was independent of phylogeny. These results suggest that NRG-1 is an important factor contributing to divergent species MLSP through its role in maintaining neuronal integrity.     

Computational analysis of molecular basis of 1:1 interactions of NRG-1beta wild-type and variants with ErbB3 and ErbB4

The neuregulin/ErbB system is a growth factor/receptor cascade that has been proven to be essential in the development of the heart and the sympathetic nervous system. However, the basis of the specificity of ligand-receptor recognition remains to be elucidated. In this study, the structures of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes were modeled based on the available structures of the homologous proteins. The binding free energies of NRG-1beta to ErbB3 and ErbB4 were calculated using the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) computational method. In addition, computational alanine-scanning mutagenesis was performed in the binding site of NRG-1beta and the difference in the binding free energies between NRG-1beta mutants and the receptors was calculated. The results specify the contribution of each residue at the interaction interfaces to the binding affinity of NRG-1beta with ErbB3 and ErbB4, identifying several important interaction residue pairs that are in agreement with previously acquired experimental data. This indicates that the presented structural models of NRG-1beta/ErbB3 and NRG-1beta/ErbB4 complexes are reliable and could be used to guide future studies, such as performing desirable mutations on NRG-1beta to increase the binding affinity and selectivity to the receptor and discovering new therapeutic agents for the treatment of heart failure.     

Visualizing the Distribution of Synapses from Individual Neurons in the Mouse Brain

Background

Proper function of the mammalian brain relies on the establishment of highly specific synaptic connections among billions of neurons. To understand how complex neural circuits function, it is crucial to precisely describe neuronal connectivity and the distributions of synapses to and from individual neurons.

Methods and Findings

In this study, we present a new genetic synaptic labeling method that relies on expression of a presynaptic marker, synaptophysin-GFP (Syp-GFP) in individual neurons in vivo. We assess the reliability of this method and use it to analyze the spatial patterning of synapses in developing and mature cerebellar granule cells (GCs). In immature GCs, Syp-GFP is distributed in both axonal and dendritic regions. Upon maturation, it becomes strongly enriched in axons. In mature GCs, we analyzed synapses along their ascending segments and parallel fibers. We observe no differences in presynaptic distribution between GCs born at different developmental time points and thus having varied depths of projections in the molecular layer. We found that the mean densities of synapses along the parallel fiber and the ascending segment above the Purkinje cell (PC) layer are statistically indistinguishable, and higher than previous estimates. Interestingly, presynaptic terminals were also found in the ascending segments of GCs below and within the PC layer, with the mean densities two-fold lower than that above the PC layer. The difference in the density of synapses in these parts of the ascending segment likely reflects the regional differences in postsynaptic target cells of GCs.

Conclusions

The ability to visualize synapses of single neurons in vivo is valuable for studying synaptogenesis and synaptic plasticity within individual neurons as well as information flow in neural circuits.     

Role of neuregulin-1/ErbB2 signaling in endothelium-cardiomyocyte cross-talk

Neuregulin-1 (NRG-1), a cardioactive growth factor released from endothelial cells, has been shown to be indispensable for the normal function of the adult heart by binding to ErbB4 receptors on cardiomyocytes. In the present study, we have investigated to what extent ErbB2, the favored co-factor of ErbB4 for heterodimerization, participates in the cardiac effects of endothelium-derived NRG-1. In addition, in view of our previously described anti-adrenergic effects of NRG-1, we have studied which neurohormonal stimuli affect endothelial NRG-1 expression and release and how this may fit into a broader frame of cardiovascular physiology. Immunohistochemical staining of rat heart and aorta showed that NRG-1 expression was restricted to the endocardial endothelium and the cardiac microvascular endothelium (CMVE); by contrast, NRG-1 expression was absent in larger coronary arteries and veins and in aortic endothelium. In rat CMVE in culture, NRG-1 mRNA and protein expression was down-regulated by angiotensin II and phenylephrine and up-regulated by endothelin-1 and mechanical strain. CMVE-derived NRG-1 was shown to phosphorylate cardiomyocyte ErbB2, an event prevented by a 24-h preincubation of myocytes with monoclonal ErbB2 antibodies. Pretreating cardiomyocytes with these inhibitory anti-ErbB2 antibodies significantly attenuated CMVE-induced cardiomyocyte hypertrophy and abolished the protective actions of CMVE against cardiomyocyte apoptosis. Accordingly, ErbB2 signaling participated in the paracrine survival and growth controlling effects of NRG-1 on cardiomyocytes in vitro, explaining the cardiotoxicity of ErbB2 antibodies in patients. Cardiac NRG-1 synthesis occurs in endothelial cells adjacent to cardiac myocytes and is sensitive to factors related to the regulation of blood pressure.     

A natural ErbB4 isoform that does not activate phosphoinositide 3-kinase mediates proliferation but not survival or chemotaxis

ErbB4 is a member of the epidermal growth factor receptor (ErbB) family that mediates cellular responses activated by neuregulins (NRG) and other epidermal growth factor-like growth factors. Two naturally occurring ErbB4 isoforms, ErbB4 CYT-1 and ErbB4 CYT-2, have previously been identified. Unlike ErbB4 CYT-1, ErbB4 CYT-2 lacks a phosphoinositide 3-kinase (PI3-K)-binding site and is incapable of activating PI3-K. We have now examined the consequences of the inability of this isoform to activate PI3-K on cell proliferation, survival, and chemotaxis in response to NRG-1beta: (i) NRG-1beta stimulated proliferation of cells expressing either ErbB4 CYT-1 or ErbB4 CYT-2. Consistent with the mitogenic responsiveness, analysis of downstream signaling showed that Shc and MAPK were phosphorylated after stimulating either isoform with NRG-1beta. (ii) NRG-1beta protected cells expressing ErbB4 CYT-1 but not cells expressing ErbB4 CYT-2 from starvation-induced apoptosis as measured by effects on cell number and 4', 6-diamidino-2-phenylindole staining. Furthermore, in cells expressing ErbB4 CYT-2, Akt, a protein kinase that mediates cell survival, was not phosphorylated. (iii) NRG-1beta stimulated chemotaxis and membrane ruffling in cells expressing ErbB4 CYT-1 but not in cells expressing ErbB4 CYT-2. In summary, ErbB4 CYT-2 can mediate proliferation but not chemotaxis or survival. These results suggest a novel mechanism by which cellular responses such as chemotaxis and survival may be regulated by the expression of alternative receptor-tyrosine kinase isoforms that differ in their coupling to PI3-K signaling.     

Pro-BDNF–induced synaptic depression and retraction at developing neuromuscular synapses

Postsynaptic cells generate positive and negative signals that retrogradely modulate presynaptic function. At developing neuromuscular synapses, prolonged stimulation of muscle cells induces sustained synaptic depression. We provide evidence that pro–brain-derived neurotrophic factor (BDNF) is a negative retrograde signal that can be converted into a positive signal by metalloproteases at the synaptic junctions. Application of pro-BDNF induces a dramatic decrease in synaptic efficacy followed by a retraction of presynaptic terminals, and these effects are mediated by presynaptic pan-neurotrophin receptor p75 (p75^NTR), the pro-BDNF receptor. A brief stimulation of myocytes expressing cleavable or uncleavable pro-BDNF elicits synaptic potentiation or depression, respectively. Extracellular application of metalloprotease inhibitors, which inhibits the cleavage of endogenous pro-BDNF, facilitates the muscle stimulation–induced synaptic depression. Inhibition of presynaptic p75^NTR or postsynaptic BDNF expression also blocks the activity-dependent synaptic depression and retraction. These results support a model in which postsynaptic secretion of a single molecule, pro-BDNF, may stabilize or eliminate presynaptic terminals depending on its proteolytic conversion at the synapses.     

Cardiac endothelial cells regulate reactive oxygen species-induced cardiomyocyte apoptosis through neuregulin-1beta/erbB4 signaling

Neuregulin (NRG)-1beta has a prosurvival effect on cardiac myocytes via the phosphatidylinositol-3-kinase/Akt pathway, but the physiological regulators of this system in the intact heart are unknown. In this study, we tested the hypothesis that reactive oxygen species regulate NRG/erbB signaling. We used isolated adult rat ventricular myocytes (ARVMs) or cardiac microvascular endothelial cells (CMECs) in monoculture, or together in coculture. H2O2 induced NRG-1beta release from CMECs in a concentration-dependent manner, and conditioned medium from H2O2-treated CMEC activated ARVM erbB4. NRG-1beta release occurred via proteolytic cleavage of 115-kDa transmembrane NRG-1beta and was inhibited by the metalloproteinase inhibitor 1,10-phenanthroline. In myocyte monoculture, H2O2 induced erbB4-dependent, but NRG-independent, activation of Akt. To elucidate the bioactivity of CMEC-derived NRG-1beta on ARVMs, we examined H2O2-induced myocyte apoptosis in co-culture using an antibody to NRG-1beta. The percentages of terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positive cells were significantly higher in the anti-NRG-1beta group than in the control group. The change in apoptosis induced by anti-NRG-1beta in co-culture was similar in magnitude to the protection of myocytes by addition of recombinant NRG-1beta to ARVM monocultures. Activation of NRG/erbB paracrine signaling was also seen in the intact heart subjected to oxidative stress by ischemia-reperfusion injury. Isolated perfused mouse hearts subjected to 15 min of ischemia, followed by 30 min of reperfusion, showed complete proteolytic cleavage of 115-kDa NRG-1beta, with concomitant erbB4 phosphorylation. These results demonstrate that reactive oxygen species activate NRG-1beta/erbB4 paracrine signaling in the heart and suggest that this system is involved in cardiac adaptation to oxidative stress.     

Synaptic potential in the motor giant axon of the crayfish

Some electrical properties of the synapses between central giant axons (presynaptic) and the motor giant axon (postsynaptic) of the crayfish abdominal nerve cord have been investigated. Postsynaptic potential change in response to presynaptic volleys contains two components: a spike potential and a synaptic potential of very long time course. Amplitude of the synaptic potential is graded according to the number of active presynaptic axons. Conductance increase in the synaptic membrane endures over most of the period of potential change, and it is this rather than the "electrical time constant" of the membrane that in large measure determines the form of the synaptic potential. Temporal summation of synaptic potential occurs during repetitive presynaptic stimulation, and after such stimulation the rate of decay of synaptic potential is greatly slowed.     

Lack of support for surface diffusion of postsynaptic AMPARs in tuning synaptic transmission

Repetitive stimulation of excitatory synapses triggers molecular events required for signal transfer across neuronal synapses. It has been hypothesized that one of these molecular events, the diffusion of extrasynaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPARs) (i.e., the diffusion hypothesis), is necessary to help synapses recover from paired-pulse depression. To examine this presumed role of AMPAR diffusion during repetitive presynaptic stimulation, a biophysical model based on published physiological results was developed to track the localization and gating of each AMPAR. The model demonstrates that AMPAR gating in short intervals of fewer than 100 ms is controlled by their position in relation to the glutamate release site and by their recovery from desensitization, but it is negligibly influenced by their diffusion. Therefore, these simulations failed to demonstrate a role for AMPAR diffusion in helping synapses recover from paired-pulse depression.     

Intra- and extracellular signaling by endothelial neuregulin-1

Suppression of tumor growth by inhibition of ErbB receptor signaling is well documented. However, relatively little is known about the ErbB signaling system in the regulation of angiogenesis, a process necessary for tumor growth. We have previously shown that heparin-binding EGF-like growth factor (HB-EGF) is expressed by vascular endothelial cells (EC) and promotes endothelial recruitment of vascular smooth muscle cells (SMC). To assess whether other members of the EGF-family regulate angiogenesis, the expression of 10 EGF-like growth factors in primary ECs and SMCs was analyzed. In addition to HB-EGF, neuregulin-1 (NRG-1) was expressed in ECs in vitro and in vivo. Endothelial NRG-1 was constitutively processed to soluble extracellular and intracellular signaling fragments, and its expression was induced by hypoxia. NRG-1 was angiogenic in vivo in mouse corneal pocket and chicken chorioallantoic membrane (CAM) assays. However, consistent with the lack of NRG-1 receptors in several primary EC lines, NRG-1 did not directly stimulate cellular responses in cultured ECs. In contrast, NRG-1 promoted EC responses in vitro and angiogenesis in CAM in vivo by mechanisms dependent on VEGF-A and VEGFR-2. These results indicate that NRG-1 is expressed by ECs and regulates angiogenesis by mechanisms involving paracrine up-regulation of VEGF-A.     

Inhibition of endoplasmic reticulum stress by neuregulin-1 protects against myocardial ischemia/reperfusion injury

Neuregulin-1 (NRG-1), an endogenously produced polypeptide, is the ligand of cardiomyocyte ErbB receptors, with cardiovascular protective effects. In the present study, we explored whether the cardioprotective effect of NRG-1 against I/R injury is mediated by inhibiting myocardial endoplasmic reticulum (ER) stress. In vitro, NRG-1 directly inhibited the upregulation of ER stress markers such as glucose-regulated protein 78, CCAAT/enhancer binding protein homologous protein and cleaved caspase-12 induced by the ER stress inducers tunicamycin or dithiothreitol in both neonatal and adult ventricular myocytes. Attenuating ErbB signals by an ErbB inhibitor AG1478 or ErbB4 knockdown and preincubation with phosphoinositide 3-kinase inhibitors all reversed the effect of NRG-1 inhibiting ER stress in cultured neonatal rat cardiomyocytes. Concurrently, cardiomyocyte ER stress and apoptosis induced by hypoxia-reoxygenation were decreased by NRG-1 treatment in vitro. Furthermore, in an in vivo rat model of myocardium ischemia/reperfusion (I/R), intravenous NRG-1 administration significantly decreased ER stress and myocardial infarct size induced by I/R. NRG-1 could protect the heart against I/R injury by inhibiting myocardial ER stress, which might be mediated by the phosphoinositide 3-kinase/Akt signaling pathway.     

Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low‐affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin‐positive (PV) interneurons. Nrg3 and ErbB4 are located pre‐ and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo. Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short‐term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4⁺ interneurons and affects short‐term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non‐neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4⁺ interneurons.     

Proteolytic cleavage and phosphorylation of a tumor-associated ErbB4 isoform promote ligand-independent survival and cancer cell growth

The ErbB1 and ErbB2 receptors are oncogenes with therapeutic significance in human cancer, whereas the transforming potential of the related ErbB4 receptor has remained controversial. Here, we have addressed whether four alternatively spliced ErbB4 isoforms differ in regulating cellular responses relevant for tumor growth. We show that the two tumor necrosis factor-α converting enzyme (TACE)-cleavable ErbB4 isoforms (the juxtamembrane [JM]-a isoforms) were overexpressed in a subset of primary human breast cancers together with TACE. The overexpression of the JM-a cytoplasmic (CYT)-2 ErbB4 isoform promoted ErbB4 phosphorylation, survival of interleukin-3-dependent cells, and proliferation of breast cancer cells even in the absence of ligand stimulation, whereas activation of the other three ErbB4 isoforms required ligand stimulation. Ligand-independent cellular responses to ErbB4 JM-a CYT-2 overexpression were regulated by both tyrosine kinase activity and a two-step proteolytic generation of an intracellular receptor fragment involving first a TACE-like proteinase, followed by γ-secretase activity. These data suggest a novel transforming mechanism for the ErbB4 receptor in human breast cancer that is 1) specific for a single receptor isoform and 2) depends on proteinase cleavage and kinase activity but not ligand activation of the receptor.     

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.     

Long-term depression-inducing stimuli promote cleavage of the synaptic adhesion molecule NGL-3 through NMDA receptors,matrix metalloproteinases and presenilin/γ-secretase

Long-term depression (LTD) reduces the functional strength of excitatory synapses through mechanisms that include the removal of AMPA glutamate receptors from the postsynaptic membrane. LTD induction is also known to result in structural changes at excitatory synapses, including the shrinkage of dendritic spines. Synaptic adhesion molecules are thought to contribute to the development, function and plasticity of neuronal synapses largely through their trans-synaptic adhesions. However, little is known about how synaptic adhesion molecules are altered during LTD. We report here that NGL-3 (netrin-G ligand-3), a postsynaptic adhesion molecule that trans-synaptically interacts with the LAR family of receptor tyrosine phosphatases and intracellularly with the postsynaptic scaffolding protein PSD-95, undergoes a proteolytic cleavage process. NGL-3 cleavage is induced by NMDA treatment in cultured neurons and low-frequency stimulation in brain slices and requires the activities of NMDA glutamate receptors, matrix metalloproteinases (MMPs) and presenilin/γ-secretase. These results suggest that NGL-3 is a novel substrate of MMPs and γ-secretase and that NGL-3 cleavage may regulate synaptic adhesion during LTD.