NRG1 Protein Research Articles

Boosting neuregulin 1 type-III expression hastens SMA motor axon maturation

Intercellular communication between axons and Schwann cells is critical for attaining the complex morphological steps necessary for axon maturation. In the early onset motor neuron disease spinal muscular atrophy (SMA), many motor axons are not ensheathed by Schwann cells nor grow sufficiently in radial diameter to become myelinated. These developmentally arrested motor axons are dysfunctional and vulnerable to rapid degeneration, limiting efficacy of current SMA therapeutics. We hypothesized that accelerating SMA motor axon maturation would improve their function and reduce disease features. A principle regulator of peripheral axon development is neuregulin 1 type III (NRG1-III). Expressed on axon surfaces, it interacts with Schwann cell receptors to mediate axon ensheathment and myelination. We examined NRG1 mRNA and protein expression levels in human and mouse SMA tissues and observed reduced expression in SMA spinal cord and in ventral, but not dorsal root axons. To determine the impact of neuronal NRG1-III overexpression on SMA motor axon development, we bred NRG1-III overexpressing mice to SMA∆7 mice. Neonatally, elevated NRG1-III expression increased SMA ventral root size as well as axon segregation, diameter, and myelination resulting in improved motor axon conduction velocities. NRG1-III was not able to prevent distal axonal degeneration nor improve axon electrophysiology, motor behavior, or survival of older mice. Together these findings demonstrate that early SMA motor axon developmental impairments can be ameliorated by a molecular strategy independent of SMN replacement providing hope for future SMA combinatorial therapeutic approaches.

Apoptotic Factors, CaNma111 and CaYbh3, Function in Candida albicans Filamentation by Regulating the Hyphal Suppressors, Nrg1 and Tup1.

The morphological switch from the yeast to hyphal form is a key virulence attribute of the opportunistic fungal pathogen, Candida albicans. Our recent report showed that deletion of the newly identified apoptotic factor, CaNma111 or CaYbh3, leads to hyperfilamentation and increased virulence in a mouse infection model. CaNma111 and CaYbh3 are homologs of the pro-apoptotic protease, HtrA2/Omi, and BH3-only protein, respectively. In this study, we examined the effects of CaNMA111 and CaYBH3 deletion mutations on the expression levels of the hypha-specific transcription factors, Cph1 (a hyphal activator), Nrg1 (a hyphal repressor), and Tup1 (a hyphal repressor). The protein levels of Nrg1 were decreased in Caybh3/Caybh3 cells while those of Tup1 were decreased in both Canma111/Canma111 and Caybh3/Caybh3 cells. These effects on Nrg1 and Tup1 proteins were retained during serum-induced filamentation and appear to explain the hyperfilamentation phenotypes of the CaNMA111 and CaYBH3 deletion mutants. Treatment with the apoptosis-inducing dose of farnesol decreased the Nrg1 protein levels in the wild-type strain and more evidently in Canma111/Canma111 and Caybh3/Caybh3 mutant strains. Together, our results suggest that CaNma111 and CaYbh3 are key regulators of Nrg1 and Tup1 protein levels in C. albicans.

Prenatal di-(2-ethylhexyl) phthalate exposure induced myocardial cytotoxicity via the regulation of the NRG1-dependent ErbB2/ErbB4-PI3K/AKT signaling pathway in fetal mice

Environmental sanitation of maternal contact during pregnancy is extremely important for the development of different fetal tissues and organs. In particular, during early pregnancy, any adverse exposure may cause abnormal fetal growth or inhibit the development of embryogenic organs. The potential risks of phthalate exposure, which affects the development of humans and animals, are becoming a serious concern worldwide. However, the specific molecular mechanism of di-(2-ethylhexyl) phthalate (DEHP)-induced cardiotoxicity in fetal mice remains unclear. In this study, animal models of DEHP gavage at concentrations of 250, 500, and 1000 mg/kg/day within 8.5–18.5 days of pregnancy were established. The cell proliferation, survival, and apoptosis rates were evaluated using CCK8, EdU, TUNEL and flow cytometry. The molecular mechanism was assessed via transcriptome sequencing, immunohistochemistry, immunofluorescence, reverse transcription-quantitative polymerase chain reaction, and Western blot analysis. In vivo, DEHP increased apoptosis, decreased Ki67 and CD31 expression, reduced heart weight and area, slowed down myocardial sarcomere development, and caused cardiac septal defect in fetal mice heart. Transcriptome sequencing showed that DEHP decreased NRG1 expression and downregulated the ErbB2/ErbB4-PI3K/AKT signaling pathway-related target genes. In vitro, primary cardiomyocytes were cultured with DEHP at a concentration of 150 μg/mL combined with ErbB inhibitor (AG1478, 10 μmol/L) and/or NRG1 protein (100 ng/mL) for 72 h. After DEHP intervention, the expression of NRG1 and the phosphorylation level of ErbB2, ErbB4, PI3K, and AKT decreased, and the apoptosis-related protein levels increased. Moreover, the apoptosis rate increased. After adding exogenous NRG1, the phosphorylation level of the NRG1/ERbB2/ERbB4-PI3K/AKT pathway increased, and the apoptosis-related protein levels decreased. Further, the apoptosis rate reduced. Interestingly, after exposure to DEHP and AG1478 + NRG1, the anti-apoptotic effect of NRG1 and cardiomyocyte proliferation decreased by inhibiting the NRG1/ERbB2/ERbB4-PI3K/AKT pathway. Hence, the NRG1-dependent regulation of the ERbB2/ERbB4-PI3K/AKT signaling pathway may be a key mechanism of DEHP-induced myocardial cytotoxicity.

Induction of Nrg1 and ErbB4 Is Specific to Virus Induced Injury of the Myocardium and May be Detected during Pathogenesis of Viral Myocarditis as Blood‐Based Biomarkers for Diagnosis

BackgroundMyocarditis, inflammation of the myocardium, globally affects >3 million people annually. Heterogeneous histological findings and clinical presentation ranging from flu‐like illness to acute cardiogenic shock make diagnosis exceedingly difficult. The current gold standard for diagnosis requires histological examination of invasive endomyocardial biopsies, which provides a sensitivity of <30% in independently published studies. Although etiologies are diverse, viruses are the most prominent causes of myocarditis. Our studies demonstrated that the coxsackievirus B3 (CVB3), a common pathogen associated with viral myocarditis, manipulates the expression of Neuregulin 1 (Nrg1) and its ligand, receptor tyrosine‐protein kinase, ErbB4. Moreover, previous studies in other viruses have demonstrated preferential expression of the higher affinity Nrg1β versus the less potent Nrg1α isoform. The goal of this research is to determine the specificity of Nrg1 and ErbB4 induction to viral myocarditis as compared to other forms of acute myocardial injury, particularly myocardial infarction (m.i.). Additionally, we aim to analyze expression, tissue and isoform specificity of Nrg1 and ErbB4 in blood and heart tissue during the pathogenesis of viral myocarditis towards developing a non‐invasive blood‐based diagnostic assay via novel biomarkers.Methods4‐week old male A/J mice were sham or CVB3 infected. Blood and tissue were harvested at time points corresponding to the acute, sub‐acute and chronic phases of disease. Specimens were analyzed for mRNA and protein expression and sub‐cellular localization using RT‐qPCR, Western blotting and confocal microscopy, respectively. Expression of Nrg1 and ErbB4 in viral myocarditis models was compared to murine myocardial infarction models via confocal microscopy.ResultsUpregulation (p<0.05) of Nrg1 and ErbB4 protein fragments were observed in murine heart tissue at different phases of viral pathogenesis as compared to m.i. and non‐infected controls. The observed upregulation was specific to the heart and not observed in the infected pancreas or lung. In addition, ~35 kDa and ~26 kDa fragments of Nrg1 and ErbB4 respectively were detected in the plasma of infected mice, while absent in the non‐infected controls. Confocal microscopy revealed that Nrg1 localized to the nuclear periphery while a diffuse increase in cellular ErbB4 expression was observed during infection. mRNA levels of Nrg1β were significantly upregulated (~3‐fold) at the acute phase (6–7 dpi) of viral myocarditis, while the differences in expression of Nrg1α were not significant.ConclusionTissue specific upregulation and fragments detected in the plasma of both ErbB4 and Nrg1, with preferential expression of Nrg1β, were observed as a result of CVB3 infection. Moreover, upregulation was not observed in an ischemic model of acute myocardial injury, indicating a viral specific process. These composite observations support the concept that these biomarkers may ultimately yield a non‐invasive blood‐based diagnostic assay for viral myocarditis.Support or Funding InformationThis research is supported by the Myocarditis Foundation and the Michael Smith Foundation for Health Research.

Effect of Nrg1 Repressor on NTH1 Transcription and Molecular Docking of Nrg1 on NTH1 Promoter

The amount of intracellular trehalose increases in response to environmental stress in yeast (Saccharomyces cerevisiae). When that stress is terminated, the accumulated trehalose rapidly degrades into glucose rapidly. Synthesis of trehalose is fulfilled by the Trehalose Phosphate Synthase (TPS) enzyme complex, whereas the degradation of trehalose is done by the neutral trehalase enzyme. Under different stress conditions, transcription of the NTH1 gene is activated and Stress Response Elements (STRE) are required for this activation. Nrg1 protein can bind promoters including STRE and PDS elements. Because of the presence of three possible Nrg1 repressor binding sites on the NTH1 promoter, the NTH1 gene may be regulated by the Nrg1 repressor. In order to test this hypothesis, Δnrg1 mutant yeast and its isogenic wild-type yeast strain were used to analyze the transcriptional activation of the NTH1 gene under nitrogen starving conditions. Nth1 transcription of the mutant yeast was seven-fold higher than that of the wild-type under growth conditions, and was not changed during nitrogen starvation. The protein-DNA docking analysis also supported the possibility of Nrg1 binding to the NTH1 promoter. These results revealed that NTH1 gene expression is constitutive in the absence of the Nrg1 repressor protein, hence the transcription of NTH1 is repressed by the Nrg1 protein.

Investigating Nrg1 Signaling in the Regenerating Axolotl Spinal Cord Using Multiplexed FISH.

Amputation of a salamander tail leads to functional spinal cord regeneration through activation of endogenous stem cells. Identifying the signaling pathways that control cell proliferation in these neural stem cells will help elucidate the mechanisms underlying the salamander's regenerative ability. Here, we show that neuregulin 1 (Nrg1)/ErbB2 signaling is an important pathway in the regulation of neural stem cell proliferation in the spinal cord of the axolotl salamander (Ambystoma mexicanum). Simultaneous localization of nrg1 mRNA and Nrg1 protein was performed by utilizing a hybridization chain reaction fluorescence in situ hybridization (FISH) methodology in tissue sections. Multiplexed FISH also permitted the phenotyping of multiple cell types on a single fixed section allowing the characterization of mRNA expression, protein expression, and tissue architecture. Pharmacological inhibition of ErbB2 showed that intact Nrg1/ErbB2 signaling is critical for adult homeostatic regeneration as well as for injury-induced spinal cord regeneration. Overall, our results highlight the importance of the NRG1/ErbB2 signaling pathway in neural stem cell proliferation in the axolotl.

Pathological analysis of ErbB family and NRG-1 protein in progressive supranuclear palsy

Pathological analysis of ErbB family and NRG-1 protein in progressive supranuclear palsy

Amyloid precursor protein and amyloid precursor-like protein 2 have distinct roles in modulating myelination, demyelination, and remyelination of axons.

The identification of factors that regulate myelination provides important insight into the molecular mechanisms that coordinate nervous system development and myelin regeneration after injury. In this study, we investigated the role of amyloid precursor protein (APP) and its paralogue amyloid precursor-like protein 2 (APLP2) in myelination using APP and APLP2 knockout (KO) mice. Given that BACE1 regulates myelination and myelin sheath thickness in both the peripheral and central nervous systems, we sought to determine if APP and APLP2, as alternate BACE1 substrates, also modulate myelination, and therefore provide a better understanding of the events regulating axonal myelination. In the peripheral nervous system, we identified that adult, but not juvenile KO mice, have lower densities of myelinated axons in their sciatic nerves while in the central nervous system, axons within both the optic nerves and corpus callosum of both KO mice were significantly hypomyelinated compared to wild-type (WT) controls. Biochemical analysis demonstrated significant increases in BACE1 and myelin oligodendrocyte glycoprotein and decreased NRG1 and proteolipid protein levels in both KO brain tissue. The acute cuprizone model of demyelination/remyelination revealed that whereas axons in the corpus callosum of WT and APLP2-KO mice underwent similar degrees of demyelination and subsequent remyelination, the myelinated callosal axons in APP-KO mice were less susceptible to cuprizone-induced demyelination and showed a failure in remyelination after cuprizone withdrawal. These data identified APP and APLP2 as modulators of normal myelination and demyelination/remyelination conditions. Deletion of APP and APLP2 identifies novel interplays between the BACE1 substrates in the regulation of myelination.

Hyphal induction under the condition without inoculation in Candida albicans is triggered by Brg1-mediated removal of NRG1 inhibition.

Candida albicans can switch between yeast and hyphae growth forms, which is critical for its pathogenesis. Diluting from saturated cells into fresh medium at 37°C is routinely used to induce hyphae, which depends on the cAMP-PKA pathway-activated transcriptional down-regulation of NRG1 and degradation of Nrg1 protein triggered by inoculation. It is reported that N-acetylglucosamine (GlcNAc), serum or neutral pH could stimulate filamentation in log phase cells, whereas how C. albicans develops hyphae without inoculation remains unknown. Here, we show that NRG1 down-regulation is necessary for hyphal growth under this condition. Instead of cAMP-PKA pathway, GlcNAc sensor Ngs1 is responsible for the down-regulation of NRG1 upon GlcNAc induction in log phase cells through its N-acetyltransferase activity. From a genetic screen, Brg1 is found to be essential for hyphal development without inoculation. Ngs1 binds to BRG1 promoter to induce its expression in GlcNAc. Importantly, constitutively expressed BRG1 induces NRG1 down-regulation even in the absence of GlcNAc or Ngs1. Serum or neutral pH-induced filamentation in log phase cells is also through Brg1-mediated NRG1 down-regulation. Our study provides a molecular mechanism for how C. albicans forms hyphae in different cell states. This flexibility may facilitate C. albicans to adapt varied host environment during infection.

Increased expression of neuregulin 1 in the urothelium of rat bladder with partial bladder outlet obstruction

BackgroundThis study determined whether changes in the expression of neuregulin (NRG) 1, erbB2 tyrosine kinase (ErbB2) and the M2 muscarinic receptor in the urothelium and detrusor muscle of the rat bladder were associated with partial bladder outlet obstruction (PBOO).MethodsMale Sprague-Dawley rats (body weight 250–300 g) were used and subdivided into control (n = 10) and PBOO groups (n = 20). PBOO was induced for 21 days, and the expression of NRG1, ErbB2 and M2 muscarinic receptor mRNA and protein was evaluated using reverse transcriptase-polymerase chain reaction (RT-PCR) and western blotting, respectively.ResultsIn the urothelium of rat bladder samples, protein expression and mRNA expression of NRG1, ErbB2 and M2 muscarinic receptor were significantly increased in the PBOO group compared to the control group (p < 0.05). Only mRNA expression levels of NRG1/ ErbB2 were higher in the detrusor muscle of the PBOO group compared to the control group (p < 0.05).ConclusionsOur study demonstrated remarkable changes in the expression of NRG1/ErbB2 receptor mRNA and protein in the urothelium and muscle layer. These results suggest that NRG1 overexpression plays some kind of role against the PBOO-induced upregulated muscarinic receptors in detrusor overactivity.

Overexpression of Neuregulin 1 Type III Confers Hippocampal mRNA Alterations and Schizophrenia-Like Behaviors in Mice.

Neuregulin 1 (NRG1) is a schizophrenia candidate gene whose protein product is involved in neuronal migration, survival, and synaptic plasticity via production of specific isoforms. Importantly, NRG1 type III (NRG1 III) mRNA is increased in humans inheriting a schizophrenia risk haplotype for the NRG1 gene (HapICE), and NRG1 protein levels can be elevated in schizophrenia. The nature by which NRG1 type III overexpression results in schizophrenia-like behavior and brain pathology remains unclear, therefore we constructed a transgenic mouse with Nrg1 III overexpression in forebrain neurons (CamKII kinase+). Here, we demonstrate construct validity for this mouse model, as juvenile and adult Nrg1 III transgenic mice exhibit an overexpression of Nrg1 III mRNA and Nrg1 protein in multiple brain regions. Furthermore, Nrg1 III transgenic mice have face validity as they exhibit schizophrenia-relevant behavioral phenotypes including deficits in social preference, impaired fear-associated memory, and reduced prepulse inhibition. Additionally, microarray assay of hippocampal mRNA uncovered transcriptional alterations downstream of Nrg1 III overexpression, including changes in serotonin receptor 2C and angiotensin-converting enzyme. Transgenic mice did not exhibit other schizophrenia-relevant behaviors including hyperactivity, social withdrawal, or an increased vulnerability to the effects of MK-801 malate. Our results indicate that this novel Nrg1 III mouse is valid for modeling potential pathological mechanisms of some schizophrenia-like behaviors, for determining what other neurobiological changes may be downstream of elevated NRG1 III levels and for preclinically testing therapeutic strategies that may be specifically efficacious in patients with the NRG1 (HapICE) risk genotype.

Neuregulin-1 protects mouse cerebellum against oxidative stress and neuroinflammation

Cerebellum undergoes degenerative changes in neurodegenerative diseases. Two main factors including oxidative stress and neuroinflammation mediate neurodegeneration. Neuregulin-1 (Nrg1) has been implicated in many neurodegenerative diseases, while the underlying mechanisms are unknown. We hypothesized that Nrg1 prevents oxidative stress and neuroinflammation in neurodegeneration. We found a positive correlation between Nrg1 protein levels and ErbB4 and ErbB2 receptor phosphorylation in microarrays of normal human cerebellar tissue. In addition, Nrg1 was also co-localized with pErbB4 and pErbB2. Primary mouse cerebellar granule neurons (CGNs) were treated with H2O2 or LPS combined with recombinant Nrg1β (rNrg1β). Western blot analysis and immunofluorescence revealed that H2O2 and LPS-induced neuronal toxicity down-regulated the activation of ErbB receptors and Akt1, and the ratio of Bcl2/Bax, which was reversed by rNrg1β. In vivo studies showed that LPS-induced neuroinflammation in mouse cerebellum down-regulated pErbB4, pErbB2, pAkt1/Akt1 and Bcl2/Bax levels, whereas rNrg1β treatment reversed the changes. Immunohistochemistry and Western blot analysis showed that rNrg1β alleviates neuroinflammation by reducing the number of microglial cells and astrocytes and the expression of IL1β. Our results indicate that Nrg1 protects against oxidative stress and neuroinflammation in mouse cerebellum, suggesting potential therapeutic application in neuroinflammation associated with neurodegenerative diseases.

Abstract P6-12-09: Pan-HER, an antibody mixture with antitumor activity against drug-resistant HER2-overexpressing breast cancers with high ERBB ligand expression

Abstract Background: Amplification/overexpression of ERBB receptors and/or ligands has been associated with resistance to anti-HER2 therapies. Pan-HER is a mixture of six antibodies targeting each of the ERBB receptors, EGFR, HER2 and HER3, with synergistic pairs of antibodies. Each pair of antibodies simultaneously blocks ligand binding and/or induces target degradation, thus preventing compensatory mechanisms to anti-ERBB therapies. We examined the antitumor activity of Pan-HER against drug-sensitive and -resistant HER2+ breast cancer cells and xenografts. Results: Pan-HER exhibited potent growth inhibitory activity against a panel of HER2+ breast cancer cells (BT474, MDA-453, MDA-361, SUM190, HCC1954, UACC893 and SKBR3). Growth inhibition was associated with internalization and degradation of EGFR, HER2 and HER3. Pan-HER was superior to the combination of trastuzumab/pertuzumab (TP) against HER2+/PIK3CA mutant MDA-361, HCC1954, UACC893 and MDA-453 cells. We next compared the effect of Pan-HER against BT474, HCC1954 and MDA-361 xenografts established in nude mice to that of trastuzumab/lapatinib (TL), TP and T-DM1. All treatments were effective across the panel of xenografts. In mice with MDA-361 tumors, Pan-HER and TP were superior to TL. Immunoblot analysis showed significant downregulation of EGFR, HER2 and HER3 only in tumors treated with Pan-HER. After a complete response, treatment was discontinued. Among mice with BT474 xenografts treated with TP, TL and T-DM1, 25-50% of mice exhibited a tumor recurrence within 50 weeks of follow-up, while no recurrences were registered in mice treated with Pan-HER. Tumors recurring after TP and T-DM1 expressed significantly higher HER3 and P-HER3 protein levels and NRG1 mRNA levels. HCC1954 xenografts recurring after T-DM1 also overexpressed NRG1 mRNA compared to tumors before therapy. We next examined the effect of Pan-HER against trastuzumab-resistant HR6 (BT474) cells (Ritter et al. CCR 2007) and HCC1954 and UACC893 cells with acquired resistance to T-DM1 (TDR; IC50 &amp;gt;5-, &amp;gt;6- and 600-fold in HR6, UACC893-TDR and HCC1954-TDR cells, respectively, vs. parental cells). All T-DM1-resistant cells expressed significantly higher HER3 and P-HER3 protein levels and NRG1 mRNA and protein levels. Treatment with the HER3 neutralizing antibody LJM716 resensitized HR6 and HCC1954-TDR cells to T-DM1, suggesting a causal association between the NRG1-HER3 axis and drug resistance. Mice with HR6 tumors were treated with Pan-HER, TL, TP and T-DM1. Only Pan-HER arrested HR6 tumor growth and downregulated EGFR, HER2, HER3, P-HER3 and P-AKT. Finally, HCC1954-TDR tumors rapidly grew in vivo despite treatment with T-DM1. Administration of Pan-HER to mice bearing HCC1954-TDR xenografts growing in the presence of T-DM1, induced rapid tumor regressions. Conclusions: These data suggest that multitarget therapeutic interventions, such as Pan-HER, which simultaneously remove and/or block all ERBB receptors and ligands, are a feasible and effective approach against HER2-overexpressing cancers both sensitive and resistant to anti-HER2 therapies. Citation Format: Schwarz LJ, Hutchinson KE, Estrada MV, Sanders ME, Dugger TC, Formisano L, Guerrero AL, Red-Brewer M, Young CD, Lantto J, Pedersen MW, Kragh M, Horak ID, Arteaga CL. Pan-HER, an antibody mixture with antitumor activity against drug-resistant HER2-overexpressing breast cancers with high ERBB ligand expression [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-09.

Abstract 4669: Druggable oncogene fusions in lung invasive mucinous adenocarcinoma

Abstract Invasive mucinous adenocarcinoma (IMA) of lung, a subtype of lung adenocarcinoma, is known to be strongly associated with KRAS mutation. Genetic aberration is unknown in IMA without KRAS mutation. From an IMA cohort of 90 cases, consisting of 56 cases (62%) with KRAS mutations, we conducted whole-transcriptome sequencing on 32 IMAs, including 27 cases without KRAS mutation. We identified oncogenic fusions that occurred mutually exclusively with KRAS mutations: CD74-NRG1, SLC3A2-NRG1 (NRG1 fusions: 6/90), EZR-ERBB4, TRIM24-BRAF, and KIAA1468-RET. The CD74-NRG1 and SLC3A2-NRG1 fusion proteins contained the transmembrane domain of CD74 and SLC3A2 and retained the EGF-like domain of the NRG1 protein (NRG1 III-β3 form). Membrane-tethered EGF-like domain might activate juxtacrine signaling through HER2:HER3 receptors. To reveal the mechanism of the oncogenicity, exposing EFM-19 cells which express HER2 and HER3 to conditioned media from H1299 cells expressing exogenous CD74-NRG1 fusion protein resulted in phosphorylation of HER2 and HER3, and downstream mitogen-activated kinase and AKT. The phosphorylation was suppressed by lapatinib and afatinib. When EZR-ERBB4 or TRIM24-BRAF cDNA was exogenously expressed in NIH3T3 cells, ERBB4 or downstream mediator of BRAF was phosphorylated under serum-free condition. The expression of fusion genes induced anchorage-independent growth. The phosphorylation and growth were suppressed by the kinase inhibitors targeting fusion-induced signaling. These results show that the NRG1, ERBB4 and BRAF fusions are novel driver mutations and potential targets. About 15% (13/90; 14.4%) of IMAs harbor druggable aberrations, taken together a small fraction of known driver aberrations. Citation Format: Takashi Nakaoku, Koji Tsuta, Hitoshi Ichikawa, Kouya Shiraishi, Hiromi Sakamoto, Masato Enari, Koh Furuta, Yoko Shimada, Hideaki Ogiwara, Shun-ichi Watanabe, Hiroshi Nokihara, Kazuki Yasuda, Masaki Hiramoto, Takao Nammo, Young Hak Kim, Michiaki Mishima, Jun Yokota, Teruhiko Yoshida, Takashi Kohno. Druggable oncogene fusions in lung invasive mucinous adenocarcinoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4669. doi:10.1158/1538-7445.AM2015-4669

Neuregulin 1 expression and electrophysiological abnormalities in the Neuregulin 1 transmembrane domain heterozygous mutant mouse.

BackgroundThe Neuregulin 1 transmembrane domain heterozygous mutant (Nrg1 TM HET) mouse is used to investigate the role of Nrg1 in brain function and schizophrenia-like behavioural phenotypes. However, the molecular alterations in brain Nrg1 expression that underpin the behavioural observations have been assumed, but not directly determined. Here we comprehensively characterise mRNA Nrg1 transcripts throughout development of the Nrg1 TM HET mouse. In addition, we investigate the regulation of high-frequency (gamma) electrophysiological oscillations in this mutant mouse to associate molecular changes in Nrg1 with a schizophrenia-relevant neurophysiological profile.MethodsUsing exonic probes spanning the cysteine-rich, epidermal growth factor (EGF)-like, transmembrane and intracellular domain encoding regions of Nrg1, mRNA levels were measured using qPCR in hippocampus and frontal cortex from male and female Nrg1 TM HET and wild type-like (WT) mice throughout development. We also performed electrophysiological recordings in adult mice and analysed gamma oscillatory at baseline, in responses to auditory stimuli and to ketamine.ResultsIn both hippocampus and cortex, Nrg1 TM HET mice show significantly reduced expression of the exon encoding the transmembrane domain of Nrg1 compared with WT, but unaltered mRNA expression encoding the extracellular bioactive EGF-like and the cysteine-rich (type III) domains, and development-specific and region-specific reductions in the mRNA encoding the intracellular domain. Hippocampal Nrg1 protein expression was not altered, but NMDA receptor NR2B subunit phosphorylation was lower in Nrg1 TM HET mice. We identified elevated ongoing and reduced sensory-evoked gamma power in Nrg1 TM HET mice.InterpretationWe found no evidence to support the claim that the Nrg1 TM HET mouse represents a simple haploinsufficient model. Further research is required to explore the possibility that mutation results in a gain of Nrg1 function.

Effects of schizophrenia risk variation in the NRG1 gene on NRG1-IV splicing during fetal and early postnatal human neocortical development.

Neuregulin 1 (NRG1) is a multifunctional neurotrophin that mediates neurodevelopment and schizophrenia risk. The NRG1 gene undergoes extensive alternative splicing, and association of brain NRG1 type IV isoform expression with the schizophrenia-risk polymorphism rs6994992 is a potential mechanism of risk. Novel splice variants of NRG1-IV (NRG1-IVNV), with predicted unique signaling capabilities, have been cloned in fetal brain tissue. The authors investigated the temporal dynamics of transcription of NRG1-IVNV, compared with the major NRG1 isoforms, across human prenatal and postnatal prefrontal cortical development, and they examined the association of rs6994992 with NRG1-IVNV expression. NRG1 type I-IV and NRG1-IVNV isoforms were evaluated with quantitative real-time polymerase chain reaction in human postmortem prefrontal cortex tissue samples at 14 to 39 weeks gestation and postnatal ages 0-83 years. The association of rs6994992 genotype with NRG1-IVNV expression and the subcellular distribution and proteolytic processing of NRG1-IVNV isoforms were also determined. Expression of NRG1 types I, II, and III was temporally regulated during prenatal and postnatal neocortical development. NRG1-IVNV was expressed from 16 weeks gestation until age 3. Homozygosity for the schizophrenia risk allele (T) of rs6994992 conferred lower cortical NRG1-IVNV levels. Assays showed that NRG1-IVNV is a novel nuclear-enriched, truncated NRG1 protein resistant to proteolytic processing. To the authors' knowledge, this study provides the first quantitative map of NRG1 isoform expression during human neocortical development and aging. It identifies a potential mechanism of early developmental risk for schizophrenia at the NRG1 locus, involving a novel class of NRG1 proteins.

Fabrication of Aligned Carbon Nanotube/Polycaprolactone/Gelatin Nanofibrous Matrices for Schwann Cell Immobilization

In this study, we utilized a mandrel rotating collector consisting of two parallel, electrically conductive pieces of tape to fabricate aligned electrospun polycaprolactone/gelatin (PG) and carbon nanotube/polycaprolactone/gelatin (PGC) nanofibrous matrices. Furthermore, we examined the biological performance of the PGC nanofibrous and film matrices using an in vitro culture of RT4‐D6P2T rat Schwann cells. Using cell adhesion tests, we found that carbon nanotube inhibited Schwann cell attachment on PGC nanofibrous and film matrices. However, the proliferation rates of Schwann cells were higher when they were immobilized on PGC nanofibrous matrices compared to PGC film matrices. Using western blot analysis, we found that NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PG nanofibrous matrices. However, the carbon nanotube inhibited NRG1 and P0 protein expression in cells immobilized on PGC film matrices. Moreover, the NRG1 and P0 protein expression levels were higher for cells immobilized on PGC nanofibrous matrices compared to PGC film matrices. We found that the matrix topography and composition influenced Schwann cell behavior.

Electrospun Hyaluronan‐Gelatin Nanofibrous Matrix for Nerve Tissue Engineering

Schwann cells play a critical role in the repair of the peripheral nerve. The goal of this study was to fabricate electrospun gelatin (Gel) and hyaluronan‐gelatin (HA‐Gel) composite nanofibers to provide a suitable growth environment for Schwann cells. The fiber diameters of Gel, 0.5 HA‐Gel, 1 HA‐Gel, and 1.5 HA‐Gel were 130 ± 30 nm, 294 ± 87 nm, 362 ± 129 nm, and 224 ± 54 nm, respectively. The biological performance of Gel and HA‐Gel was evaluated using an in vitro culture of RT4‐D6P2T rat Schwann cells. We found that the cell attachment and proliferation rates were not significantly different on these matrices. However, the Schwann cells displayed better organized F‐actin on HA‐Gel than on Gel. Moreover, the expression levels of several genes, including Nrg1, GFAP, and P0, were significantly higher on HA‐Gel than on Gel. In addition, the levels of Nrg1 and P0 protein expression were also higher on the HA‐Gel than on Gel. These results indicate that the hyaluronan‐gelatin composite nanofibrous matrix could potentially be used in peripheral nerve repair.

Comprehensive analysis of NRG1 common and rare variants in Hirschsprung patients.

Hirschsprung disease (HSCR, OMIM 142623) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract, which results in tonic contraction of the aganglionic gut segment and functional intestinal obstruction. The RET proto-oncogene is the major gene for HSCR with differential contributions of its rare and common, coding and noncoding mutations to the multifactorial nature of this pathology. Many other genes have been described to be associated with the pathology, as NRG1 gene (8p12), encoding neuregulin 1, which is implicated in the development of the enteric nervous system (ENS), and seems to contribute by both common and rare variants. Here we present the results of a comprehensive analysis of the NRG1 gene in the context of the disease in a series of 207 Spanish HSCR patients, by both mutational screening of its coding sequence and evaluation of 3 common tag SNPs as low penetrance susceptibility factors, finding some potentially damaging variants which we have functionally characterized. All of them were found to be associated with a significant reduction of the normal NRG1 protein levels. The fact that those mutations analyzed alter NRG1 protein would suggest that they would be related with HSCR disease not only in Chinese but also in a Caucasian population, which reinforces the implication of NRG1 gene in this pathology.

Expressions of Neuregulin 1βand ErbB4 in Prefrontal Cortex and Hippocampus of a Rat Schizophrenia Model Induced by Chronic MK-801 Administration

Recent human genetic studies and postmortem brain examinations of schizophrenia patients strongly indicate that dysregulation of NRG1 and ErbB4 may be important pathogenic factors of schizophrenia. However, this hypothesis has not been validated and fully investigated in animal models of schizophrenia. In this study we quantitatively examined NRG1 and ErbB4 protein expressions by immunohistochemistry and Western blot in the brain of a rat schizophrenia model induced by chronic administration of MK-801 (a noncompetitive NMDA receptor antagonist). Our data showed that NRG1β and ErbB4 expressions were significantly increased in the rat prefrontal cortex and hippocampus but in different subregions. These findings suggest that altered expressions of NRG1 and ErbB4 might be attributed to the schizophrenia. Further study in the role and mechanism of NRG1 and ErbB4 may lead to better understanding of the pathophysiology for this disorder.