NRG1 Expression Research Articles

Abstract 1553: Biomarker evaluation in a randomized phase 2 study of MEHD7945A (MEHD) versus cetuximab (Cet) in ≥2 line recurrent/metastatic (R/M) squamous cell carcinomas of the head and neck (SCCHN) [MEHGAN

Abstract Background MEHD is a novel dual-action humanized IgG1 antibody that blocks ligand binding to EGFR and HER3, inhibiting all major ligand-dependent HER complex signaling. Preclinical and Phase 1a clinical data suggested ligand-driven HER3 signaling as a promising target for therapy in a subset of patients with SCCHN. Results from the MEHGAN study showed comparable objective response rates and PFS for MEHD and Cet (Fayette et al, ESMO 2014). Here we report the results of comprehensive and comparative biomarker analyses from that study. Methods Archival and fresh (as available) tumor tissues were evaluated to characterize the biology of anti-HER therapy in SCCHN, and to identify potential predictive biomarkers for improved outcomes with MEHD compared to Cet, with particular attention to the HER3 ligand NRG1. NRG1 and ERBB3 RNA expression was measured by both ISH (data analysis are ongoing) and qRT-PCR. Additionally, extensive gene expression analyses and HPV detection were performed by qRT-PCR. Results Of 121 randomized patients in MEHGAN, 107 had archival tissues with sufficient tumor content and quality for biomarker analyses. Key findings include: 1) Most patients with CT RECIST responses on either treatment arm had higher (≥ median) tumor expression levels of NRG1 as measured by qRT-PCR 2) EGFR ligands such as amphiregulin were co-expressed with NRG1, consistent with preclinical analysis in an independent panel of SCCHN tumor samples (Genentech data on file). 3) 24 HPV (+) patients (20%) were identified, consistent with published prevalence reports. 4) Higher EGFR and HER3 ligand expression was observed in HPV (-) samples relative to HPV (+) samples and, moreover, no responses were seen in HPV (+) patients. These results are consistent with prior correlative ligand observations and may point to differential roles for HER signaling biology in HPV (-) versus HPV (+) SCCHN. Conclusions NRG1 expression did not predict enhanced responsiveness to MEHD versus Cet or, conversely, resistance to Cet. NRG1 and EGFR ligands appear to have similar expression patterns in SCCHN. Higher levels of NRG1 and EGFR ligands were associated with greater activity for both MEHD and Cet, and were consistently observed in HPV (-) SCCHN versus HPV (+) SCCHN. These data suggest distinct HER signaling biology in these 2 patient groups and warrant further evaluation to potentially inform treatment approaches in SCCHN. * We would like acknowledge and thank all of the MEHGAN study investigators and patients. Citation Format: Elicia Penuel, Amy V. Kapp, An Do, Rachel Tam, Teiko Sumiyoshi, Chaitra Marathe, Susan Sa, Franklin Peale, Mark Lackner, Scott Holden, Tanguy Seiwert, Andrea Pirzkall. Biomarker evaluation in a randomized phase 2 study of MEHD7945A (MEHD) versus cetuximab (Cet) in ≥2 line recurrent/metastatic (R/M) squamous cell carcinomas of the head and neck (SCCHN) [MEHGAN]. [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 1553. doi:10.1158/1538-7445.AM2015-1553

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.

Rapid transient isoform-specific neuregulin1 transcription in motor neurons is regulated by neurotrophic factors and axon-target interactions.

The neuregulins (NRGs) are a family of alternatively spliced factors that play important roles in nervous system development and disease. In motor neurons, NRG1 expression is regulated by activity and neurotrophic factors, however, little is known about what controls isoform-specific transcription. Here we show that NRG1 expression in the chick embryo increases in motor neurons that have extended their axons and that limb bud ablation before motor axon outgrowth prevents this induction, suggesting a trophic role from the developing limb. Consistently, NRG1 induction after limb bud ablation can be rescued by adding back the neurotrophic factors BDNF and GDNF. Mechanistically, BDNF induces a rapid and transient increase in type I and type III NRG1 mRNAs that peak at 4h in rat embryonic ventral spinal cord cultures. Blocking MAPK or PI3K signaling or blocking transcription with Actinomycin D blocks BDNF induced NRG1 gene induction. BDNF had no effect on mRNA degradation, suggesting that transcriptional activation rather than message stability is important. Furthermore, BDNF activates a reporter construct that includes 700bp upstream of the type I NRG1 start site. Protein synthesis is also required for type I NRG1 mRNA transcription as cycloheximide produced a super-induction of type I, but not type III NRG1 mRNA, possibly through a mechanism involving sustained activation of MAPK and PI3K. These results reveal the existence of highly responsive, transient transcriptional regulatory mechanisms that differentially modulate NRG1 isoform expression as a function of extracellular and intracellular signaling cascades and mediated by neurotrophic factors and axon-target interactions.

Nrg1 is an injury-induced cardiomyocyte mitogen for the endogenous heart regeneration program in zebrafish.

Heart regeneration is limited in adult mammals but occurs naturally in adult zebrafish through the activation of cardiomyocyte division. Several components of the cardiac injury microenvironment have been identified, yet no factor on its own is known to stimulate overt myocardial hyperplasia in a mature, uninjured animal. In this study, we find evidence that Neuregulin1 (Nrg1), previously shown to have mitogenic effects on mammalian cardiomyocytes, is sharply induced in perivascular cells after injury to the adult zebrafish heart. Inhibition of Erbb2, an Nrg1 co-receptor, disrupts cardiomyocyte proliferation in response to injury, whereas myocardial Nrg1 overexpression enhances this proliferation. In uninjured zebrafish, the reactivation of Nrg1 expression induces cardiomyocyte dedifferentiation, overt muscle hyperplasia, epicardial activation, increased vascularization, and causes cardiomegaly through persistent addition of wall myocardium. Our findings identify Nrg1 as a potent, induced mitogen for the endogenous adult heart regeneration program.

23 Invited lecture: Tumor–stroma interactions promote operational flexibility in cancer progression

Malignant cancer cells do not act as lone wolves to achieve metastasis, as they exist within a complex ecosystem consisting of an extracellular matrix scaffold populated by carcinoma-associated fibroblasts (CAFs), endothelial cells and immune cells (De Wever and Mareel, J Pathol, 2003; De Wever et al., Int J Cancer, 2008; De Wever et al., Semin Cancer Biol., 2014). Paracrine interactions between CAFs, also termed myofibroblasts, and cancer cells stimulate invasion of colon cancer cells in matrix scaffolds. The pro-invasive activity was found also with the secretome and we identified the combined action of Scatter Factor/Hepatocyte Growth Factor (SF/HGF) and tenascin-C (TNC) to promote invasion (De Wever et al., FASEBJ, 2004). Secretome proteomics revealed the presence of multiple other pro-metastatic proteins suggesting that the molecular characteristics of a CAF remain in evolution (De Boeck et al., Proteomics, 2013). CAFs show operational flexibility; eg their ability to respond dynamically to a cancer cell’s fluctuating demands by shifting profitable signals necessary in metastasis. Local, tissue-resident fibroblasts and mesenchymal stem cells (MSCs) coming from reservoir sites such as bone marrow (BM) are the main progenitor cells of CAFs (De Boeck et al., Oral Oncol, 2010). A successful invader must survive in an ectopic environment and CAFs provide advantageous signals to cancer cells to accomplish survival through AKT activation. Neuregulin (NRG)-1 secreted by CAFs increase cancer cell number through HER2/HER3-dependent activation of the PI3K/AKT pathway. Translational data demonstrate transmembrane NRG-1 expression in CAF in primary colorectal cancer and in liver metastasis, with the strongest expression in the immediate vicinity of HER2/HER3 expressing cancer cells. Moreover, high transmembrane NRG-1 expression is a poor prognosis marker in colorectal cancer (De Boeck et al., GUT, 2013). The odds that an aggressive cancer cell will form a metastatic colony in some organ are vanishingly small. Nevertheless, metastasis is responsible for more than 90% of cancer-associated mortality; thus the clinical need to prevent or target metastasis is high. Viewing cancer within the context of ecosystems may provide opportunities to exploit CAFs to create tumor-environment bio-mimetics to deceive and redirect metastatic cancer cells.

Filament condition-specific response elements control the expression of NRG1 and UME6, key transcriptional regulators of morphology and virulence in Candida albicans.

Candida albicans is the most frequently isolated human fungal pathogen and can cause a range of mucosal and systemic infections in immunocompromised individuals. Morphogenesis, the ability to undergo a reversible transition from budding yeast to elongated filaments, is an essential virulence trait. The yeast-to-filament transition is associated with expression of genes specifically important for filamentation as well as other virulence-related processes, and is controlled, in part, by the key transcriptional regulators Nrg1 and Ume6. Both of these regulators are themselves controlled at the transcriptional level by filament-inducing environmental cues, although little is known about how this process occurs. In order to address this question and determine whether environmental signals regulate transcription of UME6 and NRG1 via distinct and/or common promoter elements, we performed promoter deletion analyses. Strains bearing promoter deletion constructs were induced to form filaments in YEPD plus 10% serum at 37°C, Spider medium (nitrogen and carbon starvation) and/or Lee’s medium pH 6.8 (neutral pH) and reporter gene expression was measured. In the NRG1 promoter we identified several distinct condition-specific response elements for YEPD plus 10% serum at 37°C and Spider medium. In the UME6 promoter we also identified response elements for YEPD plus 10% serum at 37°C. While a few of these elements are distinct, others overlap with those which respond to Lee’s pH 6.8 medium. Consistent with UME6 possessing a very long 5’ UTR, many response elements in the UME6 promoter are located significantly upstream from the coding sequence. Our data indicate that certain distinct condition-specific elements can control expression of C. albicans UME6 and NRG1 in response to key filament-inducing environmental cues. Because C. albicans encounters a variety of host microenvironments during infection, our results suggest that UME6 and NRG1 expression can be differentially modulated by multiple signaling pathways to control filamentation and virulence in vivo.

The Common Genetic Variant rs944289 on Chromosome 14q13.3 Associates with Risk of Both Malignant and Benign Thyroid Tumors in the Japanese Population

Several single nucleotide polymorphisms (SNP) have been identified to be associated with the risk for differentiated thyroid cancer in populations of distinct ethnic background. The relationship of these genetic markers to a benign tumor of the thyroid, follicular adenoma (FA), is not well established. In a multicenter retrospective case-control study, five thyroid cancer-related SNPs-rs966513 (9q22.33, FOXE1), rs944289 (14q13.3, PTCSC3), rs2439302 (8p12, NRG1), rs1867277 (9q22.23, FOXE1), and rs6983267 (8q24, POU5F1B)-were genotyped in 959 cases of histologically verified FA, 535 papillary thyroid carcinomas (PTC), and 2766 population controls. A significant association was found between FA and rs944289 (p=0.002; OR 1.176 [CI 1.064-1.316]), and suggestively with rs2439302 (p=0.033; OR 1.149 [CI 1.010-1.315]). In PTC, significant associations were confirmed for rs965513 (p=4.21E-04; OR 1.587 [CI 1.235-2.000]) and rs944289 (p=0.003; OR 1.234 [CI 1.075-1.408]), newly found for rs2439302 (p=0.003; OR 1.266 [CI 1.087-1.493]) and rs1867277 (p=1.17E-04; OR 1.492 [CI 1.235-1.818]), and was not replicated for rs6983267 (p=0.082; OR 1.136 [CI 0.980-1.316]) in this series. A significant correlation between rs2439302 genotype and relative expression of NRG1 was detected in normal and tumor counterparts of PTC (about 10% decrease per each risk allele). NRG1 expression also significantly correlated with that of PTCSC3. Association of rs944289, which was previously known to confer risk for thyroid cancer, with FA, and the correlation between PTCSC3 and NRG1 expression demonstrates that predisposing genetic factors are partly common for benign and malignant thyroid tumors, and imply broader roles of the pathways they underlie in thyroid tumorigenesis, not limited to carcinogenesis.

Abstract 2612: ljm716, an anti her3 antibody that inhibits her3 dimerization, displays anti-tumor activity in esophageal squamous cell cancer

Abstract Background: Esophageal cancer is the eighth most common cancer and the sixth most common cause of cancer related death in the world. Squamous cell carcinoma is the predominant histologic subtype of esophageal cancer, particularly in Asia. Currently, no targeted therapy has been approved for treating this type of disease. Platinum-based combination chemotherapy remains to be the standard care for first line treatment of advanced or metastatic esophageal cancer with limited efficacy. Activation of EGFR/HER3/PI3K pathway is frequently observed in esophageal cancer, however, their therapeutic value remains to be further tested. Methods: In this study, we evaluated the anti-tumor activity of LJM716, a fully human anti-HER3 antibody, either alone or in combination with anti-EGFR antibody cetuximab or PI3K alpha subunit inhibitor BYL719 in esophageal squamous cancer. LJM716, cetuximab and BYL719 single agent as well as their pair-wised combination activity were assessed in 24 esophageal squamous cancer cell (ESCC) lines using the CellTiter-Glo® Luminescent Cell Viability Assay. Single agent and combination anti-tumor activities of these reagents were also tested in selected cell line xenograft and patient derived xenograft mouse models. Results: NRG1 expression (60%), PIK3CA mutation (8%), moderate PIK3CA (30%) and EGFR (30%) amplification were detected in a panel of 24 ESCC lines. Anti-HER3 antibody LJM716 potently inhibited HER3 phosphorylation and decreased pAKT level in these cell lines. In vitro, LJM716 inhibited proliferation of a sub-set of ESCC lines. LJM716 further enhanced and broadened the anti-tumor activities of both BYL719 and cetuximab in ESCC lines. In mouse xenograft models, LJM716 single agent treatment induced tumor stasis while combination with either BYL719 or cetuximab induced model dependent tumor regression. Conclusion: NRG1-HER3-PI3K pathway activation is commonly observed in esophageal squamous cell cancer. Activation of HER3 is a potential mechanism leading to resistance to EGFR or PI3K targeted therapies, and combining anti-HER3 therapies with therapies targeting PI3K/EGFR may provide new therapeutic strategy for patients with esophageal squamous cell cancer. Citation Format: Qing Sheng, Hui-Qin Wang, rita das, yan chen, jinsheng liang, Fiona Xu, zongyao Wang, Z. Alexander Cao, youzhen Wang, alan huang. ljm716, an anti her3 antibody that inhibits her3 dimerization, displays anti-tumor activity in esophageal squamous cell cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2014-2612

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.

Genetic Mouse Models of Neuregulin 1: Gene Dosage Effects, Isoform-Specific Functions, and Relevance to Schizophrenia

The neuregulins (NRG1–NRG4) are a family of structurally related intercellular signaling proteins, members of the epidermal growth factor class of neurotrophins and ligands for the ErbB family of receptor tyrosine kinases. NRG1 is perhaps the most studied at the biological level because of its genetic involvement in human cancers, cardiac development, and schizophrenia ( 1 Harrison P.J. Law A.J. Neuregulin 1 and schizophrenia: Genetics, gene expression, and neurobiology. Biol Psychiatry. 2006; 60: 132-140 Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar ). The NRG1 gene maps to 8p21-p12, a chromosomal locus showing evidence of genome-wide linkage to schizophrenia, and common polymorphic variation in NRG1 has been repeatedly associated with increased disease risk ( 1 Harrison P.J. Law A.J. Neuregulin 1 and schizophrenia: Genetics, gene expression, and neurobiology. Biol Psychiatry. 2006; 60: 132-140 Abstract Full Text Full Text PDF PubMed Scopus (376) Google Scholar , 2 Stefansson H. Sigurdsson E. Steinthorsdottir V. Bjornsdottir S. Sigmundsson T. Ghosh S. et al. Neuregulin 1 and susceptibility to schizophrenia. Am J Hum Genet. 2002; 71: 877-892 Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar ). Genetic mouse models of NRG1 have revolutionized understanding of the role of NRG1/ErbB signaling in nervous system development and function, revealing that NRG1 is a critical mediator of neuronal and glial cell differentiation, proliferation, and survival as well as a key regulator of myelination, neurotransmitter function, and synaptic plasticity. Despite progress in understanding of the basic neurobiological role of NRG1, ambiguity about the causative molecular basis of involvement of NRG1 in schizophrenia (i.e., gain or loss of function vs. isoform-specific dysregulation) has complicated the development of disease-relevant genetic mouse models. To date, there has been considerable emphasis on the role of NRG1 haploinsufficiency in rodents for the development of cognition and behaviors related to schizophrenia ( 2 Stefansson H. Sigurdsson E. Steinthorsdottir V. Bjornsdottir S. Sigmundsson T. Ghosh S. et al. Neuregulin 1 and susceptibility to schizophrenia. Am J Hum Genet. 2002; 71: 877-892 Abstract Full Text Full Text PDF PubMed Scopus (1414) Google Scholar , 3 O’Tuathaigh C.M. Harte M. O’Leary C. O’Sullivan G.J. Blau C. Lai D. et al. Schizophrenia-related endophenotypes in heterozygous neuregulin-1 ‘knockout’ mice. Eur J Neurosci. 2010; 31: 349-358 Crossref PubMed Scopus (66) Google Scholar ), even though evidence of NRG1 downregulation in schizophrenia is minimal. In comparison, less attention has been paid to the neurobiological effects of increased NRG1 expression and the role of isoform-specific dysregulation, for which evidence of a pathogenic role in schizophrenia is considerable. More recent evidence regarding potential molecular mechanisms of risk at the NRG1 locus in schizophrenia has created a gradual shift from focus on NRG1 haploinsuffiency to the development of more sophisticated, disease-relevant transgenic mouse models of NRG1 isoform dysregulation. Reversible Overexpression of Bace1-Cleaved Neuregulin-1 N-Terminal Fragment Induces Schizophrenia-Like Phenotypes in MiceBiological PsychiatryVol. 76Issue 2PreviewNeuregulin-1 (Nrg1) is a pleiotropic signaling molecule that regulates neural development, and mutation of Nrg1 is a risk factor for schizophrenia. Cleavage of type I β1 Nrg1 isoform by Bace1 releases a secreted N-terminal fragment (Nrg1-ntfβ), which can bind to a cognate ErbB receptor to activate the specific signaling cascade. This study aimed to determine whether increased expression of Nrg1 is beneficial for brain development and functions. Full-Text PDF

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.

Neuregulin 1-β regulates cell adhesion molecule L1 expression in the cortex and hippocampus of mice

Neuregulin 1 (Nrg1) functions in neuronal migration, survival and differentiation as well as synaptogenesis during ontogenetic development and maintenance of synaptic functions in the adult mammalian brain. The neural adhesion molecule L1 (L1CAM) functions in similar overlapping, but also non-overlapping roles in the nervous system. In the present study, we therefore investigated some aspects of the functional relationship between Nrg1 and L1 in mammalian neural cells. Nrg1 regulates the expression of L1 in cultures of both human neuroblastoma SK-N-SH cells and mouse cortical and hippocampal neurons. To analyze the role of Nrg1 on L1 expression in vivo, young adult male mice received intraperitoneal injections of Nrg1 or PBS (vehicle control). The correlation between Nrg1 and L1 expression was tested by qPCR, Western blot analysis, and immunocytology. Our data indicate that neuregulin 1-β (Nrg1β) increases L1 expression in neurons of the cerebral cortex, and decreases expression in neurons of the hippocampus in vitro and in vivo. In addition, Nrg1 induces phosphorylation of its receptors, ErbB2 and ErbB4, the predominant ErbB receptors in the nervous system. These results show that Nrg1β affects expression of L1 in the central nervous system and in parallel activates the ErbB receptors for Nrg1, suggesting a crosstalk between molecules that are of prime importance for nervous system functions.

Complexity of trophic factor signaling in experimental autoimmune encephalomyelitis: Differential expression of neurotrophic and gliotrophic factors

Soluble factors that promote survival and differentiation of glia and neurons during development are likely to play key roles in neurodegeneration and demyelinating diseases such as multiple sclerosis (MS) and have the potential to be important therapeutic targets. We examined the effect of TrkB signaling and the expression patterns of neurotrophic and gliotrophic factors in the mouse brain in MOG-induced experimental allergic encephalomyelitis (EAE). With induction of mild disease, TrkB heterozygous mice were more severely affected compared to their wild type littermates. However, with more potent disease induction, TrkB heterozygotes fared similar to their wild type littermates, suggesting complex modulatory roles for TrkB signaling. One possible explanation for this difference is that the expression patterns of neurotrophic factors correlate with disease severity in individual mice with mild disease, but not in more severe disease. With the less potent induction in C57BL/6 mice, we found that BDNF was consistently increased at EAE onset, while the soluble gliotrophic factor neuregulin (NRG1) was increased only in the chronic phase of the disease. Treatment of these animals with glatiramer acetate (GA) to decrease disease severity resulted in lower levels of both BDNF and NRG1 expression in some mice at 35days after immunization compared to those in untreated EAE mice, but had no direct effect on these factors in the absence of EAE. Our results suggest a complex interplay between neurotrophic and gliotrophic factors in EAE that is dependent on disease stage and severity. While signaling by BDNF through TrkB is protective in mild disease, this effect was not seen in more severe disease. The late induction of NRG1 in the chronic stage of disease could also worsen disease severity through its known ability to activate microglial, inflammatory pathways. While complex, these studies begin to define underlying axoglial trophic activities that are likely involved in both disease pathogenesis and repair.

Reversal of Behavioral Deficits and Synaptic Dysfunction in Mice Overexpressing Neuregulin 1

Neuregulin 1 (Nrg1) is a susceptibility gene of schizophrenia, a disabling mental illness that affects 1% of the general population. Here, we show that ctoNrg1 mice, which mimic high levels of NRG1 observed in forebrain regions of schizophrenic patients, exhibit behavioral deficits and hypofunction of glutamatergic and GABAergic pathways. Intriguingly, these deficits were diminished when NRG1 expression returned to normal in adult mice, suggesting that damage which occurred during development is recoverable. Conversely, increase of NRG1 in adulthood was sufficient to cause glutamatergic impairment and behavioral deficits. We found that the glutamatergic impairment by NRG1 overexpression required LIM domain kinase 1 (LIMK1), which was activated in mutant mice, identifying a pathological mechanism. These observations demonstrate that synaptic dysfunction and behavioral deficits in ctoNrg1 mice require continuous NRG1 abnormality in adulthood, suggesting that relevant schizophrenia may benefit from therapeutic intervention to restore NRG1 signaling.

Neuregulin 1 Enhances Cell Adhesion Molecule L1 Expression in Human Glioma Cells and Promotes Their Migration as a Function of Malignancy

Similar functions of L1, a cell adhesion molecule, and the cytokine neuregulin 1 (Nrg1) have been suggested in tumorigenesis and the promotion of metastasis. We studied the relationships of Nrg1 and L1 expression in human gliomas. Using immunofluorescence staining on a human glioma tissue microarray, we found a positive correlation between levels of L1 and Nrg1α or Nrg1β expression; expression tended to increase with increasing WHO (World Health Organization) tumor grade. L1 was also found to colocalize with either Nrg1 isoform. In cultures of U87-MG human glioblastoma and human U251 and SHG-44 glioma cells, the base levels of full-length L1 expression were increased by the 2 Nrg1 molecules in the nanomolar range, and Nrg1 siRNA downregulated full-length L1 expression in these tumor cell lines. U87-MG cells treated with either Nrg1 isoform also showed enhanced migration when compared with that treated with vehicle control. In addition, administration of either lapatinib (a dual inhibitor of both the epidermal growth factor receptor and ErbB-2) or erlotinib (an inhibitor of the epidermal growth factor receptor) in combination with either Nrg1α or Nrg1β inhibited the L1 expression elicited by these cytokines in U87-MG cells. Together, our data suggest that Nrg1 regulates L1 expression in gliomas, and that Nrg1 may contribute to malignancy by upregulating the L1 expression in glioblastoma cells, thereby enhancing their migration.

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.

LH and hCG Action on the Same Receptor Results in Quantitatively and Qualitatively Different Intracellular Signalling

Human luteinizing hormone (hLH) and chorionic gonadotropin (hCG) act on the same receptor (LHCGR) but it is not known whether they elicit the same cellular and molecular response. This study compares for the first time the activation of cell-signalling pathways and gene expression in response to hLH and hCG. Using recombinant hLH and recombinant hCG we evaluated the kinetics of cAMP production in COS-7 and hGL5 cells permanently expressing LHCGR (COS-7/LHCGR, hGL5/LHCGR), as well as cAMP, ERK1/2, AKT activation and progesterone production in primary human granulosa cells (hGLC). The expression of selected target genes was measured in the presence or absence of ERK- or AKT-pathways inhibitors. In COS-7/LHCGR cells, hCG is 5-fold more potent than hLH (cAMP ED50: 107.1±14.3 pM and 530.0±51.2 pM, respectively). hLH maximal effect was significantly faster (10 minutes by hLH; 1 hour by hCG). In hGLC continuous exposure to equipotent doses of gonadotropins up to 36 hours revealed that intracellular cAMP production is oscillating and significantly higher by hCG versus hLH. Conversely, phospho-ERK1/2 and -AKT activation was more potent and sustained by hLH versus hCG. ERK1/2 and AKT inhibition removed the inhibitory effect on NRG1 (neuregulin) expression by hLH but not by hCG; ERK1/2 inhibition significantly increased hLH- but not hCG-stimulated CYP19A1 (aromatase) expression. We conclude that: i) hCG is more potent on cAMP production, while hLH is more potent on ERK and AKT activation; ii) hGLC respond to equipotent, constant hLH or hCG stimulation with a fluctuating cAMP production and progressive progesterone secretion; and iii) the expression of hLH and hCG target genes partly involves the activation of different pathways depending on the ligand. Therefore, the LHCGR is able to differentiate the activity of hLH and hCG.

Fronto-temporal-mesolimbic gene expression and heritable differences in amphetamine-disrupted sensorimotor gating in rats

Differences in sensitivity to the prepulse inhibition (PPI)-disruptive effects of D2-family agonists in Sprague-Dawley (SD) vs. Long Evans (LE) rats are heritable, reflect differential activation of DA signaling in the nucleus accumbens (NAC), and are associated with differences in expression of specific NAC genes. These differences may inform us about the biology of PPI deficits in disorders such as schizophrenia. After confirming these strain-based PPI differences, we measured expression of four genes in NAC and other regions that regulate PPI: medial prefrontal cortex and ventral hippocampus (VH). Startle and PPI were assessed in SD and LE rats administered D-amphetamine (0 vs. 4.5 mg/kg, sc). Two weeks later, brain tissue was processed for comt, nrg1, grid2, and csnk1e expression; blood comt expression was also tested. Data confirmed expected PPI phenotypes. Gene expression levels differed across strains, sexes, and brain regions, with LE > SD expression in most genes and regions, and female > male expression for all NAC genes. Within any brain region, expression of the four genes was highly inter-correlated; across regions, correlations were less robust, reflecting distinct strain- or sex-based subgroups. PPI amphetamine sensitivity at 120 ms correlated significantly with NAC nrg1 expression, while amphetamine sensitivity for 30 ms PPI and startle magnitude correlated significantly with VH nrg1 and blood comt expression. Rat strains differing in a schizophrenia-linked phenotype also differ in expression levels of genes associated both with that phenotype, and with schizophrenia, within brain regions associated with that phenotype and schizophrenia.

Neuregulins are essential for spermatogonial proliferation and meiotic initiation in neonatal mouse testis

The transition from mitosis to meiosis is unique to germ cells. In murine embryonic ovaries and juvenile testes, retinoic acid (RA) induces meiosis via the stimulated by retinoic acid gene 8 (Stra8), but its molecular pathway requires elucidation. We present genetic evidence in vivo and in vitro that neuregulins (NRGs) are essential for the proliferation of spermatogonia and the initiation of meiosis. Tamoxifen (TAM) was injected into 14-day post-partum (dpp) Sertoli cell-specific conditional Nrg1(Ser-/-) mutant mice. TAM induced testis degeneration, suppressed BrdU incorporation into spermatogonia and pre-leptotene primary spermatocytes, and decreased and increased the number of STRA8-positive and TUNEL-positive cells, respectively. In testicular organ cultures from 5-6 dpp wild-type mice and cultures of their re-aggregated spermatogonia and Sertoli cells, FSH, RA [all-trans-retinoic acid (ATRA), AM580, 9-cis-RA] and NRG1 promoted spermatogonial proliferation and meiotic initiation. However, TAM treatment of testicular organ cultures from the Nrg1(Ser-/-) mutants suppressed spermatogonial proliferation and meiotic initiation that was promoted by FSH or AM580. In re-aggregated cultures of purified spermatogonia, NRG1, NRG3, ATRA and 9-cis-RA promoted their proliferation and meiotic initiation, but neither AM580 nor FSH did. In addition, FSH, RAs and NRG1 promoted Nrg1 and Nrg3 mRNA expression in Sertoli cells. These results indicate that in juvenile testes RA and FSH induced meiosis indirectly through Sertoli cells when NRG1 and NRG3 were upregulated, as NRG1 amplified itself and NRG3. The amplified NRG1 and NRG3 directly induced meiosis in spermatogonia. In addition, ATRA and 9-cis-RA activated spermatogonia directly and promoted their proliferation and eventually meiotic initiation.

Expression of NRG1 and its receptors in human bladder cancer

Background:Therapies targeting ERBB2 have shown success in the clinic. However, response is not determined solely by expression of ERBB2. Levels of ERBB3, its preferred heterodimerisation partner and ERBB ligands may also have a role.Methods:We measured NRG1 expression by real-time quantitative RT–PCR and ERBB receptors by western blotting and immunohistochemistry in bladder tumours and cell lines.Results:NRG1α and NRG1β showed significant coordinate expression. NRG1β was upregulated in 78% of cell lines. In tumours, there was a greater range of expression with a trend towards increased NRG1α with higher stage and grade. Increased expression of ERBB proteins was detected in 15% (EGFR), 20% (ERBB2), 41% (ERBB3) and 0% (ERBB4) of cell lines. High EGFR expression was detected in 28% of tumours, associated with grade and stage (P=0.05; P=0.04). Moderate or high expression of ERBB2 was detected in 22% and was associated with stage (P=0.025). Cytoplasmic ERBB3 was associated with high tumour grade (P=0.01) and with ERBB2 positivity. In cell lines, NRG1β expression was significantly inversely related to ERBB3, but this was not confirmed in tumours.Conclusion:There is a wide spectrum of NRG1 and ERBB receptor expression in bladder cancer. In advanced tumours, EGFR, ERBB2 and ERBB3 upregulation is common and there is a relationship between expression of ERBB2 and ERBB3 but not the NRG1 ligand.