Preclinical and Clinical Characterization of Fibroblast-derived Neuregulin-1 on Trastuzumab and Pertuzumab Activity in HER2-positive Breast Cancer.

PURPOSE: To characterize expression of neuregulin-1 (NRG1), an HER3 ligand, in HER2-positive breast cancer and its relation with the efficacy of trastuzumab with or without pertuzumab. EXPERIMENTAL DESIGN: Characterization of NRG1 expression in tumor cell lines, in tumor specimens, and in cancer-associated fibroblasts (CAFs). Patient-derived CAFs were used to investigate NRG1 impact on the activity of trastuzumab with or without pertuzumab in HER2-positive breast cancer cells. The relationship between NRG1 expression and pathologic response to anti-HER2-based neoadjuvant therapy was assessed in a retrospective patient cohort and in the NeoSphere trial. RESULTS: NRG1 was expressed in HER2-positive breast cancer-derived fibroblasts at significantly higher levels than in cancer cells. NRG1 and the conditioned media (CM) from CAFs phosphorylated HER3 and AKT in cancer cells and mediated trastuzumab resistance. Stable genetic depletion of NRG1 from CAFs overcame trastuzumab resistance. Pertuzumab effectively suppressed trastuzumab resistance mediated by either NRG1 or CAF's CM. NRG1 engaged an epithelial-to-mesenchymal transition that was prevented by trastuzumab and pertuzumab. In clinical samples, stromal and/or tumor cell expression of NRG1 determined by immunohistochemistry was uncommon (13.2%) yet significantly linked with residual disease following trastuzumab-based neoadjuvant therapy. In the NeoSphere trial, the magnitude of the difference of pathologic complete response rates favoring the pertuzumab arm was higher in the NRG1-high group. CONCLUSIONS: CAF-derived NRG1 mediates trastuzumab resistance through HER3/AKT, which might be reverted by pertuzumab. In patients with HER2-positive breast cancer, high expression of NRG1 was associated to poor response to trastuzumab, but not in combination with pertuzumab.

Human endothelial colony-forming cells provide trophic support for pluripotent stem cell-derived cardiomyocytes via distinctively high expression of neuregulin-1.

The search for a source of endothelial cells (ECs) with translational therapeutic potential remains crucial in regenerative medicine. Human blood-derived endothelial colony-forming cells (ECFCs) represent a promising source of autologous ECs due to their robust capacity to form vascular networks in vivo and their easy accessibility from peripheral blood. However, whether ECFCs have distinct characteristics with translational value compared to other ECs remains unclear. Here, we show that vascular networks generated with human ECFCs exhibited robust paracrine support for human pluripotent stem cell-derived cardiomyocytes (iCMs), significantly improving protection against drug-induced cardiac injury and enhancing engraftment at ectopic (subcutaneous) and orthotopic (cardiac) sites. In contrast, iCM support was notably absent in grafts with vessels lined by mature-ECs. This differential trophic ability was due to a unique high constitutive expression of the cardioprotective growth factor neuregulin-1 (NRG1). ECFCs, but not mature-ECs, were capable of actively releasing NRG1, which, in turn, reduced apoptosis and increased the proliferation of iCMs via the PI3K/Akt signaling pathway. Transcriptional silencing of NRG1 abrogated these cardioprotective effects. Our study suggests that ECFCs are uniquely suited to support human iCMs, making these progenitor cells ideal for cardiovascular regenerative medicine.

Upregulated neuregulin-1 protects against optic nerve injury by regulating the RhoA/cofilin/F-actin axis.

OBJECTIVE: In recent years, the roles of Neuregulin-1 (NRG-1) in optic nerve injury and retinal cells have been investigated. However, the molecular mechanism by which NRG-1 affects optic nerve injury remains elusive and merits deeper exploration. Hence, this study examined the specific function of NRG-1 in the RhoA/cofilin/F-actin axis in optic nerve injury. METHODS: Retinal cells were isolated and identified for subsequent experimental uses. Reverse transcription quantitative polymerase chain reaction and Western blot assays were performed to measure NRG-1 expression in retinal cells which were cultured under elevated pressure. TUNEL staining was used to detect the cell apoptosis rate, and Western blot assay was performed to detect the expression of related genes. The axon growth was examined by immunofluorescence. The effects of NRG-1 on RhoA activity, cofilin phosphorylation, and F-actin were detected by Western blot assay. In other studies we established a rat model of acute optic nerve injury, and tested for beneficial effects of NRG-1 in vivo. RESULTS: High expression of NRG-1 was evident in the retinal tissues of rats with optic nerve injury. Overexpressing NRG-1 successfully inhibited RhoA activity and the phosphorylation of cofilin and promoted F-actin expression. In cell experiments, overexpressed NRG-1 suppressed the apoptosis of retinal cells and promoted axon growth through the RhoA/cofilin/F-actin axis. In animal experiments, overexpressed NRG-1 relieved retinal injury. CONCLUSION: Our results strongly suggest that overexpressed NRG-1 is highly effective in the protection of normal optic nerve function by suppressing RhoA activity and the phosphorylation of cofilin and rescuing F-actin function.

The lncRNA Ftx/miR-382-5p/Nrg1 axis improves the inflammation response of microglia and spinal cord injury repair.

During spinal cord injury (SCI), a quick and sustained decline of Neuregulin-1 (Nrg1) has been observed, exerting a significant positive effect in modulating the proliferation of astrocytes and the formation of glial scars within the damaged spinal cord. In this study, we revealed the abnormal downregulation of lncRNA Ftx and Nrg1 and upregulation of miR-382-5p after SCI, which contributed to the inflammatory response in microglial cells and affected SCI repair. Ftx overexpression was significantly reduced, and Ftx knockdown further promoted LPS effects on the inflammatory factors, indicating that lncRNA Ftx might affect the microglial inflammatory response. miR-382-5p targeted both lncRNA Ftx and Nrg1, and lncRNA Ftx competed with Nrg1 for miR-382-5p binding to act as a ceRNA, therefore counteracting miR-382-5p-mediated inhibition of Nrg1. miR-382-5p overexpression was significantly enhanced, and Nrg1 overexpression attenuated LPS effects on inflammatory factors within the microglia. Under LPS stimulation, the effects of Ftx overexpression were significantly reversed by overexpression of miR-382-5p, and the effects of miR-382-5p overexpression were significantly reversed by Nrg1 overexpression. In summary, the lncRNA Ftx/miR-382-5p/Nrg1 axis improves the inflammation response of the microglia, which might improve SCI repair.

Therapeutic Role of Neuregulin 1 Type III in SOD1-Linked Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron (Mn) disease without effective cure currently available. Death of MNs in ALS is preceded by failure of neuromuscular junctions and axonal retraction. Neuregulin 1 (NRG1) is a neurotrophic factor highly expressed in MNs and neuromuscular junctions that support axonal and neuromuscular development and maintenance. NRG1 and its ErbB receptors are involved in ALS. Reduced NRG1 expression has been found in ALS patients and in the ALS SOD1G93A mouse model; however, the expression of the isoforms of NRG1 and its receptors is still controversial. Due to the reduced levels of NRG1 type III (NRG1-III) in the spinal cord of ALS patients, we used gene therapy based on intrathecal administration of adeno-associated virus to overexpress NRG1-III in SOD1G93A mice. The mice were evaluated from 9 to 16 weeks of age by electrophysiology and rotarod tests. At 16 weeks, samples were harvested for histological and molecular analyses. Our results indicate that overexpression of NRG1-III is able to preserve neuromuscular function of the hindlimbs, improve locomotor performance, increase the number of surviving MNs, and reduce glial reactivity in the treated female SOD1G93A mice. Furthermore, the NRG1-III/ErbB4 axis appears to regulate MN excitability by modulating the chloride transporter KCC2 and reduces the expression of the MN vulnerability marker MMP-9. However, NRG1-III did not have a significant effect on male mice, indicating relevant sex differences. These findings indicate that increasing NRG1-III at the spinal cord is a promising approach for promoting MN protection and functional improvement in ALS.

Neuregulin and ErbB expression is regulated by development and sensory experience in mouse visual cortex.

Neuregulins (NRGs) are protein ligands that impact neural development and circuit function. NRGs signal through the ErbB receptor tyrosine kinase family. NRG1/ErbB4 signaling in parvalbumin-expressing (PV) inhibitory interneurons is critical for visual cortical plasticity. There are multiple types of NRGs and ErbBs that can potentially contribute to visual cortical plasticity at different developmental stages. Thus, it is important to understand the normal developmental expression profiles of NRGs and ErbBs in specific neuron types in the visual cortex, and to study whether and how their expression changes in PV inhibitory neurons and excitatory neurons track with sensory perturbation. Cell type-specific translating ribosome affinity purification and qPCR was used to compare mRNA expression of nrg1,2,3,4 and erbB1,2,3,4 in PV and excitatory neurons in mouse visual cortex. We show that the expression of nrg1 and nrg3 decreases in PV neurons at the critical period peak, postnatal day 28 (P28) after monocular deprivation and dark rearing, and in the adult cortex (at P104) after 2-week long dark exposure. In contrast, nrg1 expression by excitatory neurons is unchanged at P28 and P104 following sensory deprivation, whereas nrg3 expression by excitatory neurons shows changes depending on the age and the mode of sensory deprivation. ErbB4 expression in PV neurons remains consistently high and does not appear to change in response to sensory deprivation. These data provide new important details of cell type-specific NRG/ErbB expression in the visual cortex and support that NRG1/ErbB4 signaling is implicated in both critical period and adult visual cortical plasticity.

D2 receptor-mediated miRNA-143 expression is associated with the effects of antipsychotic drugs on phencyclidine-induced schizophrenia-related locomotor hyperactivity and with Neuregulin-1 expression in mice.

Non-coding RNA molecules such as miRNAs have emerged as critical regulators of neuronal functions. The present study investigates a role for miRNA-143, a highly conserved miRNA, in locomotorhyperactivity induced bythe psychotomimetic phencyclidine (PCP), a non-selective antagonist of the N-methyl-d-aspartate (NMDA) glutamate receptor. Following acute PCP administration to mice, the content of miRNA-143 was reduced in plasma, prefrontal cortex (PFC) and hippocampus, reaching a minimum after 2 h. The antipsychotics haloperidol and clozapine attenuated hyperlocomotion and the decrease in miR-143 expression induced by PCP, as did the selective D2 dopamine receptor antagonist eticlopride but not the selective D1 antagonist SCH23390. To further confirm D2 receptor-mediated miRNA-143 expression, HT-22 neuronal cell line and primary cortical cultured neuronswere studied. Stimulation of D2 receptors with the selective D2 receptor agonist quinpirole decreased expression of miRNA-143 in a time-dependent manner. This inhibition was blocked by pretreatment with eticlopride, indicating that the D2 receptor directly regulates the expression of miRNA-143. We further demonstrated that miRNA-143 directly targeted to the 3' un-translated region of neuregulin-1 (NRG1) mRNA to reduce protein expression of NRG1 in HT-22 cells and that administration of the D2 receptor agonist quinpirole to mice enhanced expression of NRG1 in PFC. The present data provide the first evidence that D2 receptors are involved in the expression ofmiRNA-143 in association with antipsychotic drug action and the developmental regulator NRG1.

Neuregulin 1 (NRG1) gene expression predicts functional outcomes in individuals at clinical high-risk for psychosis.

Little is known about valid predictive markers for functional outcomes in an at-risk for psychosis population. In a cohort of 185 individuals (age: 13-35 years) at high risk (HR) and ultra-high risk (UHR), we assessed pan-NRG1 mRNA levels across good functional status (GFS) and poor functional status (PFS) at baseline, and good functional outcome (GFO) and poor functional outcome (PFO) at 12-month follow-up. NRG1 mRNA levels were significantly higher in individuals with PFO than individuals with GFO at 12-month follow-up. Our findings suggest that NRG1 might emerge as a predictive marker for functional outcomes in at-risk for psychosis population.

Clinical significance of overexpression of NRG1 and its receptors, HER3 and HER4, in gastric cancer patients.

BACKGROUND: Neuregulin 1 (NRG1), a ligand for human epidermal growth factor (HER) 3 and HER4, can activates cell signaling pathways to promote carcinogenesis and metastasis. METHODS: To investigate the clinicopathologic significance of NRG1 and its receptors, immunohistochemistry was performed for NRG1, HER3, and HER4 in 502 consecutive gastric cancers (GCs). Furthermore, HER2, microsatellite instability (MSI), and Epstein-Barr virus (EBV) status were investigated. NRG1 gene copy number (GCN) was determined by dual-color fluorescence in situ hybridization (FISH) in 388 available GCs. RESULTS: NRG1 overexpression was observed in 141 (28.1%) GCs and closely correlated with HER3 (P = 0.034) and HER4 (P < 0.001) expression. NRG1 overexpression was significantly associated with aggressive features, including infiltrative tumor growth, lymphovascular, and neural invasion, high pathologic stage, and poor prognosis (all P < 0.05), but not associated with EBV, MSI, or HER2 status. Multivariate analysis identified NRG1 overexpression as an independent prognostic factor for survival (P = 0.040). HER3 and HER4 expressions were observed in 157 (31.3%) and 277 (55.2%), respectively. In contrast to NRG1, expression of these proteins was not associated with survival. NRG1 GCN gain (GCN ≥ 2.5) was detected in 14.7% patients, including two cases of amplification, and was moderately correlated with NRG1 overexpression (κ, 0.459; P < 0.001). CONCLUSIONS: Although our results indicate a lack of prognostic significance of HER3 and HER4 overexpression in GC, overexpression of their ligand, NRG1, was associated with aggressive clinical features and represented an independent unfavorable prognostic factor. Therefore, NRG1 is a potential prognostic and therapeutic biomarker in GC patients.

The Role of NRG1 in the Predisposition to Papillary Thyroid Carcinoma.

Context: Previous genome-wide association studies have shown that single-nucleotide polymorphism (SNP) rs2439302 in chromosome 8p12 is significantly associated with papillary thyroid carcinoma (PTC) risk and dysregulated NRG1 expression. The underlying mechanisms remain to be discovered. Objective: To evaluate the expression of NRG1 isoforms, candidate functional variants, and potential genes downstream of NRG1 in thyroid tissue. Methods: Quantitative reverse transcription polymerase chain reaction was applied for gene expression analysis. SNaPshot assay, haplotype, and computer analyses were performed to evaluate candidate functional variants. Other functional assays [chromatin immunoprecipitation (ChIP) assay, luciferase assay, small interfering RNA knockdown, and RNA sequencing] were performed. Results: Three NRG1 isoforms (NM_004495, NM_013958, and NM_001160008) tested were highly expressed in thyroid tissue. The expression levels of the three isoforms were significantly correlated with the genotypes of rs2439302. A DNA block of ~32 kb containing the risk G allele of rs2439302 was revealed, harboring multiple candidate functional variants. ChIP assay for active chromatin markers indicated at least nine regions in the DNA block showing strong H3Kme1 and H3K27Ac signals in thyroid tissue. Luciferase reporter assays revealed differential allelic activities associated with seven SNPs. Knocking down NRG1 in primary thyroid cells revealed downstream or interacting genes related to NRG1. Conclusions: Our data suggest a role for transcriptional regulation of NRG1 in the predisposition to PTC.

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

BACKGROUND: This 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). METHODS: Male 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. RESULTS: In 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). CONCLUSIONS: Our 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.

Functional prediction of miR-3144-5p in human cardiac myocytes based on transcriptome sequencing and bioinformatics.

BACKGROUND: RAN guanine nucleotide release factor (RANGRF) encoding protein MOG1 plays an important role in cardiac arrhythmia, so we intended to investigate the regulatory miRNA of RANGRF and explore its potential regulatory mechanism in arrhythmogenesis. METHODS: Based on bioinformatic analysis, miR-3144-5p was predicted to be a regulatory miRNA of RANGRF, which were then validated through a dual-luciferase reporter plasmid assay. Subsequently, the expression level of miR-3144-5p in human cardiac myocytes (HCMs) was detected, followed by cell transfection with miR-3144-5p mimics. Transcriptome sequencing was then performed in HCMs with or without transfection. The sequencing results were subjected to bioinformatic analyses, including differentially expressed gene (DEG) analysis, functional enrichment analysis, protein-protein interaction (PPI) network analysis, miRNA-target gene analysis, and miRNA-transcription factor (TF)-target gene coregulatory network analysis. RESULTS: There really existed a regulatory relation between miR-3144-5p and RANGRF. The expression level of miR-3144-5p was low in HCMs. After cell transfection, miR-3144-5p expression level significantly increased in HCMs. Bioinformatic analyses of the transcriptome sequencing results identified 300 upregulated and 271 downregulated DEGs between miR-3144-5p mimic and control group. The upregulated genes ISL1 and neuregulin 1 (NRG1) were significantly enriched in cardiac muscle cell myoblast differentiation (GO:0060379). CCL21 was one of the hub genes in the PPI network and also a target gene of miR-3144-5p. Moreover, the TF of v-Myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN) was involved in the miR-3144-5p-TF-target gene coregulatory network and interacted with the target genes of miR-3144-5p. CONCLUSION: ISL1, NRG1, CCL21, and MYCN were differentially expressed in the miR-3144-5p mimic group, suggesting that miR-3144-5p overexpression plays a role in HCMs by regulating these genes and TF. This study may provide new insight into the mechanisms behind the progression of cardiac arrhythmia.

Prevalence and influence on outcome of HER2/neu, HER3 and NRG1 expression in patients with metastatic colorectal cancer.

Our aim was to explore the impact of the HER2/neu, HER3 receptor as well as their ligands' neuregulin (NRG1) expression on the outcome of patients with metastatic colorectal cancer (mCRC). NRG1, HER2/neu and HER3 expression was evaluated in 208 patients with mCRC receiving 5-FU/LV plus irinotecan or irinotecan plus oxaliplatin as the first-line treatment. Biomarker expression was correlated with the outcome of patients. NRG1 (low: 192 vs. high: 16), HER2/neu (low: 201 vs. high: 7) and HER3 (low: 69 vs. high: 139) expressions were assessed in 208 patients. High versus low NRG1 expression significantly affected progression-free survival (PFS) [4.7 vs. 8.2 months, hazard ratio (HR): 2.45; 95% confidence interval (CI): 1.45-4.13; P=0.001], but not overall survival (OS) (15.5 vs. 20.7 months, HR: 1.33; 95% CI: 0.76-2.35; P=0.32). High versus low HER3 expression (PFS: 7.1 vs. 8.8 months, HR: 1.11; 95% CI: 0.82-1.50; P=0.50; OS: 19.8 vs. 21.1 months, HR: 0.95; 95% CI: 0.70-1.30; P=0.75) and high compared with low HER2/neu expression (PFS: 7.7 vs. 8.0 months, HR: 1.07; 95% CI: 0.71-1.60; P=0.75; OS: 16.6 vs. 21.1 months, HR: 1.13; 95% CI: 0.75-1.71; P=0.57) did not influence outcome. High NRG1 expression was associated with inferior PFS in the FIRE-1 trial. We did not detect a prognostic impact of HER2/neu and HER3 overexpression in mCRC. The frequency of overexpression was comparable with other studies.

Myocardial ischemia/reperfusion upregulates the transcription of the Neuregulin1 receptor ErbB3, but only postconditioning preserves protein translation: Role in oxidative stress.

BACKGROUND: Neuregulin1 (Nrg1) and its receptors ErbB are crucial for heart development and for adult heart structural maintenance and function and Nrg1 has been proposed for heart failure treatment. Infarct size is the major determinant of heart failure and the mechanism of action and the role of each ErbB receptor remain obscure, especially in the post-ischemic myocardium. We hypothesized that Nrg1 and ErbB are affected at transcriptional level early after ischemia/reperfusion (I/R) injury, and that the protective postconditioning procedure (PostC, brief cycles of ischemia/reperfusion carried out after a sustained ischemia) can influence this pathway. METHODS AND RESULTS: The Langendorff's heart was used as an ex-vivo model to mimic an I/R injury in the whole rat heart; after 30min of ischemia and 2h of reperfusion, with or without PostC, Nrg1 and ErbB expression were analysed by quantitative real-time PCR and Western blot. While no changes occur for ErbB2, ErbB4 and Nrg1, an increase of ErbB3 expression occurs after I/R injury, with and without PostC. However, I/R reduces ErbB3 protein, whereas PostC preserves it. An in vitro analysis with H9c2 cells exposed to redox-stress indicated that the transient over-expression of ErbB3 alone is able to increase cell survival (MTT assay), limiting mitochondrial dysfunction (JC-1 probe) and apoptotic signals (Bax/Bcl-2 ratio). CONCLUSIONS: This study suggests ErbB3 as a protective factor against death pathways activated by redox stress and supports an involvement of this receptor in the pro-survival responses.

Chronic administration of calcitriol enhanced neuregulin-1/ErbB signaling in rat myocardium.

Although emerging evidence suggests that vitamin D has beneficial effects in the cardiovascular health, the underlying mechanisms are far from fully elucidated. Given the indispensable role of neuregulin-1 (NRG1)/ErbB signaling in the cardiovascular system, the present study investigated the influences of prolonged administration of calcitriol, the active form of vitamin D, on the NRG1/ErbB system. We examined the protein expression of NRG1, ErbB receptors (ErbB2 and ErbB4) and their phosphorylated forms in the myocardium of rats following 6-week administration of calcitriol (50 ng/kg/day or 100 ng/kg/day). We further assessed the myocardial vitamin D receptor (VDR) to confirm the effect of calcitriol treatment. Additionally, serum neuregulin-1 level was also analyzed. Generally, calcitriol enhanced myocardial VDR expression and NRG1/ErbB signaling. Calcitriol increased NRG1 protein level at the higher dose, while both doses promoted ErbB2 and phosphorylated ErbB2 expression. Although calcitriol has no significant influence on ErbB4 expression, phosphorylated ErbB4 receptors were enhanced at the higher dose. Furthermore, the serum neuregulin-1 concentration was increased at both doses. Overall, our data firstly showed that chronic calcitriol administration enhanced NRG1/ErbB signaling in the heart, indicating a novel mechanism underlying the cardiac effects of vitamin D.

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

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

Elucidation of Resistance Mechanisms to Second-Generation ALK Inhibitors Alectinib and Ceritinib in Non-Small Cell Lung Cancer Cells.

Crizotinib is the first anaplastic lymphoma kinase (ALK) inhibitor to have been approved for the treatment of non-small cell lung cancer (NSCLC) harboring an ALK fusion gene, but it has been found that, in the clinic, patients develop resistance to it. Alectinib and ceritinib are second-generation ALK inhibitors which show remarkable clinical responses in both crizotinib-naive and crizotinib-resistant NSCLC patients harboring an ALK fusion gene. Despite their impressive activity, clinical resistance to alectinib and ceritinib has also emerged. In the current study, we elucidated the resistance mechanisms to these second-generation ALK inhibitors in the H3122 NSCLC cell line harboring the EML4-ALK variant 1 fusion in vitro. Prolonged treatment of the parental H3122 cells with alectinib and ceritinib led to two cell lines which are 10 times less sensitive to alectinib and ceritinib than the parental H3122 cell line. Although mutations of ALK in its kinase domain are a common resistance mechanism for crizotinib, we did not detect any ALK mutation in these resistant cell lines. Rather, overexpression of phospho-ALK and alternative receptor tyrosine kinases such as phospho-EGFR, phospho-HER3, and phospho-IGFR-1R was observed in both resistant cell lines. Additionally, NRG1, a ligand for HER3, is upregulated and responsible for resistance by activating the EGFR family pathways through the NRG1-HER3-EGFR axis. Combination treatment with EGFR inhibitors, in particular afatinib, was shown to be effective at overcoming resistance. Our study provides new mechanistic insights into adaptive resistance to second-generation ALK inhibitors and suggests a potential clinical strategy to combat resistance to these second-generation ALK inhibitors in NSCLC.

Neuregulin-1 controls an endogenous repair mechanism after spinal cord injury.

Following traumatic spinal cord injury, acute demyelination of spinal axons is followed by a period of spontaneous remyelination. However, this endogenous repair response is suboptimal and may account for the persistently compromised function of surviving axons. Spontaneous remyelination is largely mediated by Schwann cells, where demyelinated central axons, particularly in the dorsal columns, become associated with peripheral myelin. The molecular control, functional role and origin of these central remyelinating Schwann cells is currently unknown. The growth factor neuregulin-1 (Nrg1, encoded by NRG1) is a key signalling factor controlling myelination in the peripheral nervous system, via signalling through ErbB tyrosine kinase receptors. Here we examined whether Nrg1 is required for Schwann cell-mediated remyelination of central dorsal column axons and whether Nrg1 ablation influences the degree of spontaneous remyelination and functional recovery following spinal cord injury. In contused adult mice with conditional ablation of Nrg1, we found an absence of Schwann cells within the spinal cord and profound demyelination of dorsal column axons. There was no compensatory increase in oligodendrocyte remyelination. Removal of peripheral input to the spinal cord and proliferation studies demonstrated that the majority of remyelinating Schwann cells originated within the injured spinal cord. We also examined the role of specific Nrg1 isoforms, using mutant mice in which only the immunoglobulin-containing isoforms of Nrg1 (types I and II) were conditionally ablated, leaving the type III Nrg1 intact. We found that the immunoglobulin Nrg1 isoforms were dispensable for Schwann cell-mediated remyelination of central axons after spinal cord injury. When functional effects were examined, both global Nrg1 and immunoglobulin-specific Nrg1 mutants demonstrated reduced spontaneous locomotor recovery compared to injured controls, although global Nrg1 mutants were more impaired in tests requiring co-ordination, balance and proprioception. Furthermore, electrophysiological assessments revealed severely impaired axonal conduction in the dorsal columns of global Nrg1 mutants (where Schwann cell-mediated remyelination is prevented), but not immunoglobulin-specific mutants (where Schwann cell-mediated remyelination remains intact), providing robust evidence that the profound demyelinating phenotype observed in the dorsal columns of Nrg1 mutant mice is related to conduction failure. Our data provide novel mechanistic insight into endogenous regenerative processes after spinal cord injury, demonstrating that Nrg1 signalling regulates central axon remyelination and functional repair and drives the trans-differentiation of central precursor cells into peripheral nervous system-like Schwann cells that remyelinate spinal axons after injury. Manipulation of the Nrg1 system could therefore be exploited to enhance spontaneous repair after spinal cord injury and other central nervous system disorders with a demyelinating pathology.media-1vid110.1093/brain/aww039_video_abstractaww039_video_abstract.

Exercise training activates neuregulin 1/ErbB signaling and promotes cardiac repair in a rat myocardial infarction model.

AIMS: Exercise training (ET) has a cardioprotective effect and can alter the molecular response to myocardial infarction (MI). The Neuregulin 1 (NRG1)/ErbB signaling plays a critical role in cardiac repair and regeneration in the failing heart. We sought to investigate whether ET following MI could activate the NRG1/ErbB signaling and promote cardiac repair and regeneration. MAIN METHODS: Male Sprague-Dawley rats were used to establish the MI model. Exercise-trained animals were subjected to four weeks of exercise (16m/min, 50min/d, 5d/wk) following the surgery. AG1478 was used as an inhibitor of ErbB (1mg/kg body weight, administered i.v. every other day during the process of training). NRG1/ErbB signaling activation, cardiomyocyte (CM) proliferation and apoptosis were evaluated. KEY FINDINGS: In the exercise-trained rats, NRG1 expression was up-regulated and ErbB/PI3K/Akt signaling was activated compared with the MI group. In addition, ET preserved heart function accompanied with increased numbers of BrdU(+) CMs, PCNA(+) CMs and c-kit(+) cells, and reduced apoptosis level in the MI rats. In contrast, blocking ErbB signaling by AG1478 attenuated the ET-induced cardiac repair and regeneration. SIGNIFICANCE: ET up-regulates NRG1 expression and activates ErbB2, ErbB4 and PI3K/Akt signal transduction to promote cardiac repair through endogenous regeneration. Activation of ErbB may be an underlying mechanism for the ET-induced cardiac repair and regeneration following MI.

A Transient Translaminar GABAergic Interneuron Circuit Connects Thalamocortical Recipient Layers in Neonatal Somatosensory Cortex.

GABAergic activity is thought to influence developing neocortical sensory circuits. Yet the late postnatal maturation of local layer (L)4 circuits suggests alternate sources of GABAergic control in nascent thalamocortical networks. We show that a population of L5b, somatostatin (SST)-positive interneuron receives early thalamic synaptic input and, using laser-scanning photostimulation, identify an early transient circuit between these cells and L4 spiny stellates (SSNs) that disappears by the end of the L4 critical period. Sensory perturbation disrupts the transition to a local GABAergic circuit, suggesting a link between translaminar and local control of SSNs. Conditional silencing of SST+ interneurons or conversely biasing the circuit toward local inhibition by overexpression of neuregulin-1 type 1 results in an absence of early L5b GABAergic input in mutants and delayed thalamic innervation of SSNs. These data identify a role for L5b SST+ interneurons in the control of SSNs in the early postnatal neocortex.