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The neuregulin/ErbB signaling network is genetically associated with schizophrenia and modulates hippocampal γ oscillations—a type of neuronal network activity important for higher brain processes and altered in psychiatric disorders. Because neuregulin-1 (NRG-1) dramatically increases extracellular dopamine levels in the hippocampus, we investigated the relationship between NRG/ErbB and dopamine signaling in hippocampal γ oscillations. Using agonists for different D1- and D2-type dopamine receptors, we found that the D4 receptor (D4R) agonist PD168077, but not D1/D5 and D2/D3 agonists, increases γ oscillation power, and its effect is blocked by the highly specific D4R antagonist L-745,870. Using double in situ hybridization and immunofluorescence histochemistry, we show that hippocampal D4R mRNA and protein are more highly expressed in GAD67-positive GABAergic interneurons, many of which express the NRG-1 receptor ErbB4. Importantly, D4 and ErbB4 receptors are coexpressed in parvalbumin-positive basket cells that are critical for γ oscillations. Last, we report that D4R activation is essential for the effects of NRG-1 on network activity because L-745,870 and the atypical antipsychotic clozapine dramatically reduce the NRG-1–induced increase in γ oscillation power. This unique link between D4R and ErbB4 signaling on γ oscillation power, and their coexpression in parvalbumin-expressing interneurons, suggests a cellular mechanism that may be compromised in different psychiatric disorders affecting cognitive control. These findings are important given the association of a DRD4 polymorphism with alterations in attention, working memory, and γ oscillations, and suggest potential benefits of D4R modulators for targeting cognitive deficits.

This study comprehensively addresses the phenotype, function, and whole transcriptome of primary human and rodent Schwann cells (SCs) and highlights key species-specific features beyond the expected donor variability that account for the differential ability of human SCs to proliferate, differentiate, and interact with axons in vitro. Contrary to rat SCs, human SCs were insensitive to mitogenic factors other than neuregulin and presented phenotypic variants at various stages of differentiation, along with a mixture of proliferating and senescent cells, under optimal growth-promoting conditions. The responses of human SCs to cAMP-induced differentiation featured morphological changes and cell cycle exit without a concomitant increase in myelin-related proteins and lipids. Human SCs efficiently extended processes along those of other SCs (human or rat) but failed to do so when placed in co-culture with sensory neurons under conditions supportive of myelination. Indeed, axon contact-dependent human SC alignment, proliferation, and differentiation were not observed and could not be overcome by growth factor supplementation. Strikingly, RNA-seq data revealed that ~ 44 of the transcriptome contained differentially expressed genes in human and rat SCs. A bioinformatics approach further highlighted that representative SC-specific transcripts encoding myelin-related and axon growth-promoting proteins were significantly affected and that a deficient expression of key transducers of cAMP and adhesion signaling explained the fairly limited potential of human SCs to differentiate and respond to axonal cues. These results confirmed the significance of combining traditional bioassays and high-resolution genomics methods to characterize human SCs and identify genes predictive of cell function and therapeutic value.

Introduction Human epidermal growth factor receptor HER3 has been implicated in promoting the aggressiveness and metastatic potential of breast cancer. Upregulation of HER3 has been found to be a major mechanism underlying drug resistance to EGFR and HER2 tyrosine kinase inhibitors and to endocrine therapy in the treatment of breast cancer. Thus, agents that reduce HER3 expression at the plasma membrane may synergize with current therapies and offer a novel therapeutic strategy to improve treatment. Methods We devised an image-based screening platform using membrane localized HER3-YFP to identify small molecules that promote HER3 internalization and degradation. In vitro and in vivo tumor models were used to characterize the signaling effects of perhexiline, an anti-anginal drug, identified by the screening platform. Results We found perhexiline, an anti-anginal drug, selectively internalized HER3, decreased HER3 expression, and subsequently inhibited signaling downstream of HER3. Consistent with these results, perhexiline inhibited breast cancer cell proliferation in vitro and tumor growth in vivo. Conclusions This is the first demonstration that HER3 can be targeted with small molecules by eliminating it from the cell membrane. The novel approach used here led to the discovery that perhexiline ablates HER3 expression, and offers an opportunity to identify HER3 ablation modulators as innovative therapeutics to improve survival in breast cancer patients.

Schizophrenia is a severely debilitating neurodevelopmental disorder. Establishing a causal link between circuit dysfunction and particular behavioral traits that are relevant to schizophrenia is crucial to shed new light on the mechanisms underlying the pathology. We studied an animal model of the human 22q11 deletion syndrome, the mutation that represents the highest genetic risk of developing schizophrenia. We observed a desynchronization of hippocampal neuronal assemblies that resulted from parvalbumin interneuron hypoexcitability. Rescuing parvalbumin interneuron excitability with pharmacological or chemogenetic approaches was sufficient to restore wild-type-like CA1 network dynamics and hippocampal-dependent behavior during adulthood. In conclusion, our data provide insights into the network dysfunction underlying schizophrenia and highlight the use of reverse engineering to restore physiological and behavioral phenotypes in an animal model of neurodevelopmental disorder.

Neuregulin, previously known as ARIA, is a signaling protein involved in cell survival, synaptic plasticity, cell communication and differentiation. Neuregulin has also been described as a potent inducer of acetylcholine receptor transcription in muscle and although both neuregulin and acetylcholine have been individually described to have neuroprotective roles, their relationship in the cholinergic anti-inflammatory pathway of the brain has not been examined. Using three cell lines, BV-2, EOC-20 and RAW 264.7, we investigated the role that neuregulin signaling through the Erb family of tyrosine kinases may play in the anti-inflammatory process mediated by the α7 nicotinic acetylcholine receptors. Here we show that ErbB4 is expressed in all of our cell lines and is phosphorylated upon treatment with neuregulin. Neuregulin treatment further increases the expression of α7 nicotinic acetylcholine receptors in the microglial lines tested. Given the central role of α7 nicotinic acetylcholine receptors in regulating system inflammation we analyzed the expression of several pro-inflammatory cytokines in our system. Using ELISAs for TNF-α and IL-6 we show that treatment with NRG can produce a nearly a 33% decrease in the levels of tumor necrosis factor-α secreted by activated microglia and a nearly 88% decrease in IL-6. Given these results we propose a neuroprotective role for neuregulin wherein it modulates the expression of TNF-α and thus inflammation in the CNS via the upregulation of α7 nicotinic acetylcholine receptor expression in microglia in vitro. We suggest that the disregulation of neuregulin expression may be pivotal in neurological disorders characterized by inflammation.

Growth factors activating the ErbB receptors have been described in prostate tumors. The androgen dependent prostate cancer cell line, LNCaP, expresses the ErbB-1, ErbB-2 and ErbB-3 receptor tyrosine kinases. Previously, it was demonstrated that NRG activates ErbB-2/ErbB-3 heterodimers to induce LNCaP cell death, whereas, EGF activates ErbB-1/ErbB-1 or ErbB-1/ErbB-2 dimers to induce cell growth and survival. It was also demonstrated that PI3K inhibitors repressed this cell death suggesting that in androgen deprived LNCaP cells, NRG activates a PI3K-dependent pathway associated with cell death. In the present study we demonstrate that NRG induces autophagy in LNCaP cells, using LC3 as a marker. However, the autophagy induced by NRG may be incomplete since p62 levels elevate. We also demonstrated that NRG- induced autophagy is independent of mammalian target of rapamycin (mTOR) inhibition since NRG induces Akt and S6K activation. Interestingly, inhibition of reactive oxygen species (ROS) by N-acetylcysteine (NAC), inhibited NRG-induced autophagy and cell death. Our study also identified JNK and Beclin 1 as important components in NRG-induced autophagy and cell death. NRG induced elevation in JNK phosphorylation that was inhibited by NAC. Moreover, inhibitor of JNK inhibited NRG-induced autophagy and cell death. Also, in cells overexpressing Bcl-2 or cells expressing sh-RNA against Beclin 1, the effects of NRG, namely induction of autophagy and cell death, were inhibited. Thus, in LNCaP cells, NRG-induces incomplete autophagy and cell death that depend on ROS levels. These effects of NRG are mediated by signaling pathway that activates JNK and Beclin 1, but is independent of mTOR inhibition.

During nervous system development different cell-to-cell communication mechanisms operate in parallel guiding migrating neurons and growing axons to generate complex arrays of neural circuits. How such a system works in coordination is not well understood. Cross-regulatory interactions between different signalling pathways and redundancy between them can increase precision and fidelity of guidance systems. Immunoglobulin superfamily proteins of the NCAM and L1 families couple specific substrate recognition and cell adhesion with the activation of receptor tyrosine kinases. Thus it has been shown that L1CAM-mediated cell adhesion promotes the activation of the EGFR (erbB1) from Drosophila to humans. Here we explore the specificity of the molecular interaction between L1CAM and the erbB receptor family. We show that L1CAM binds physically erbB receptors in both heterologous systems and the mammalian developing brain. Different Ig-like domains located in the extracellular part of L1CAM can support this interaction. Interestingly, binding of L1CAM to erbB enhances its response to neuregulins. During development this may synergize with the activation of erbB receptors through L1CAM homophilic interactions, conferring diffusible neuregulins specificity for cells or axons that interact with the substrate through L1CAM.

Activation of ErbB2/4 receptor tyrosine kinases in cardiomyocytes by neuregulin treatment is associated with improvement in cardiac function, supporting its use in human patients with heart failure despite the lack of a specific mechanism. Neuregulin infusion in rodents increases cardiac myosin light chain kinase (cMLCK) expression and cardiac myosin regulatory light chain (RLC) phosphorylation which may improve actin-myosin interactions for contraction. We generated a cMLCK knockout mouse to test the hypothesis that cMLCK is necessary for neuregulin-induced improvement in cardiac function by increasing RLC phosphorylation. The cMLCK knockout mice have attenuated RLC phosphorylation and decreased cardiac performance measured as fractional shortening. Neuregulin infusion for seven days in wildtype mice increased cardiac cMLCK protein expression and RLC phosphorylation while increasing Akt phosphorylation and decreasing phospholamban phosphorylation. There was no change in fractional shortening. In contrast, neuregulin infusion in cMLCK knockout animals increased cardiac performance in the absence of cMLCK without increasing RLC phosphorylation. In addition, CaMKII signaling appeared to be enhanced in neuregulin-treated knockout mice. Thus, Neuregulin may improve cardiac performance in the failing heart without increasing cMLCK and RLC phosphorylation by activating other signaling pathways.

The final signal for triggering the formation of lamellipodia that initiate directional migration of mammalian cells is binding of the Wiskott–Aldrich syndrome (WASP)/WASP family verproline-homologous protein 2 (WAVE2) to the actin-related protein 2 and 3 (Arp2/3) complex. This WAVE2–Arp2/3 signal is suggested to be enhanced in some breast cancers, facilitating invasion, and/or metastasis. Here, we demonstrated one cause of the enhanced signal using four breast cancer cell lines (SKBR3, AU565, MCF7, and MDA-MB-231). The WAVE2–Arp2/3 signal was estimated semi-quantitatively by counting the number of lamellipodia expressing both WAVE2 and Arp2 using high-power confocal laser microscopy. Higher expression of the WAVE2–Arp2/3 signal was detected in SKBR3 and AU565, which have HER2 gene amplification, than in the other two cell lines that lack HER2 gene amplification. Trastuzumab suppressed both the formation of lamellipodia and migration in a Boyden chamber experiment in SKBR3 and AU565. When the HER2 gene was transfected into MCF7, the number of both lamellipodia and migrated cells was increased. This enhancement of migration did not occur in the presence of extracellular matrix, and zymographic analysis showed no clear difference between HER2 gene-transfected cells and MCF7 cells. Immunohistochemical analysis of 115 cases of breast cancer revealed that coexpression of WAVE2 and Arp2 was significantly correlated with HER2-overexpression ( P  < 0.0001). These data indicate that an abnormal signal resulting from HER2 gene amplification activates lamellipodia formation in breast cancer cells, which initiates their metalloproteinase-independent migration.

The molecular signals required for initiating myelination and maintenance of the myelin internode are not known. Several growth factor families have been implicated in promoting oligodendrocyte survival or differentiation and may have consequences on formation of myelin. We developed a reliable assay for detecting ensheathment of neurites by oligodendrocytes in spinal cord explants. This system was used to assay the effect of selected growth factors on myelin internode formation. We examined the influence on myelination of the polypeptide growth factors neuregulin (NRG), platelet-derived growth factor (PDGF), leukemia inhibitory factor (LIF), and the thyroid hormone T 3 . We found that NRG, PDGF, and T 3 treatments enhanced myelination while LIF treatment inhibited it. We furthermore found that the most potent combination of factors to enhance myelination was NRG and T 3 . Our results demonstrate that the role of growth factors on CNS myelination can be reliably studied in a controlled in vitro environment and that the impact of individual or combinations of growth factors on myelination cannot be predicted by their known effects on oligodendrocyte survival, proliferation, or differentiation.