CXCR4 expression is associated with proneural-to-mesenchymal transition in glioblastoma.

Glioblastoma (GBM) is the most common primary intracranial malignant tumor and consists of three molecular subtypes: proneural (PN), mesenchymal (MES) and classical (CL). Transition between PN to MES subtypes (PMT) is the glioma analog of the epithelial-mesenchymal transition (EMT) in carcinomas and is associated with resistance to therapy. CXCR4 signaling increases the expression of MES genes in glioma cell lines and promotes EMT in other cancers. RNA sequencing (RNAseq) data of PN GBMs in The Cancer Genome Atlas (TCGA) and secondary high-grade gliomas (HGGs) from an internal cohort were examined for correlation between CXCR4 expression and survival as well as expression of MES markers. Publicly available single-cell RNA sequencing (scRNAseq) data was analyzed for cell type specific CXCR4 expression. These results were validated in a genetic mouse model of PN GBM. Higher CXCR4 expression was associated with significantly reduced survival and increased expression of MES markers in TCGA and internal cohorts. CXCR4 was expressed in immune and tumor cells based on scRNAseq analysis. Higher CXCR4 expression within tumor cells on scRNAseq was associated with increased MES phenotype, suggesting a cell-autonomous effect. In a genetically engineered mouse model, tumors induced with CXCR4 exhibited a mesenchymal phenotype and shortened survival. These results suggest that CXCR4 signaling promotes PMT and shortens survival in GBM and highlights its inhibition as a potential therapeutic strategy.

Negr1 controls adult hippocampal neurogenesis and affective behaviors.

Recent genome-wide association studies on major depressive disorder have implicated neuronal growth regulator 1 (Negr1), a GPI-anchored cell adhesion molecule in the immunoglobulin LON family. Although Negr1 has been shown to regulate neurite outgrowth and synapse formation, the mechanism through which this protein affects mood disorders is still largely unknown. In this research, we characterized Negr1-deficient (negr1-/-) mice to elucidate the function of Negr1 in anxiety and depression. We found that anxiety- and depression-like behaviors increased in negr1-/- mice compared with wild-type mice. In addition, negr1-/- mice had decreased adult hippocampal neurogenesis compared to wild-type mice. Concurrently, both LTP and mEPSC in the dentate gyrus (DG) region were severely compromised in negr1-/- mice. In our effort to elucidate the underlying molecular mechanisms, we found that lipocalin-2 (Lcn2) expression was decreased in the hippocampus of negr1-/- mice compared to wild-type mice. Heterologous Lcn2 expression in the hippocampal DG of negr1-/- mice rescued anxiety- and depression-like behaviors and restored neurogenesis and mEPSC frequency to their normal levels in these mice. Furthermore, we discovered that Negr1 interacts with leukemia inhibitory factor receptor (LIFR) and modulates LIF-induced Lcn2 expression. Taken together, our data uncovered a novel mechanism of mood regulation by Negr1 involving an interaction between Negr1 and LIFR along with Lcn2 expression.

Comparison of microRNA expressions for the identification of chemical hazards in in vivo and in vitro hepatic injury models.

Biofluid-based biomarkers provide an efficient tool for hazard identification of chemicals. Here, we explored the potential of microRNAs (miRNAs) as biomarkers for hepatotoxicity of chemicals by linking in vitro to in vivo animal models. A search of the literature identified candidate circulating miRNA biomarkers of chemical-induced hepatotoxicity. The expression of candidate miRNAs (miR-122, miR-151a, miR-192, miR-193a, miR-194, miR-21, miR-29c), was determined by real-time reverse transcription-polymerase chain reaction in in vivo acute liver injury induced by acetaminophen, and then were further compared with those of in vitro cell assays. Candidate miRNAs, except miR-29c, were significantly or biologically upregulated by acetaminophen, at a dose that caused acute liver injury as confirmed by hepatocellular necrosis. Except miR-122 and miR-193a, other miRNAs elevated in in vivo models were confirmed by in vitro models using HepG2 cells, whereas they failed by in vitro models using human primary hepatocytes. These findings indicate that certain miRNAs may still have the potential of toxicological biomarkers in linking in vitro to in vivo hepatotoxicity.

Postinjury Induction of Activated ErbB2 Selectively Hyperactivates Denervated Schwann Cells and Promotes Robust Dorsal Root Axon Regeneration.

Following nerve injury, denervated Schwann cells (SCs) convert to repair SCs, which enable regeneration of peripheral axons. However, the repair capacity of SCs and the regenerative capacity of peripheral axons are limited. In the present studies we examined a potential therapeutic strategy to enhance the repair capacity of SCs, and tested its efficacy in enhancing regeneration of dorsal root (DR) axons, whose regenerative capacity is particularly weak. We used male and female mice of a doxycycline-inducible transgenic line to induce expression of constitutively active ErbB2 (caErbB2) selectively in SCs after DR crush or transection. Two weeks after injury, injured DRs of induced animals contained far more SCs and SC processes. These SCs had not redifferentiated and continued to proliferate. Injured DRs of induced animals also contained far more axons that regrew along SC processes past the transection or crush site. Remarkably, SCs and axons in uninjured DRs remained quiescent, indicating that caErbB2 enhanced regeneration of injured DRs, without aberrantly activating SCs and axons in intact nerves. We also found that intraspinally expressed glial cell line-derived neurotrophic factor (GDNF), but not the removal of chondroitin sulfate proteoglycans, greatly enhanced the intraspinal migration of caErbB2-expressing SCs, enabling robust penetration of DR axons into the spinal cord. These findings indicate that SC-selective, post-injury activation of ErbB2 provides a novel strategy to powerfully enhance the repair capacity of SCs and axon regeneration, without substantial off-target damage. They also highlight that promoting directed migration of caErbB2-expressing SCs by GDNF might be useful to enable axon regrowth in a non-permissive environment.SIGNIFICANCE STATEMENT Repair of injured peripheral nerves remains a critical clinical problem. We currently lack a therapy that potently enhances axon regeneration in patients with traumatic nerve injury. It is extremely challenging to substantially increase the regenerative capacity of damaged nerves without deleterious off-target effects. It was therefore of great interest to discover that caErbB2 markedly enhances regeneration of damaged dorsal roots, while evoking little change in intact roots. To our knowledge, these findings are the first demonstration that repair capacity of denervated SCs can be efficaciously enhanced without altering innervated SCs. Our study also demonstrates that oncogenic ErbB2 signaling can be activated in SCs but not impede transdifferentiation of denervated SCs to regeneration-promoting repair SCs.

Steroid receptor coactivator-2 (SRC-2) coordinates cardiomyocyte paracrine signaling to promote pressure overload-induced angiogenesis.

Pressure overload-induced cardiac stress induces left ventricular hypertrophy driven by increased cardiomyocyte mass. The increased energetic demand and cardiomyocyte size during hypertrophy necessitate increased fuel and oxygen delivery and stimulate angiogenesis in the left ventricular wall. We have previously shown that the transcriptional regulator steroid receptor coactivator-2 (SRC-2) controls activation of several key cardiac transcription factors and that SRC-2 loss results in extensive cardiac transcriptional remodeling. Pressure overload in mice lacking SRC-2 induces an abrogated hypertrophic response and decreases sustained cardiac function, but the cardiomyocyte-specific effects of SRC-2 in these changes are unknown. Here, we report that cardiomyocyte-specific loss of SRC-2 (SRC-2 CKO) results in a blunted hypertrophy accompanied by a rapid, progressive decrease in cardiac function. We found that SRC-2 CKO mice exhibit markedly decreased left ventricular vasculature in response to transverse aortic constriction, corresponding to decreased expression of the angiogenic factor VEGF. Of note, SRC-2 knockdown in cardiomyocytes decreased VEGF expression and secretion to levels sufficient to blunt in vitro tube formation and proliferation of endothelial cells. During pressure overload, both hypertrophic and hypoxic signals can stimulate angiogenesis, both of which stimulated SRC-2 expression in vitro Furthermore, SRC-2 coactivated the transcription factors GATA-binding protein 4 (GATA-4) and hypoxia-inducible factor (HIF)-1α and -2α in response to angiotensin II and hypoxia, respectively, which drive VEGF expression. These results suggest that SRC-2 coordinates cardiomyocyte secretion of VEGF downstream of the two major angiogenic stimuli occurring during pressure overload bridging both hypertrophic and hypoxia-stimulated paracrine signaling.

Highly Pathogenic Avian Influenza A(H5N6) in Domestic Cats, South Korea.

In December 2016, highly pathogenic avian influenza (HPAI) infection with systemic pathologic lesions was found in cats in South Korea. Genetic analyses indicated that the feline isolates were similar to HPAI H5N6 viruses isolated in chicken farms nearby. This finding highlights the need for monitoring of domestic mammals during HPAI outbreaks.

Association of TRPV1 and TLR4 through the TIR domain potentiates TRPV1 activity by blocking activation-induced desensitization.

BACKGROUND: We have previously reported that histamine-induced pruritus was attenuated in toll-like receptor 4 (TLR4) knockout mice due to decreased transient receptor potential V1 (TRPV1) sensitivity. Our results implied that TLR4 potentiated TRPV1 activation in sensory neurons; however, the molecular mechanism has yet to be elucidated. In this study, we investigated the molecular mechanisms of TLR4-mediated TRPV1 potentiation using TLR4-deficient sensory neurons and a heterologous expression system. METHODS: Primary sensory neurons were obtained from wild-type or TLR4 knockout mice, and HEK293T cells expressing TRPV1 and TLR4 were prepared by transient transfection. TRPV1 activity was analyzed by calcium imaging, fluorophotometry, and patch-clamp recording. Subcellular protein distribution was tested by immunocytochemistry and cell surface biotinylation assay. Protein interaction was assessed by western blot and immunoprecipitation assay. RESULTS: Direct association between TRPV1 and TLR4 was detected in HEK293T cells upon heterologous TRPV1 and TLR4 expression. In an immunoprecipitation assay using TLR4-deletion mutants and soluble toll/interleukin-1 receptor (TIR) protein, the cytoplasmic TIR domain of TLR4 was required for TLR4-TRPV1 association and TRPV1 potentiation. In TLR4-deficient sensory neurons, the activation-induced desensitization of TRPV1 increased, accompanied by enhanced TRPV1 clearance from the cell membrane upon activation compared to wild-type neurons. In addition, heterologous TLR4 expression inhibited activation-induced TRPV1 endocytosis and lysosomal degradation in HEK293T cells. CONCLUSION: Our data show that direct association between TRPV1 and TLR4 through the TIR domain enhances TRPV1 activity by blocking activation-induced TRPV1 desensitization.

Toll-Like Receptor 3 Contributes to Wallerian Degeneration after Peripheral Nerve Injury.

OBJECTIVE: It is well known that Schwann cells play an important role in Wallerian degeneration after peripheral nerve injury. Previously, we reported that toll-like receptor 3 (TLR3) is expressed on Schwann cells, implicating its role in Schwann cell activation during Wallerian degeneration. In this study, we tested this possibility using TLR3 knock-out mice. METHODS: Sciatic nerve-crush injury was induced in wild-type and TLR3 knock-out mice. Histological sections of the sciatic nerve were analyzed for Wallerian degeneration on days 3 and 7 after injury. The level of macrophage infiltration was measured by real-time RT-PCR, flow cytometry and immunohistochemistry. The macrophage-recruiting chemokine gene expressions in the injured nerve were determined by real-time RT-PCR. RESULTS: In TLR3 knock-out mice, the nerve injury-induced axonal degeneration and subsequent axonal debris clearance were reduced compared to in wild-type mice. In addition, nerve injury-induced macrophage infiltration into injury sites was attenuated in TLR3 knock-out mice and was accompanied by reduced expression of macrophage-recruiting chemokines such as CC-chemokine ligands (CCL)2/MCP-1, CCL4/MIP-1ß and CCL5/RANTES. These macrophage-recruiting chemokines were induced in primary Schwann cells upon TLR3 stimulation. Finally, intraneural injection of polyinosinic-polycytidylic acid, a synthetic TLR3 agonist, induced macrophage infiltration into the sciatic nerve in vivo. CONCLUSION: These data show that TLR3 signaling contributes to Wallerian degeneration after peripheral nerve injury by affecting Schwann cell activation and macrophage recruitment to injured nerves.

Brief Isolation Changes Nociceptive Behaviors and Compromises Drug Tests in Mice.

Herding with a litter is known to comfort rodents, whereas isolation and grouping with noncagemates provoke stress. The effects of stress induced by isolation and grouping with noncagemates on pain responses, and their underlying mechanisms remain elusive. We assessed the effect of isolation, a common condition during behavioral tests, and of grouping on defecation and pain behaviors of mice. Fecal pellets were counted 2 hours after exposure to the test chamber. It is significantly more in the isolated mice than in the grouped mice. Hindpaw withdrawal threshold and withdrawal latency were adopted as the indicatives of mechanical and thermal pain sensitivities, respectively. Interestingly, isolated mice showed higher pain thresholds than mice grouping with cagemates, and even those with noncagemates, indicating analgesic effects. Such effects were reduced by intrathecal injection of 0.01 mg/kg of naloxone (opioid receptor antagonist), atosiban (oxytocin and vasopressin receptor antagonist), and ketanserin (5-HT receptor antagonist). Intraperitoneal delivery of 1 mg/kg of naloxone and atosiban, but not ketanserin, also alleviated the isolation-induced analgesic effects. In contrast, these drugs at the same dose had no significant effect on the mice grouping with cagemates. In addition, the effect of morphine on thermal pain was more robust in the mice grouping with cagemates than in the isolated mice. These data demonstrated that brief isolation caused analgesia, mediated by endogenous opioidergic, oxytocinergic, and serotonergic pathways. These results indicate that isolation during pain behavioral tests can affect pain responses and the efficacy of drugs; thus, nociception tests should be conducted in grouping.

Pathologic Changes in Wild Birds Infected with Highly Pathogenic Avian Influenza A(H5N8) Viruses, South Korea, 2014.

In January 2014, an outbreak of infection with highly pathogenic avian influenza (HPAI) A(H5N8) virus began on a duck farm in South Korea and spread to other poultry farms nearby. During this outbreak, many sick or dead wild birds were found around habitats frequented by migratory birds. To determine the causes of death, we examined 771 wild bird carcasses and identified HPAI A(H5N8) virus in 167. Gross and histologic lesions were observed in pancreas, lung, brain, and kidney of Baikal teals, bean geese, and whooper swans but not mallard ducks. Such lesions are consistent with lethal HPAI A(H5N8) virus infection. However, some HPAI-positive birds had died of gunshot wounds, peritonitis, or agrochemical poisoning rather than virus infection. These findings suggest that susceptibility to HPAI A(H5N8) virus varies among species of migratory birds and that asymptomatic migratory birds could be carriers of this virus.