Probes for INS

In contrast to mice or zebrafish, trout exhibits post-larval muscle growth through hypertrophy and formation of new myofibers (hyperplasia). The muscle fibers are formed by the fusion of mononucleated cells (myoblasts) regulated by several muscle-specific proteins such as Myomaker or Myomixer. In this work, we identified a unique gene encoding a Myomixer protein of 77 amino acids (aa) in the trout genome. Sequence analysis and phylogenetic tree showed moderate conservation of the overall protein sequence across teleost fish (61% of aa identity between trout and zebrafish Myomixer sequences). Nevertheless, the functionally essential motif, AxLyCxL is perfectly conserved in all studied sequences of vertebrates. Using in situ hybridization, we observed that myomixer was highly expressed in the embryonic myotome, particularly in the hyperplasic area. Moreover, myomixer remained readily expressed in white muscle of juvenile (1 and 20 g) although its expression decreased in mature fish. We also showed that myomixer is up-regulated during muscle regeneration and in vitro myoblasts differentiation. Together, these data indicate that myomixer expression is consistently associated with the formation of new myofibers during somitogenesis, post-larval growth and muscle regeneration in trout.

Neuropathic pain is a major health problem that affects up to 7-10% of the population worldwide. Currently, neuropathic pain is difficult to treat due to its elusive mechanisms. Here we report that orphan G protein-coupled receptor 151 (GPR151) in nociceptive sensory neurons controls neuropathic pain induced by nerve injury. GPR151 was mainly expressed in nonpeptidergic C-fiber dorsal root ganglion (DRG) neurons and highly upregulated after nerve injury. Importantly, conditional knockout of Gpr151 in adult nociceptive sensory neurons significantly alleviated chronic constriction injury (CCI)-induced neuropathic pain-like behavior but did not affect basal nociception. Moreover, GPR151 in DRG neurons was required for CCI-induced neuronal hyperexcitability and upregulation of colony-stimulating factor 1 (CSF1), which is necessary for microglial activation in the spinal cord after nerve injury. Mechanistically, GPR151 coupled with P2X3 ion channels and promoted their functional activities in neuropathic pain-like hypersensitivity. Knockout of Gpr151 suppressed P2X3-mediated calcium elevation and spontaneous pain behavior in CCI mice. Conversely, overexpression of Gpr151 significantly enhanced P2X3-mediated calcium elevation and DRG neuronal excitability. Furthermore, knockdown of P2X3 in DRGs reversed CCI-induced CSF1 upregulation, spinal microglial activation, and neuropathic pain-like behavior. Finally, the co-expression of GPR151 and P2X3 was confirmed in small-diameter human DRG neurons, indicating the clinical relevance of our findings. Together, our results suggest that GPR151 in nociceptive DRG neurons plays a key role in the pathogenesis of neuropathic pain and could be a potential target for treating neuropathic pain.

The navigation cue netrin-1 is well-documented for its key role in cancer development and represents a promising therapeutic target currently under clinical investigation. Phase 1 and 2 clinical trials are ongoing with NP137, a humanized monoclonal antibody against netrin-1. Interestingly, the epitope recognized by NP137 in netrin-1 shares 90% homology with its counterpart in netrin-3, the closest member to netrin-1 in humans, for which little is known in the field of cancer. Here, we unveiled that netrin-3 appears to be expressed specifically in human neuroblastoma (NB) and small cell lung cancer (SCLC), two subtypes of neuroectodermal/neuroendocrine lineages. Netrin-3 and netrin-1 expression are mutually exclusive, and the former is driven by the MYCN oncogene in NB, and the ASCL-1 or NeuroD1 transcription factors in SCLC. Netrin-3 expression is correlated with disease stage, aggressiveness, and overall survival in NB. Mechanistically, we confirmed the high affinity of netrin-3 for netrin-1 receptors and we demonstrated that netrin-3 genetic silencing or interference using NP137, delayed tumor engraftment, and reduced tumor growth in animal models. Altogether, these data support the targeting of netrin-3 in NB and SCLC.

Kynurenine 3-monooxygenase (KMO) regulates the levels of neuroactive metabolites in the kynurenine pathway (KP), dysregulation of which is associated with Huntington's disease (HD) pathogenesis. KMO inhibition leads to increased levels of neuroprotective relative to neurotoxic metabolites, and has been found to ameliorate disease-relevant phenotypes in several HD models. Here, we crossed KMO knockout mice to R6/2 HD mice to examine the effect of KMO depletion in the brain and periphery. KP genes were dysregulated in peripheral tissues from R6/2 mice and KMO ablation normalised levels of a subset of these. KP metabolites were also assessed, and KMO depletion led to increased levels of neuroprotective kynurenic acid in brain and periphery, and dramatically reduced neurotoxic 3-hydroxykunurenine levels in striatum and cortex. Notably, the increased levels of pro-inflammatory cytokines TNFa, IL1β, IL4 and IL6 found in R6/2 plasma were normalised upon KMO deletion. Despite these improvements in KP dysregulation and peripheral inflammation, KMO ablation had no effect upon several behavioural phenotypes. Therefore, although genetic inhibition of KMO in R6/2 mice modulates several metabolic and inflammatory parameters, these do not translate to improvements in primary disease indicators-observations which will likely be relevant for other interventions targeted at peripheral inflammation in HD.

Opioid dependence is accompanied by neuroplastic changes in reward circuitry leading to a negative affective state contributing to addictive behaviors and risk of relapse. The current study presents a neuroimmune mechanism through which chronic opioids disrupt the ventral tegmental area (VTA) dopaminergic circuitry that contributes to impaired reward behavior. Opioid dependence was induced in rodents by treatment with escalating doses of morphine. Microglial activation was observed in the VTA following spontaneous withdrawal from chronic morphine treatment. Opioid-induced microglial activation resulted in an increase in brain-derived neurotrophic factor (BDNF) expression and a reduction in the expression and function of the K+Cl- co-transporter KCC2 within VTA GABAergic neurons. Inhibition of microglial activation or interfering with BDNF signaling prevented the loss of Cl- extrusion capacity and restored the rewarding effects of cocaine in opioid-dependent animals. Consistent with a microglial-derived BDNF-induced disruption of reward, intra-VTA injection of BDNF or a KCC2 inhibitor resulted in a loss of cocaine-induced place preference in opioid-naïve animals. The loss of the extracellular Cl- gradient undermines GABAA-mediated inhibition, and represents a mechanism by which chronic opioid treatments can result in blunted reward circuitry. This study directly implicates microglial-derived BDNF as a negative regulator of reward in opioid-dependent states, identifying new therapeutic targets for opiate addictive behaviors.

Spatial mapping of heterogeneity in gene expression in cancer tissues can improve our understanding of cancers and help in the rapid detection of cancers with high accuracy and reliability. Significant advancements have been made in recent years in OMICS technologies, which possess the strong potential to be applied in the spatial mapping of biopsy tissue samples and their molecular profiling to a single-cell level. The clinical application of OMICS technologies in spatial profiling of cancer tissues is also advancing. The current review presents recent advancements and prospects of applying OMICS technologies to the spatial mapping of various analytes in cancer tissues. We benchmark the current state of the art in the field to advance existing OMICS technologies for high throughput spatial profiling. The factors taken into consideration include spatial resolution, types of biomolecules, numbers of different biomolecules detected from the same assay, labeled versus label-free approaches, and approximate time required for each assay. Further advancements are still needed for the widespread application of OMICs technologies in performing fast and high throughput spatial mapping of cancer tissues as well as their effective use in research and clinical applications.

Hypothalamic Kiss1 neurons control gonadotropin-releasing hormone release through the secretion of kisspeptin. Kiss1 neurons serve as a nodal center that conveys essential regulatory cues for the attainment and maintenance of reproductive function. Despite this critical role, the mechanisms that control kisspeptin synthesis and release remain largely unknown. Using Drop-Seq data from the arcuate nucleus of adult mice and in situ hybridization, we identified Nescient Helix-Loop-Helix 2 (Nhlh2), a transcription factor of the basic helix-loop-helix family, to be enriched in Kiss1 neurons. JASPAR analysis revealed several binding sites for NHLH2 in the Kiss1 and Tac2 (neurokinin B) 5' regulatory regions. In vitro luciferase assays evidenced a robust stimulatory action of NHLH2 on human KISS1 and TAC3 promoters. The recruitment of NHLH2 to the KISS1 and TAC3 promoters was further confirmed through chromatin immunoprecipitation. In vivo conditional ablation of Nhlh2 from Kiss1 neurons using Kiss1Cre:Nhlh2fl/fl mice induced a male-specific delay in puberty onset, in line with a decrease in arcuate Kiss1 expression. Females retained normal reproductive function albeit with irregular estrous cycles. Further analysis of male Kiss1Cre:Nhlh2fl/fl mice revealed higher susceptibility to metabolic challenges in the release of luteinizing hormone and impaired response to leptin. Overall, in Kiss1 neurons, Nhlh2 contributes to the metabolic regulation of kisspeptin and NKB synthesis and release, with implications for the timing of puberty onset and regulation of fertility in male mice.

Studies have found that circular RNAs (circRNAs) play key roles in cardiovascular diseases. However, the function of circROBO2 in acute myocardial infarction (AMI) is unclear. This study aimed to investigate the pathogenesis of circROBO2 in AMI. qRT-PCR and Western blot were used to determine the expression levels of circROBO2, miR-1184, and TRADD in AMI and sham-operated mouse models at mRNA and protein level, respectively. The relationship among miR-1184, circROBO2 and TRADD was evaluated by RNA immunoprecipitation (RIP) analysis and luciferase reporter gene analysis. The roles of circROBO2, miR-1184, and TRADD in myocardial cell apoptosis were evaluated using flow cytometry. Ultrasound echocardiography, serum creatine kinase MB (CK-MB) and lactate dehydrogenase (LDH), myocardial infarction area, and myocardial cell apoptosis were measured to examine the effects of circROBO2 on myocardial injury. The expression levels of miR-1184 were significantly reduced, and the expression levels of circROBO2 and TRADD were significantly increased in MI group. CircROBO2 acted as a sponge for miR-1184 by upregulating the expression of TRADD. In addition, overexpression of miR-1184 enhanced the protective effect of knockdown of circROBO2 by partially inhibiting the expression of TRADD in vivo and in vitro. Knockdown of circROBO2 reduced the apoptosis of cardiomyocytes by increasing the expression levels of miR-1184, which in turn decreased the expression levels of TRADD in the myocardium post-MI.

The Ezh2 gene encodes a histone methyltransferase of the Polycomb Repressive Complex 2 that methylates histone H3 lysine 27. In this work we asked whether EZH2 has a role in the development of the pharyngeal apparatus and whether it regulates the expression of the Tbx1 gene, which encodes a key transcription factor required in pharyngeal development. To these ends, we performed genetic in vivo experiments with mouse embryos and we used mouse embryonic stem cell (ESC)-based protocols to probe endoderm and cardiogenic mesoderm differentiation. Results showed that EZH2 occupies the Tbx1 gene locus in mouse embryos, and that suppression of EZH2 was associated with reduced expression of Tbx1 in differentiated mESCs. Conditional deletion of Ezh2 in the Tbx1 expression domain, which includes the pharyngeal endoderm, did not cause cardiac defects but revealed that the gene has an important role in the morphogenesis of the 3rd pharyngeal pouch (PP). We found that in conditionally deleted embryos the 3rd PP was hypoplastic, had reduced expression of Tbx1, lacked the expression of Gcm2, a gene that marks the parathyroid domain, but expressed FoxN1, a gene marking the thymic domain. Consistently, the parathyroids did not develop, and the thymus was hypoplastic. Thus, Ezh2 is required for parathyroid and thymic development, probably through a function in the pouch endoderm. This discovery also provides a novel interpretational key for the finding of Ezh2 activating mutations in hyperparathyroidism and parathyroid cancer.

Calcium-dependent secretion activator 2 (CAPS2) regulates the trafficking and exocytosis of neuropeptide-containing dense-core vesicles (DCVs). CAPS2 is prominently expressed in the medial habenula (MHb), which is related to depressive behavior; however, how MHb neurons cause depressive symptoms and the role of CAPS2 remains unclear. We hypothesized that dysfunction of MHb CAPS neurons might cause defects in neuropeptide secretion and the activity of monoaminergic centers, resulting in depressive-like behaviors. In this study, we examined (1) CAPS2 expression in the habenula of depression animal models and major depressive disorder patients and (2) the effects of down-regulation of MHb CAPS2 on the animal behaviors, synaptic transmission in the interpeduncular nucleus (IPN), and neuronal activity of monoamine centers. Habenular CAPS2 expression was decreased in the rat chronic restraint stress model, mouse learned helplessness model, and showed tendency to decrease in depression patients who died by suicide. Knockdown of CAPS2 in the mouse habenula evoked despair-like behavior and a reduction of the release of DCVs in the IPN. Neuronal activity of IPN and monoaminergic centers was also reduced. These results implicate MHb CAPS2 as playing a pivotal role in depressive behavior through the regulation of neuropeptide secretion of the MHb-IPN pathway and the activity of monoaminergic centers.

Most seven transmembrane receptors (7TMRs) are G protein-coupled receptors; however, some 7TMRs evoke intracellular signals through β-arrestin as a biased receptor. As several β-arrestin-biased agonists have been reported to be cardioprotective, we examined the role of the chemokine receptor CXCR7 as a β-arrestin-biased receptor in the heart. Among 510 7TMR genes examined, Cxcr7 was the most abundantly expressed in the murine heart. Single-cell RNA-sequencing analysis revealed that Cxcr7 was abundantly expressed in cardiomyocytes and fibroblasts. Cardiomyocyte-specific Cxcr7 null mice showed more prominent cardiac dilatation and dysfunction than control mice 4 weeks after myocardial infarction. In contrast, there was no difference in cardiac phenotypes between fibroblast-specific Cxcr7-knockout mice and control mice even after myocardial infarction. TC14012, a specific agonist of CXCR7, significantly recruited β-arrestin to CXCR7 in CXCR7-expressing cells and activated extracellular signal-regulated kinase (ERK) in neonatal rat cardiomyocytes. Cxcr7 expression was significantly increased and ERK was activated in the border zone of the heart in control, but not Cxcr7 null mice. These results indicate that the abundantly expressed CXCR7 in cardiomyocytes may play a protective role in the heart as a β-arrestin-biased receptor and that CXCR7 may be a novel therapeutic target for myocardial infarction.

Chemotherapy-induced neuropathy (CIPN) is a common complication of paclitaxel. CIPN affects the quality of life of cancer survivors and frequently leads to discontinuation of treatment. Paclitaxel affects neuronal microtubules and induces neuronal mitochondrial dysfunction. However, there is limited clinical information regarding paclitaxel's effects on monocytes. Preclinical studies suggest that paclitaxel-induced neuronal damage is driven by monocytes/macrophages. Therefore, we evaluated whether paclitaxel selectively induces mitochondrial dysfunction and a pro-inflammatory phenotype in human circulating monocytes. We conducted studies in human primary peripheral blood mononuclear cells (PBMCs) from cancer patients being treated with paclitaxel, and in vitro analysis in PBMC cells and monocytes, and THP-1 monocytes in the presence of paclitaxel (0.1, 1, 10 uM). We used flow cytometric markers to study mitochondrial reactive oxygen species (ROS) and mitochondrial membrane potential, namely MitoSox and DIOC6(3) respectively. We also measured mRNA levels of pro- and anti-inflammatory molecules using qRT-PCR. In vitro paclitaxel induced a depolarization state in mitochondria in THP-1, human primary monocytes, and primary human PBMCs, but it did not change MitoSox. Monocytes in PBMCs cells from patients treated with paclitaxel showed significative depolarization state in mitochondria when compared to cells from control patients. In THP-1 cells, paclitaxel enhanced mRNA levels of the pro-inflammatory cytokines IL-8 and TNF alpha. In human primary PBMCs, paclitaxel reduced the anti-inflammatory factors CD163 and IL-10, and enhanced the TNF alpha, COX-2 and MCP-1 mRNA levels. Our study provides evidence that paclitaxel can induce mitochondrial dysfunction in isolated human monocytes and in monocytes present in total PBMCs cells. The observed depolarizing changes are indicative of a pro-mitophagy state, which is in accordance with the paclitaxel-induced pro-inflammatory phenotype in these cells. Early detection of mitochondria dysfunction in human monocytes could be a predictable sign to CIPN development in cancer patients. Our research was supported by the Early-Career Investigator Award W81XWH-16-1-0438 of the Department of Defense, The Pershing Square Sohn Cancer Research Alliance, Weill Cornell Medicine Funds, Department of Anesthesiology-Wake Forest School of Medicine Funds, Comprehensive Cancer Center-Wake Forest School of Medicine Funds, NIDA R21CA248106, National Center for Advancing Translational Sciences (NCATS)-NIH through Grant Award Number UL1TR001420. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.