Concise Communication

Abstract

Beta-arrestin-1 and -2 (Barr1 and Barr2, respectively) are intracellular signaling molecules that regulate many important metabolic functions. We previously demonstrated that mice lacking Barr2 selectively in pancreatic beta-cells showed pronounced metabolic impairments. Here we investigated whether Barr1 plays a similar role in regulating beta-cell function and whole body glucose homeostasis. Initially, we inactivated the Barr1 gene in beta-cells of adult mice (beta-barr1-KO mice). Beta-barr1-KO mice did not display any obvious phenotypes in a series of in vivo and in vitro metabolic tests. However, glibenclamide and tolbutamide, two widely used antidiabetic drugs of the sulfonylurea (SU) family, showed greatly reduced efficacy in stimulating insulin secretion in the KO mice in vivo and in perifused KO islets in vitro. Additional in vivo and in vitro studies demonstrated that Barr1 enhanced SU-stimulated insulin secretion by promoting SU-mediated activation of Epac2. Pull-down and co-immunoprecipitation experiments showed that Barr1 can directly interact with Epac2 and that SUs such as glibenclamide promote Barr1/Epac2 complex formation, triggering enhanced Rap1 signaling and insulin secretion. These findings suggest that strategies aimed at promoting Barr1 signaling in beta-cells may prove useful for the development of efficacious antidiabetic drugs.

Authors

Luiz F. Barella, Mario Rossi, Lu Zhu, Yinghong Cui, Fang C. Mei, Xiaodong Cheng, Wei Chen, Vsevolod V. Gurevich, Jürgen Wess

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Abstract

Poroma is a benign skin tumor exhibiting terminal sweat gland duct differentiation. The present study aimed to explore the potential role of gene fusions in the tumorigenesis of poromas. RNA sequencing and reverse transcription PCR identified highly recurrent YAP1-MAML2 and YAP1-NUTM1 fusions in poromas (92/104 lesions, 88.5%) and their rare malignant counterpart, porocarcinomas (7/11 lesions, 63.6%). A WWTR1-NUTM1 fusion was identified in a single lesion of poroma. Fluorescent in-situ hybridization confirmed genomic rearrangements involving these genetic loci. Immunohistochemical staining could readily identify the YAP1 fusion products as nuclear expression of the N-terminal portion of YAP1 with a lack of the C-terminal portion. YAP1 and WWTR1, also known as YAP and TAZ, respectively, encode paralogous transcriptional activators of TEAD, which are negatively regulated by the Hippo signaling pathway. The YAP1 and WWTR1 fusions strongly transactivated a TEAD reporter and promoted anchorage-independent growth, confirming their tumorigenic roles. Our results demonstrate the frequent presence of transforming YAP1 fusions in poromas and porocarcinomas and suggest YAP1/TEAD-dependent transcription as a candidate therapeutic target against porocarcinoma.

Authors

Shigeki Sekine, Tohru Kiyono, Eijitsu Ryo, Reiko Ogawa, Susumu Wakai, Hitoshi Ichikawa, Koyu Suzuki, Satoru Arai, Koji Tsuta, Mitsuaki Ishida, Yuko Sasajima, Naoki Goshima, Naoya Yamazaki, Taisuke Mori

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Abstract

Highly effective direct-acting antivirals against Hepatitis C virus (HCV) have created an opportunity to transplant organs from HCV-positive individuals into HCV-negative recipients, since de novo infection can be routinely cured. As this procedure is performed more widely, it becomes increasingly important to understand the biological underpinnings of virus transmission, especially the multiplicity of infection. Here, we used single genome sequencing of plasma virus in four genotype 1a HCV-positive organ donors and their seven organ recipients to assess the genetic bottleneck associated with HCV transmission following renal and cardiac transplantation. In all recipients, de novo infection was established by multiple genetically distinct viruses that reflect the full phylogenetic spectrum of replication-competent virus circulating in donor plasma. This was true in renal and cardiac transplantation and in recipients with peak viral loads ranging between 2.9 and 6.6 log10 IU/mL. The permissive transmission process characterized here contrasts sharply with sexual or injection-related transmission, which occurs less frequently per exposure and is generally associated with a stringent genetic bottleneck. These findings highlight the effectiveness of current anti-HCV regimens, while raising caution regarding the substantially higher multiplicity of infection seen in organ transplantation-associated HCV acquisition.

Authors

Muhammad N. Zahid, Shuyi Wang, Gerald H. Learn, Peter L. Abt, Emily A. Blumberg, Peter P. Reese, David S. Goldberg, George M. Shaw, Katharine J. Bar

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Abstract

Ritonavir (RTV) is on the World Health Organization's List of Essential Medicines for antiretroviral therapy, but can cause hepatotoxicity by unknown mechanisms. Multiple clinical studies found that hepatotoxicity occurred in 100% of participants who were pretreated with rifampicin or efavirenz followed by RTV-containing regimens. Both rifampicin and efavirenz are activators of the pregnane X receptor (PXR), a transcription factor with significant inter-species differences in ligand-dependent activation. Using PXR-humanized mouse models, we recapitulated the RTV hepatotoxicity observed in the clinic. PXR was found to modulate RTV hepatotoxicity through CYP3A4-dependent pathways involved in RTV bioactivation, oxidative stress, and endoplasmic reticulum stress. In summary, the current work demonstrated the essential roles of human PXR and CYP3A4 in RTV hepatotoxicity, which can be applied to guide the safe use of RTV-containing regimens in the clinic.

Authors

Amina I. Shehu, Jie Lu, Pengcheng Wang, Junjie Zhu, Yue Wang, Da Yang, Deborah McMahon, Wen Xie, Frank J. Gonzalez, Xiaochao Ma

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Abstract

Transfer RNAs (tRNAs) are a major class of noncoding RNA. Stress-induced cleavage of tRNA is highly conserved and results in tRNA fragments. Here we find specific tRNA fragments in plasma are associated with epilepsy. Small RNA sequencing of plasma samples collected during video-EEG monitoring of focal epilepsy patients identified significant differences in three tRNA fragments (5′GlyGCC, 5′AlaTGC, and 5′GluCTC) from controls. Levels of these tRNA fragments were higher in pre-seizure than post-seizure samples, suggesting they may serve as biomarkers of seizure risk in epilepsy patients. In vitro studies confirmed that production and extracellular release of tRNA fragments was lower after epileptiform-like activity in hippocampal neurons. We designed PCR-based assays to quantify tRNA fragments in a cohort of pre- and post-seizure plasma samples from focal epilepsy patients and healthy controls. Receiver operating characteristic analysis indicated that tRNA fragments potently distinguished pre- from post-seizure patients. Elevated tRNA fragments levels were not detected in patients with psychogenic non-epileptic seizures, and did not result from medication tapering. This study identifies a novel class of epilepsy biomarker and reveals the potential existence of prodromal molecular patterns in blood that could be used to predict seizure risk.

Authors

Marion C. Hogg, Rana Raoof, Hany El Naggar, Naser Monsefi, Norman Delanty, Donncha F. O'Brien, Sebastian Bauer, Felix Rosenow, David C. Henshall, Jochen H.M. Prehn

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Abstract

Deep vein thrombosis (DVT), caused by alterations in venous homeostasis is the third most common cause of cardiovascular mortality; however, key molecular determinants in venous thrombosis have not been fully elucidated. Several lines of evidence indicate that DVT occurs at the intersection of dysregulated inflammation and coagulation. The enzyme ectonucleoside tri(di)phosphohydrolase (ENTPD1, also known as CD39) is a vascular ecto-apyrase on the surface of leukocytes and the endothelium that inhibits intravascular inflammation and thrombosis by hydrolysis of phosphodiester bonds from nucleotides released by activated cells. Here, we evaluated the contribution of CD39 to venous thrombosis in a restricted-flow model of murine inferior vena cava stenosis. CD39-deficiency conferred a >2-fold increase in venous thrombogenesis, characterized by increased leukocyte engagement, neutrophil extracellular trap formation, fibrin, and local activation of tissue factor in the thrombotic milieu. This was orchestrated by increased phosphorylation of the p65 subunit of NFκB, activation of the NLRP3 inflammasome, and interleukin-1β (IL-1β) release in CD39-deficient mice. Substantiating these findings, an IL-1β-neutralizing antibody attenuated the thrombosis risk in CD39-deficient mice. These data demonstrate that IL-1β is a key accelerant of venous thrombo-inflammation, which can be suppressed by CD39. CD39 inhibits in vivo crosstalk between inflammation and coagulation pathways, and is a critical vascular checkpoint in venous thrombosis.

Authors

Vinita Yadav, Liguo Chi, Raymond Zhao, Benjamin Tourdot, Srilakshmi Yalavarthi, Benjamin N. Jacobs, Alison Banka, Hui Liao, Sharon Koonse, Anuli C. Anyanwu, Scott Visovatti, Michael Holinstat, J. Michelle Kahlenberg, Jason S. Knight, David J. Pinsky, Yogendra Kanthi

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Abstract

Aside from its catalytic function in protein synthesis, leucyl-tRNA synthetase (LRS) has a nontranslational function in regulating cell growth via the mammalian target of rapamycin (mTOR) complex 1 (mTORC1) pathway by sensing amino acid availability. mTOR also regulates skeletal myogenesis, but the signaling mechanism is distinct from that in cell growth regulation. A role of LRS in myogenesis has not been reported. Here we report that LRS negatively regulated myoblast differentiation in vitro. This function of LRS was independent of its regulation of protein synthesis, and it required leucine-binding but not tRNA charging activity of LRS. Local knock down of LRS accelerated muscle regeneration in a mouse injury model, and so did the knock down of Rag or Raptor. Further in vitro studies established a Rag-mTORC1 pathway, which inhibits the IRS1-PI3K-Akt pathway, to be the mediator of the nontranslational function of LRS in myogenesis. BC-LI-0186, an inhibitor reported to disrupt LRS-Rag interaction, promoted robust muscle regeneration with enhanced functional recovery, and this effect was abolished by cotreatment with an Akt inhibitor. Taken together, our findings revealed what we believe is a novel function for LRS in controlling the homeostasis of myogenesis, and suggested a potential therapeutic strategy to target a noncanonical function of a housekeeping protein.

Authors

Kook Son, Jae-Sung You, Mee-Sup Yoon, Chong Dai, Jong Hyun Kim, Nidhi Khanna, Aditi Banerjee, Susan A. Martinis, Gyoonhee Han, Jung Min Han, Sunghoon Kim, Jie Chen

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Abstract

Opioid use disorder (OUD) is associated with the emergence of persistent negative emotional states during drug abstinence that drive compulsive drug taking and seeking. Functional magnetic resonance imaging (fMRI) in rats identified neurocircuits that were activated by stimuli that were previously paired with heroin withdrawal. The activation of amygdala and hypothalamic circuits was related to the degree of heroin dependence, supporting the significance of conditioned negative affect in sustaining compulsive-like heroin seeking and taking and providing neurobiological insights into the drivers of the current opioid crisis.

Authors

Stephanie A. Carmack, Robin J. Keeley, Janaina C.M. Vendruscolo, Emily G. Lowery-Gionta, Hanbing Lu, George F. Koob, Elliot A. Stein, Leandro F. Vendruscolo

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Abstract

Ischemic stroke is a predominant cause of disability worldwide, with thrombolytic or mechanical removal of the occlusion being the only therapeutic option. Reperfusion bears the risk of an acute deleterious calcium-dependent breakdown of the blood-brain barrier. Its mechanism, however, is unknown. Here, we identified type 5 NADPH oxidase (NOX5), a calcium-activated, ROS-forming enzyme, as the missing link. Using a humanized knockin (KI) mouse model and in vitro organotypic cultures, we found that reoxygenation or calcium overload increased brain ROS levels in a NOX5-dependent manner. In vivo, postischemic ROS formation, infarct volume, and functional outcomes were worsened in NOX5-KI mice. Of clinical and therapeutic relevance, in a human blood-barrier model, pharmacological NOX inhibition also prevented acute reoxygenation-induced leakage. Our data support further evaluation of poststroke recanalization in the presence of NOX inhibition for limiting stroke-induced damage.

Authors

Ana I. Casas, Pamela W.M. Kleikers, Eva Geuss, Friederike Langhauser, Thure Adler, Dirk H. Busch, Valerie Gailus-Durner, Martin Hrabê de Angelis, Javier Egea, Manuela G. Lopez, Christoph Kleinschnitz, Harald H.H.W. Schmidt

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Abstract

BRAF and CRAF are critical components of the MAPK signaling pathway which is activated in many cancer types. In approximately 1% of melanomas, BRAF or CRAF are activated through structural arrangements. We describe here a metastatic melanoma with a GOLGA4-RAF1 fusion and pathogenic variants in CTNNB1 and CDKN2A. Anti-CTLA4/anti-PD1 combination immunotherapy failed to control tumor progression. In the absence of other actionable variants the patient was administered MEK inhibitor therapy on the basis of its potential action against RAF1 fusions. This resulted in a profound and clinically significant response. We demonstrated that GOLGA4-RAF1 expression was associated with ERK activation, elevated expression of the RAS/RAF downstream co-effector ETV5, and a high Ki67 index. These findings provide a rationale for the dramatic response to targeted therapy. This study shows that thorough molecular characterization of treatment-resistant cancers can identify therapeutic targets and personalize management, leading to improved patient outcomes.

Authors

Christopher R. McEvoy, Huiling Xu, Kortnye Smith, Dariush Etemadmoghadam, Huei San Leong, David Y. Choong, David J. Byrne, Amir Iravani, Sophie Beck, Linda Mileshkin, Richard W. Tothill, David D. Bowtell, Bindi M. Bates, Violeta Nastevski, Judy Browning, Anthony H. Bell, Chloe Khoo, Jayesh Desai, Andrew P. Fellowes, Stephen B. Fox, Owen W.J. Prall

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