Non-coding RNAs derived from the foot-and-mouth disease virus genome trigger broad antiviral activity against coronaviruses
Miguel Rodríguez-Pulido, Eva Calvo-Pinilla, Miryam Polo, Juan-Carlos Saiz, Raúl Fernández-González, Eva Pericuesta, Alfonso Gutiérrez-Adán, Francisco Sobrino, Miguel A. Martín-Acebes and Margarita Sáiz
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of a potentially severe respiratory disease, the coronavirus disease 2019 (COVID-19), an ongoing pandemic with limited therapeutic options. Here, we assessed the anti-coronavirus activity of synthetic RNAs mimicking speciﬁc domains in the non-coding regions of the foot-and-mouth disease virus (FMDV) genome (ncRNAs). These molecules are known to exert broad-spectrum antiviral activity in cell culture, mice and pigs effectively triggering the host innate immune response. The ncRNAs showed potent antiviral activity against SARS-CoV-2 after transfection in human intestinal Caco-2 and lung epithelium Calu-3 2B4 cells. When the in vivo efﬁcacy of the FMDV ncRNAs was assessed in K18-hACE2 mice, administration of naked ncRNA before intranasal SARS-CoV-2 infection signiﬁcantly decreased the viral load and the levels of pro-inﬂammatory cytokines in the lungs compared with untreated infected mice. The ncRNAs were also highly efﬁcacious when assayed against common human HCoV-229E and porcine transmissible gastroenteritis virus (TGEV) in hepatocyte-derived Huh-7 and swine testis ST cells, respectively. These results are a proof of concept of the pan-coronavirus antiviral activity of the FMDV ncRNAs including human and animal divergent coronaviruses and potentially enhance our ability to ﬁght future emerging variants.
The enterohemorrhagic Escherichia coli insertion sequence-excision enhancer protein is a DNA polymerase with microhomology-mediated end-joining activity
Patricia A. Calvo, Víctor Mateo-Cáceres, Silvia Díaz-Arco, Modesto Redrejo-Rodríguez and Miguel de Vega
Bacterial genomes contain an abundance of trans-posable insertion sequence (IS) elements that are essential for genome evolution and ﬁtness. Among them, IS629 is present in most strains of enterohe-morrhagic Escherichia coli O157 and accounts for many polymorphisms associated with gene inacti-vation and/or genomic deletions. The excision of IS629 from the genome is promoted by IS-excision enhancer (IEE) protein. Despite IEE has been identi-ﬁed in the most pathogenic serotypes of E. coli,its biochemical features that could explain its role in IS excision are not yet understood. We show that IEE is present in >30% of all available E. coli genome as-semblies, and is highly conserved and very abundant within enterohemorrhagic, enteropathogenic and en-terotoxigenic genomes. In vitro analysis of the re-combinant protein from E. coli O157:H7 revealed the presence of a Mn2+-dependent error-prone DNA poly-merase activity in its N-terminal archaeo-eukaryotic primase (AEP) domain able to promote dislocations of the primer and template strands. Importantly, IEE could efﬁciently perform in vitro an end-joining reac-tion of 3’-single-strand DNA overhangs with ≥4bp of homology requiring both the N-terminal AEP and C-terminal helicase domains. The proposed role for IEE in the novel IS excision mechanism is discussed.
SOCS3 deregulation contributes to aberrant activation of the JAK/STAT pathway in precursor T-cell neoplasms
Antonio Lahera, Pilar López-Nieva, Hernán Alarcón, José L. Marín-Rubio, María Á. Cobos-Fernández, Pablo Fernández-Navarro, Agustín F. Fernández, Laura Vela-Martín, Isabel Sastre, Sara Ruiz-García, Pilar Llamas, José L. López-Lorenzo, Javier Cornago, Javier Santos, José Fernández-Piqueras, María Villa-Morales
Despite the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway being frequently altered in T-ALL/LBL, no specific therapy has been approved for T-ALL/LBL patients with constitutive signalling by JAK/STAT, so there is an urgent need to identify pathway members that may be potential therapeutic targets. In the present study, we searched for JAK/STAT pathway members potentially modulated through aberrant methylation and identified SOCS3 hypermethylation as a recurrent event in T-ALL/LBL. Additionally, we explored the implications of SOCS3 deregulation in T-ALL/LBL and demonstrated that SOCS3 counteracts the constitutive activation of the JAK/STAT pathway through different molecular mechanisms. Therefore, SOCS3 emerges as a potential therapeutic target in T-ALL/LBL.
A Dual Role for FADD in Human Precursor T-Cell Neoplasms
José Luis Marín-Rubio, Laura Vela-Martín, Jack Gudgeon, Eduardo Pérez-Gómez , Frances R. Sidgwick, Matthias Trost, Debbie L. Cunningham, Javier Santos, José Fernández-Piqueras and María Villa-Morales
A reduction in FADD levels has been reported in precursor T-cell neoplasms and other tumor types. Such reduction would impact on the ability of tumor cells to undergo apoptosis and has been associated with poor clinical outcomes. However, FADD is also known to participate in non-apoptotic functions, but these mechanisms are not well-understood. Linking FADD expression to the severity of precursor T-cell neoplasms could indicate its use as a prognostic marker and may open new avenues for targeted therapeutic strategies. Using transcriptomic and clinical data from patients with precursor T-cell neoplasms, complemented by in vitro analysis of cellular functions and by high-throughput interactomics, our results allow us to propose a dual role for FADD in precursor T-cell neoplasms, whereby resisting cell death and chemotherapy would be a canonical consequence of FADD deficiency in these tumors, whereas deregulation of the cellular metabolism would be a relevant non-canonical function in patients expressing FADD. These results reveal that evaluation of FADD expression in precursor T-cell neoplasms may aid in the understanding of the biological processes that are affected in the tumor cells. The altered biological processes can be of different natures depending on the availability of FADD influencing its ability to exert its canonical or non-canonical functions. Accordingly, specific therapeutic interventions would be needed in each case.
Replication gap suppression depends on the double-strand DNA binding activity of BRCA2
Domagoj Vugic, Isaac Dumoulin, Charlotte Martin, Anna Minello, Lucia Alvaro-Aranda, Jesus Gomez-Escudero, Rady Chaaban, Rana Lebdy, Catharina von Nicolai, Virginie Boucherit, Cyril Ribeyre, Angelos Constantinou, Aura Carreira
Replication stress (RS) is a major source of genomic instability and is intrinsic to cancer cells. RS is also the consequence of chemotherapeutic drugs for treating cancer. However, adaptation to RS is also a mechanism of resistance to chemotherapy. BRCA2 deficiency results in replication stress in human cells. BRCA2 protein's main functions include DNA repair by homologous recombination (HR) both at induced DNA double-strand breaks (DSB) and spontaneous replicative lesions. At stalled replication forks, BRCA2 protects the DNA from aberrant nucleolytic degradation and is thought to limit the appearance of ssDNA gaps by arresting replication and via post-replicative HR. However, whether and how BRCA2 acts to limit the formation of ssDNA gaps or mediate their repair, remains ill-defined. Here, we use breast cancer variants affecting different domains of BRCA2 to shed light on this function. We demonstrate that the N-terminal DNA binding domain (NTD), and specifically, its dsDNA binding activity, is required to prevent and repair/fill-in ssDNA gaps upon nucleotide depletion but not to limit PARPi-induced ssDNA gaps. Thus, these findings suggest that nucleotide depletion and PARPi trigger gaps via distinct mechanisms and that the NTD of BRCA2 prevents nucleotide depletion-induced ssDNA gaps.
Nat Commun. 2023 Jan 27;14(1):446. doi: 10.1038/s41467-023-36149-0.
Treatment with the senolytics dasatinib/quercetin reduces SARS-CoV-2-related mortality in mice
Andrés Pastor-Fernández, Antonio R. Bertos, Arantzazu Sierra-Ramírez, Javier del Moral-Salmoral, Javier Merino, Ana I. de Ávila, Cristina Olagüe, Ricardo Villares, Gloria González-Aseguinolaza, María Ángeles Rodríguez, Manuel Fresno, Nuria Gironés, Matilde Bustos, Cristian Smerdou, Pablo Jose Fernandez-Marcos, Cayetano von Kobbe
The enormous societal impact of the ongoing COVID-19 pandemic has been particularly harsh for some social groups, such as the elderly. Recently, it has been suggested that senescent cells could play a central role in pathogenesis by exacerbating the pro-inflammatory immune response against SARS-CoV-2. Therefore, the selective clearance of senescent cells by senolytic drugs may be useful as a therapy to ameliorate the symptoms of COVID-19 in some cases. Using the established COVID-19 murine model K18-hACE2, we demonstrated that a combination of the senolytics dasatinib and quercetin (D/Q) significantly reduced SARS-CoV-2-related mortality, delayed its onset, and reduced the number of other clinical symptoms. The increase in senescent markers that we detected in the lungs in response to SARS-CoV-2 may be related to the post-COVID-19 sequelae described to date. These results place senescent cells as central targets for the treatment of COVID-19, and make D/Q a new and promising therapeutic tool.
Acid digestion and symbiont: Proton sharing at the origin of mitochondriogenesis?
The initial relationships between organisms leading to endosymbiosis and the first eukaryote are currently a topic of hot debate. Here, I present a theory that offers a gradual scenario in which the origins of phagocytosis and mitochondria are intertwined in such a way that the evolution of one would not be possible without the other. In this scenario, the premitochondrial bacterial symbiont became initially associated with a protophagocytic host on the basis of cooperation to kill prey with symbiont-produced toxins and reactive oxygen species (ROS). Subsequently, the cooperation was focused on the digestion stage, through the acidification of the protophagocytic cavities via exportation of protons produced by the aerobic respiration of the symbiont. The host gained an improved phagocytic capacity and the symbiont received organic compounds from prey. As the host gradually lost its membrane energetics to develop lysosomal digestion, respiration was centralized in the premitochondrial symbiont for energy production for the consortium.
Autosomal Recessive Cerebellar Atrophy and Spastic Ataxia in Patients With Pathogenic Biallelic Variants in GEMIN5
Deepa S. Rajan, Sukhleen Kour, Tyler R. Fortuna, Margot A. Cousin, Sarah S. Barnett, Zhiyv Niu, Dusica Babovic-Vuksanovic, Eric W. Klee, Brian Kirmse, Micheil Innes, Siri Lynne Rydning, Kaja K. Selmer, Magnus Dehli Vigeland, Anne Kjersti Erichsen, Andrea H. Nemeth, Francisca Millan, Catherine DeVile, Katherine Fawcett, Adrien Legendre, David Sims, Ricardo Parolin Schnekenberg, Lydie Burglen, Sandra Mercier, Somayeh Bakhtiari, Rosario Francisco-Velilla, Azman Embarc-Buh,Encarnacion Martinez-Salas, Kristen Wigby, Jerica Lenberg, Jennifer R. Friedman, Michael C. Kruer, Udai Bhan Pandey
The hereditary ataxias are a heterogenous group of disorders with an increasing number of causative genes being described. Due to the clinical and genetic heterogeneity seen in these conditions, the majority of such individuals endure a diagnostic odyssey or remain undiagnosed. Defining the molecular etiology can bring insights into the responsible molecular pathways and eventually the identification of therapeutic targets. Here, we describe the identification of biallelic variants in the GEMIN5 gene among seven unrelated families with nine affected individuals presenting with spastic ataxia and cerebellar atrophy. GEMIN5, an RNA-binding protein, has been shown to regulate transcription and translation machinery. GEMIN5 is a component of small nuclear ribonucleoprotein (snRNP) complexes and helps in the assembly of the spliceosome complexes. We found that biallelic GEMIN5 variants cause structural abnormalities in the encoded protein and reduce expression of snRNP complex proteins in patient cells compared with unaffected controls. Finally, knocking out endogenous Gemin5 in mice caused early embryonic lethality, suggesting that Gemin5 expression is crucial for normal development. Our work further expands on the phenotypic spectrum associated with GEMIN5-related disease and implicates the role of GEMIN5 among patients with spastic ataxia, cerebellar atrophy, and motor predominant developmental delay.
Gemin5-dependent RNA association with polysomes enables selective translation of ribosomal and histone mRNAs
Azman Embarc-Buh, Rosario Francisco-Velilla, Juan Antonio Garcia-Martin, Salvador Abellan, Jorge Ramajo & Encarnacion Martinez-Salas
Selective translation allows to orchestrate the expression of specific proteins in response to different signals through the concerted action of cis-acting elements and RNA-binding proteins (RBPs). Gemin5 is a ubiquitous RBP involved in snRNP assembly. In addition, Gemin5 regulates translation of different mRNAs through apparently opposite mechanisms of action. Here, we investigated the differential function of Gemin5 in translation by identifying at a genome-wide scale the mRNAs associated with polysomes. Among the mRNAs showing Gemin5-dependent enrichment in polysomal fractions, we identified a selective enhancement of specific transcripts. Comparison of the targets previously identified by CLIP methodologies with the polysome-associated transcripts revealed that only a fraction of the targets was enriched in polysomes. Two different subsets of these mRNAs carry unique cis-acting regulatory elements, the 5’ terminal oligopyrimidine tracts (5’TOP) and the histone stem-loop (hSL) structure at the 3’ end, respectively, encoding ribosomal proteins and histones. RNA-immunoprecipitation (RIP) showed that ribosomal and histone mRNAs coprecipitate with Gemin5. Furthermore, disruption of the TOP motif impaired Gemin5-RNA interaction, and functional analysis showed that Gemin5 stimulates translation of mRNA reporters bearing an intact TOP motif. Likewise, Gemin5 enhanced hSL-dependent mRNA translation. Thus, Gemin5 promotes polysome association of only a subset of its targets, and as a consequence, it favors translation of the ribosomal and the histone mRNAs. Together, the results presented here unveil Gemin5 as a novel translation regulator of mRNA subsets encoding proteins involved in fundamental cellular processes.
Structural basis for Gemin5 decamer-mediated mRNA binding
Qiong Guo, Shidong Zhao, Rosario Francisco-Velilla, Jiahai Zhang, Azman Embarc-Buh, Salvador Abellan, Mengqi Lv, Peiping Tang, Qingguo Gong, Huaizong Shen, Linfeng Sun, Xuebiao Yao, Jinrong Min, Yunyu Shi, Encarnacion Martínez-Salas, Kaiming Zhang & Chao Xu
Gemin5 in the Survival Motor Neuron (SMN) complex serves as the RNA-binding protein to deliver small nuclear RNAs (snRNAs) to the small nuclear ribonucleoprotein Sm complex via its N-terminal WD40 domain. Additionally, the C-terminal region plays an important role in regulating RNA translation by directly binding to viral RNAs and cellular mRNAs. Here, we present the three-dimensional structure of the Gemin5 C-terminal region, which adopts a homodecamer architecture comprised of a dimer of pentamers. By structural analysis, mutagenesis, and RNA-binding assays, we find that the intact pentamer/decamer is critical for the Gemin5 C-terminal region to bind cognate RNA ligands and to regulate mRNA translation. The Gemin5 high-order architecture is assembled via pentamerization, allowing binding to RNA ligands in a coordinated manner. We propose a model depicting the regulatory role of Gemin5 in selective RNA binding and translation. Therefore, our work provides insights into the SMN complex-independent function of Gemin5.
Functional and structural deficiencies of Gemin5 variants associated with neurological disorders
Rosario Francisco-Velilla, Azman Embarc-Buh, Francisco del Caño-Ochoa, Salvador Abellan, Marçal Vilar, Sara Alvarez, Alberto Fernandez-Jaen, Sukhleen Kour, Deepa S Rajan, Udai Bhan Pandey, Santiago Ramón-Maiques, Encarnacion Martinez-Salas
Dysfunction of RNA-binding proteins is often linked to a wide range of human disease, particularly with neurological conditions. Gemin5 is a member of the survival of the motor neurons (SMN) complex, a ribosome-binding protein and a translation reprogramming factor. Recently, pathogenic mutations in Gemin5 have been reported, but the functional consequences of these variants remain elusive. Here, we report functional and structural deficiencies associated with compound heterozygosity variants within the Gemin5 gene found in patients with neurodevelopmental disorders. These clinical variants are located in key domains of Gemin5, the tetratricopeptide repeat (TPR)–like dimerization module and the noncanonical RNA-binding site 1 (RBS1). We show that the TPR-like variants disrupt protein dimerization, whereas the RBS1 variant confers protein instability. All mutants are defective in the interaction with protein networks involved in translation and RNA-driven pathways. Importantly, the TPR-like variants fail to associate with native ribosomes, hampering its involvement in translation control and establishing a functional difference with the wild-type protein. Our study provides insights into the molecular basis of disease associated with malfunction of the Gemin5 protein.
Histone H1 regulates non-coding RNA turnover on chromatin in a m6A-dependent manner
José Miguel Fernández-Justel, Cristina Santa-María, Sara Martín-Vírgala, Albert Jordan, Javier F. Cáceres, María Gómez
Linker histones are highly abundant chromatin-associated proteins with well-established structural roles in chromatin and as general transcriptional repressors. In addition, it has been long proposed that histone H1 exerts context-specific effects on gene expression. Here, we identify a function of histone H1 in chromatin structure and transcription using a range of genomic approaches. In the absence of histone H1, there is an increase in the transcription of non-coding RNAs, together with reduced levels of m6A modification leading to their accumulation on chromatin and causing replication-transcription conflicts. This strongly suggests that histone H1 prevents non-coding RNA transcription and regulates non-coding transcript turnover on chromatin. Accordingly, altering the m6A RNA methylation pathway rescues the replicative phenotype of H1 loss. This work unveils unexpected regulatory roles of histone H1 on non-coding RNA turnover and m6A deposition, highlighting the intimate relationship between chromatin conformation, RNA metabolism, and DNA replication to maintain genome performance.
Pluripotency factors regulate the onset of Hox cluster activation in the early embryo
Elena Lopez-Jimenez, Julio Sainz de Aja, Claudio Badia-Careaga, Antonio Barral, Isabel Rollan, Raquel Rouco, Elisa Santos, María Tiana, Jesus Victorino, Hector Sanchez-Iranzo, Rafael D Acemel, Carlos Torroja, Javier Adan, Eduardo Andres-Leon, Jose Luis Gomez-Skarmeta, Giovanna Giovinazzo, Fatima Sanchez-Cabo, Miguel Manzanares
Pluripotent cells are a transient population present in the early mammalian embryo dependent on transcription factors, such as OCT4 and NANOG, which maintain pluripotency while simultaneously suppressing lineage specification. Interestingly, these factors are not exclusive to uncommitted cells, but are also expressed during early phases of differentiation. However, their role in the transition from pluripotency to lineage specification is largely unknown. Using genetic models for controlled Oct4 or Nanog expression during postimplantation stages, we found that pluripotency factors play a dual role in regulating key lineage specifiers, initially repressing their expression and later being required for their proper activation. We show that the HoxB cluster is coordinately regulated in this way by OCT4 binding sites located at the 3’ end of the cluster. Our results show that core pluripotency factors are not limited to maintaining the pre-committed epiblast, but are also necessary for the proper deployment of subsequent developmental programs.
Small-molecule activation of OGG1 increases oxidative DNA damage repair by gaining a new function
Maurice Michel, Carlos Benítez-Buelga, Patricia A. Calvo, Bishoy M. F. Hanna, Oliver Mortusewicz, Geoffrey Masuyer, Jonathan Davies, Olov Wallner, Kumar Sanjiv, Julian J. Albers, Sergio Castañeda-Zegarra, Ann-Sofie Jemth, Torkild Visnes, Ana Sastre-Perona, Akhilesh N. Danda, Evert J. Homan, Karthick Marimuthu, Zhao Zhenjun, Celestine N. Chi, Antonio Sarno, Elisée Wiita, Catharina von Nicolai, Anna J. Komor, Varshni Rajagopal, Sarah Müller, Emily C. Hank, Marek Varga, Emma R. Scaletti, Monica Pandey, Stella Karsten, Hanne Haslene-Hox, Simon Loevenich, Petra Marttila, Azita Rasti, Kirill Mamonov, Florian Ortis, Fritz Schömberg, Olga Loseva, Josephine Stewart, Nicholas D’Arcy-Evans, Tobias Koolmeister, Martin Henriksson, Dana Michel, Ana de Ory, Lucia Acero, Oriol Calvete, Martin Scobie, Christian Hertweck, Ivan Vilotijevic, Christina Kalderén, Ana Osorio, Rosario Perona, Alexandra Stolz, Pål Stenmark, Ulrika Warpman Berglund, Miguel de Vega, Thomas Helleday
Oxidative DNA damage is recognized by 8-oxoguanine (8-oxoG) DNA glycosylase 1 (OGG1), which excises 8-oxoG, leaving a substrate for apurinic endonuclease 1 (APE1) and initiating repair. Here, we describe a small molecule (TH10785) that interacts with the phenylalanine-319 and glycine-42 amino acids of OGG1, increases the enzyme activity 10-fold, and generates a previously undescribed β,δ-lyase enzymatic function. TH10785 controls the catalytic activity mediated by a nitrogen base within its molecular structure. In cells, TH10785 increases OGG1 recruitment to and repair of oxidative DNA damage. This alters the repair process, which no longer requires APE1 but instead is dependent on polynucleotide kinase phosphatase (PNKP1) activity. The increased repair of oxidative DNA lesions with a small molecule may have therapeutic applications in various diseases and aging.
Abrogation of Notch Signaling in Embryonic TECs Impacts Postnatal mTEC Homeostasis and Thymic Involution
María Jesús García-León, Marta Mosquera, Carmela Cela, Juan Alcain, Saulius Zuklys, Georg Holländer and María L. Toribio
Notch signaling is crucial for fate specification and maturation of thymus-seeding progenitors along the T-cell lineage. Recent studies have extended the role of Notch signaling to thymic epithelial cells (TECs), showing that Notch regulates TEC progenitor maintenance and emergence of medullary TECs (mTECs) in fetal thymopoiesis. Based on immunohistochemistry studies of spatiotemporal regulation of Notch activation in the postnatal thymus, we show that in vivo Notch activation is not confined to fetal TECs. Rather, Notch signaling, likely mediated through the Notch1 receptor, is induced in postnatal cortical and medullary TECs, and increases significantly with age in the latter, in both humans and mice, suggesting a conserved role for Notch signaling in TEC homeostasis during thymus aging. To investigate the functional impact of Notch activation in postnatal TEC biology, we used a mouse model in which RPBJκ, the transcriptional effector of canonical Notch signaling, is deleted in epithelial cells, including TECs, under the control of the transcription factor Foxn1. Immunohistochemistry and flow cytometry analyses revealed no significant differences in TEC composition in mutant (RPBJκ-KOTEC) and wild-type (WT) littermate mice at early postnatal ages. However, a significant reduction of the medullary region was observed in mutant compared to WT older thymi, which was accompanied by an accelerated decrease of postnatal mTEC numbers. Also, we found that organization and integrity of the postnatal thymic medulla critically depends on activation of the canonical Notch signaling pathway, as abrogation of Notch signaling in TECs led to the disruption of the medullary thymic microenvironment and to an accelerated thymus atrophy. These features paralleled a significant increase in the proportion of intrathymic non-T lineage cells, mostly B cells, and a slight decrease of DP thymocyte numbers compatible with a compromised thymic function in mutant mice. Therefore, impaired Notch signaling induced in embryonic development impacts postnatal TECs and leads to an accelerated mTEC degeneration and a premature thymus involution. Collectively, our data have uncovered a new role for Notch1 signaling in the control of adult mTEC homeostasis, and point toward Notch signaling manipulation as a novel strategy for thymus regeneration and functional recovery from immunosenescence.
Analysis of the metabolic proteome of lung adenocarcinomas by reverse-phase protein arrays (RPPA) emphasizes mitochondria as targets for therapy
Laura Torresano, Fulvio Santacatterina, Sonia Domínguez-Zorita, Cristina Nuevo-Tapioles, Alfonso Núñez-Salgado, Pau B. Esparza-Moltó, Lucía González-Llorente, Inés Romero-Carramiñana, Cristina Núñez de Arenas, Brenda Sánchez-Garrido, Laura Nájera, Clara Salas, Mariano Provencio & José M. Cuezva
Lung cancer is the leading cause of cancer-related death worldwide despite the success of therapies targeting oncogenic drivers and immune-checkpoint inhibitors. Although metabolic enzymes offer additional targets for therapy, the precise metabolic proteome of lung adenocarcinomas is unknown, hampering its clinical translation. Herein, we used Reverse Phase Protein Arrays to quantify the changes in enzymes of glycolysis, oxidation of pyruvate, fatty acid metabolism, oxidative phosphorylation, antioxidant response and protein oxidative damage in 128 tumors and paired non-tumor adjacent tissue of lung adenocarcinomas to profile the proteome of metabolism. Steady-state levels of mitochondrial proteins of fatty acid oxidation, oxidative phosphorylation and of the antioxidant response are independent predictors of survival and/or of disease recurrence in lung adenocarcinoma patients. Next, we addressed the mechanisms by which the overexpression of ATPase Inhibitory Factor 1, the physiological inhibitor of oxidative phosphorylation, which is an independent predictor of disease recurrence, prevents metastatic disease. We highlight that IF1 overexpression promotes a more vulnerable and less invasive phenotype in lung adenocarcinoma cells. Finally, and as proof of concept, the therapeutic potential of targeting fatty acid assimilation or oxidation in combination with an inhibitor of oxidative phosphorylation was studied in mice bearing lung adenocarcinomas. The results revealed that this therapeutic approach significantly extended the lifespan and provided better welfare to mice than cisplatin treatments, supporting mitochondrial activities as targets of therapy in lung adenocarcinoma patients.
Novel RNase H inhibitors blocking RNA-directed strand displacement DNA synthesis by HIV-1 reverse transcriptase
Samara Martín-Alonso, Dongwei Kang, Javier Martínez del Río, Joanna Luczkowiak, Estrella Frutos-Beltrán, Lina Zhang, Xiqiang Cheng, Xinyong Liu, Peng Zhan, Luis Menéndez-Arias
In retroviruses, strand displacement DNA-dependent DNA polymerization catalyzed by the viral reverse transcriptase (RT) is required to synthesize double-stranded proviral DNA. In addition, strand displacement during RNA-dependent DNA synthesis is critical to generate high-quality cDNA for use in molecular biology and biotechnology. In this work, we show that the loss of RNase H activity due to inactivating mutations in HIV-1 RT (e.g. D443N or E478Q) has no significant effect on strand displacement while copying DNA templates, but has a large impact on DNA polymerization in reactions carried out with RNA templates. Similar effects were observed with β-thujaplicinol and other RNase H active site inhibitors, including compounds with dual activity (i.e., characterized also as inhibitors of HIV-1 integrase and/or the RT DNA polymerase). Among them, dual inhibitors of HIV-1 RT DNA polymerase/RNase H activities, containing a 7-hydroxy-6-nitro-2H-chromen-2-one pharmacophore were found to be very potent and effective strand displacement inhibitors in RNA-dependent DNA polymerization reactions. These findings might be helpful in the development of transcriptomics technologies to obtain more uniform read coverages when copying long RNAs and for the construction of more representative libraries avoiding biases towards 5’ and 3’ ends, while providing valuable information for the development of novel antiretroviral agents.
Overexpression of wild type RRAS2, without oncogenic mutations, drives chronic lymphocytic leukemia
Alejandro M. Hortal, Clara L. Oeste, Claudia Cifuentes, Miguel Alcoceba, Isabel Fernández-Pisonero, Laura Clavaín, Rut Tercero, Pilar Mendoza, Verónica Domínguez, Marta García-Flores, Belén Pintado, David Abia, Carmen García-Macías, Almudena Navarro-Bailón, Xosé R. Bustelo, Marcos González & Balbino Alarcón
Chronic lymphocytic leukemia (CLL) is the most frequent, and still incurable, form of leukemia in the Western World. It is widely accepted that cancer results from an evolutionary process shaped by the acquisition of driver mutations which confer selective growth advantage to cells that harbor them. Clear examples are missense mutations in classic RAS genes (KRAS, HRAS and NRAS) that underlie the development of approximately 13% of human cancers. Although autonomous B cell antigen receptor (BCR) signaling is involved and mutations in many tumor suppressor genes and oncogenes have been identified, an oncogenic driver gene has not still been identified for CLL.
p38 activation occurs mainly in microglia in the P301S Tauopathy mouse model
Juan R. Perea, Esther García, Laura Vallés-Saiz, Raquel Cuadros, Félix Hernández, Marta Bolós & Jesús Avila
Tauopathies are a group of neurodegenerative diseases characterized by the accumulation of hyperphosphorylated tau protein in the brain. Many of these pathologies also present an inflammatory component determined by the activation of microglia, the resident immune cells of the brain. p38 MAPK is one of the molecular pathways involved in neuroinflammation. Although this kinase is expressed mainly in glia, its activation in certain neurodegenerative diseases such as Alzheimer's Disease has been associated with its ability to phosphorylate tau in neurons. Using the P301S Tauopathy mouse model, here we show that p38 activation increases during aging and that this occurs mainly in microglia of the hippocampus rather than in neurons. Furthermore, we have observed that these mice present an activated microglial variant called rod microglia. Interestingly, p38 activation in this subpopulation of microglia is decreased. On the basis of our findings, we propose that rod microglia might have a neuroprotective phenotype in the context of tau pathology.
p38 Inhibition Decreases Tau Toxicity in Microglia and Improves Their Phagocytic Function
Juan R. Perea, Marta Bolós, Raquel Cuadros, Esther García, Vega García-Escudero, Félix Hernández, Róisín M. McManus, Michael T. Heneka & Jesús Avila
Alzheimer’s disease (AD) and other tauopathies are histopathologically characterized by tau aggregation, along with a chronic inflammatory response driven by microglia. Over the past few years, the role of microglia in AD has been studied mainly in relation to amyloid-β (Aβ) pathology. Consequently, there is a substantial knowledge gap concerning the molecular mechanisms involved in tau-mediated toxicity and neuroinflammation, thus hindering the development of therapeutic strategies. We previously demonstrated that extracellular soluble tau triggers p38 MAPK activation in microglia. Given the activation of this signaling pathway in AD and its involvement in neuroinflammation processes, here we evaluated the effect of p38 inhibition on primary microglia cultures subjected to tau treatment. Our data showed that the toxic effect driven by tau in microglia was diminished through p38 inhibition. Furthermore, p38 blockade enhanced microglia-mediated tau phagocytosis, as reflected by an increase in the number of lysosomes. In conclusion, these results contribute to our understanding of the functions of p38 in the central nervous system (CNS) beyond tau phosphorylation in neurons and provide further insights into the potential of p38 inhibition as a therapeutic strategy to halt neuroinflammation in tauopathies.
Endocytosis mediated by an atypical CUBAM complex modulates slit diaphragm dynamics in nephrocytes
Alexandra Atienza-Manuel, Vicente Castillo-Mancho, Stefano De Renzis, Joaquim Culi, Mar Ruiz-Gómez
The vertebrate endocytic receptor CUBAM, consisting of three cubilin monomers complexed with a single amnionless molecule, plays a major role in protein reabsorption in the renal proximal tubule. Here, we show that Drosophila CUBAM is a tripartite complex composed of dAmnionless and two cubilin paralogues Cubilin and Cubilin-2, and that it is required for nephrocyte slit diaphragm (SD) dynamics. Loss of CUBAM-mediated endocytosis induces dramatic morphological changes in nephrocytes and promotes enlarged ingressions of the external membrane and SD mislocalisation. These phenotypes result in part from an imbalance between endocytosis, strongly impaired in CUBAM mutants, and exocytosis in these highly active cells. Noteworthy, rescuing receptor-mediated endocytosis by Megalin/LRP2 or Rab5 expression only partially restores SD-positioning in CUBAM mutants, suggesting a specific requirement of CUBAM in SD degradation and/or recycling. This finding and the reported expression of CUBAM in podocytes argue for a possible unexpected conserved role of this endocytic receptor in vertebrate SD remodelling.
Coordination between cell proliferation and apoptosis after DNA damage in Drosophila
Mireya Ruiz-Losada, Raul González, Ana Peropadre, Alejandro Gil-Gálvez, Juan J. Tena, Antonio Baonza & Carlos Estella
Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These “life” or “death” cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.
Update and latest advances in antiretroviral therapy
Luis Menéndez-Arias and Rafael Delgado
Since the first cases of AIDS appeared in 1981, human immunodeficiency virus type 1 (HIV-1) infection has reached pandemic proportions. Forty years later, research has led to the approval of more than 30 antiretroviral drugs, while combination therapies have turned HIV-1 infection into a chronic, but manageable disease. Still, drug toxicity and acquired and transmitted drug resistance remain as major threats to therapy success. In this review, we provide an overview on currently available anti-HIV drugs and the latest developments in antiretroviral therapy, focused on new antiretroviral agents acting on known and unexploited antiviral targets, prevention therapies aimed to improve available drug combinations, and research on new long-acting therapies, particularly those involving novel drug candidates such as lenacapavir or islatravir.
Mosmo Is Required for Zebrafish Craniofacial Formation
Carlos Camacho-Macorra, Marcos Sintes, Noemí Tabanera, Irene Grasa, Paola Bovolenta and Marcos J. Cardozo
Hedgehog (Hh) signaling is a highly regulated molecular pathway implicated in many developmental and homeostatic events. Mutations in genes encoding primary components or regulators of the pathway cause an array of congenital malformations or postnatal pathologies, the extent of which is not yet fully defined. Mosmo (Modulator of Smoothened) is a modulator of the Hh pathway, which encodes a membrane tetraspan protein. Studies in cell lines have shown that Mosmo promotes the internalization and degradation of the Hh signaling transducer Smoothened (Smo), thereby down-modulating pathway activation. Whether this modulation is essential for vertebrate embryonic development remains poorly explored. Here, we have addressed this question and show that in zebrafish embryos, the two mosmo paralogs, mosmoa and mosmob, are expressed in the head mesenchyme and along the entire ventral neural tube. At the cellular level, Mosmoa localizes at the plasma membrane, cytoplasmic vesicles and primary cilium in both zebrafish and chick embryos. CRISPR/Cas9 mediated inactivation of both mosmoa and mosmob in zebrafish causes frontonasal hypoplasia and craniofacial skeleton defects, which become evident in the adult fish. We thus suggest that MOSMO is a candidate to explain uncharacterized forms of human congenital craniofacial malformations, such as those present in the 16p12.1 chromosomal deletion syndrome encompassing the MOSMO locus.
Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis
Tania Moreno-Mármol, Mario Ledesma-Terrón, Noemi Tabanera, Maria Jesús Martin-Bermejo, Marcos J Cardozo, Florencia Cavodeassi, Paola Bovolenta
The vertebrate eye primordium consists of a pseudostratified neuroepithelium, the optic vesicle (OV), in which cells acquire neural retina or retinal pigment epithelium (RPE) fates. As these fates arise, the OV assumes a cup shape, influenced by mechanical forces generated within the neural retina. Whether the RPE passively adapts to retinal changes or actively contributes to OV morphogenesis remains unexplored. We generated a zebrafish Tg(E1-bhlhe40:GFP) line to track RPE morphogenesis and interrogate its participation in OV folding. We show that, in virtual absence of proliferation, RPE cells stretch and flatten, thereby matching the retinal curvature and promoting OV folding. Localized interference with the RPE cytoskeleton disrupts tissue stretching and OV folding. Thus, extreme RPE flattening and accelerated differentiation are efficient solutions adopted by fast-developing species to enable timely optic cup formation. This mechanism differs in amniotes, in which proliferation drives RPE expansion with a much-reduced need of cell flattening.
The presynaptic glycine transporter GlyT2 is regulated by the Hedgehog pathway in vitro and in vivo
Andrés de la Rocha-Muñoz, Enrique Núñez, Anjali Amrapali Vishwanath, Sergio Gómez-López, Dhanasak Dhanasobhon, Nelson Rebola, Beatriz López-Corcuera, Jaime de Juan-Sanz & Carmen Aragón
The identity of a glycinergic synapse is maintained presynaptically by the activity of a surface glycine transporter, GlyT2, which recaptures glycine back to presynaptic terminals to preserve vesicular glycine content. GlyT2 loss-of-function mutations cause Hyperekplexia, a rare neurological disease in which loss of glycinergic neurotransmission causes generalized stiffness and strong motor alterations. However, the molecular underpinnings controlling GlyT2 activity remain poorly understood. In this work, we identify the Hedgehog pathway as a robust controller of GlyT2 expression and transport activity. Modulating the activation state of the Hedgehog pathway in vitro in rodent primary spinal cord neurons or in vivo in zebrafish embryos induced a selective control in GlyT2 expression, regulating GlyT2 transport activity. Our results indicate that activation of Hedgehog reduces GlyT2 expression by increasing its ubiquitination and degradation. This work describes a new molecular link between the Hedgehog signaling pathway and presynaptic glycine availability.
Gαq activation modulates autophagy by promoting mTORC1 signaling
Sofía Cabezudo, Maria Sanz-Flores, Alvaro Caballero, Inmaculada Tasset, Elena Rebollo, Antonio Diaz, Anna M. Aragay, Ana María Cuervo, Federico Mayor Jr & Catalina Ribas
The mTORC1 node plays a major role in autophagy modulation. We report a role of the ubiquitous Gαq subunit, a known transducer of plasma membrane G protein-coupled receptors signaling, as a core modulator of mTORC1 and autophagy. Cells lacking Gαq/11 display higher basal autophagy, enhanced autophagy induction upon different types of nutrient stress along with a decreased mTORC1 activation status. They are also unable to reactivate mTORC1 and thus inactivate ongoing autophagy upon nutrient recovery. Conversely, stimulation of Gαq/11 promotes sustained mTORC1 pathway activation and reversion of autophagy promoted by serum or amino acids removal. Gαq is present in autophagic compartments and lysosomes and is part of the mTORC1 multi-molecular complex, contributing to its assembly and activation via its nutrient status-sensitive interaction with p62, which displays features of a Gαq effector. Gαq emerges as a central regulator of the autophagy machinery required to maintain cellular homeostasis upon nutrient fluctuations.
Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543
Cristina Benito-Muñoz, Almudena Perona, Raquel Felipe, Gonzalo Pérez-Siles, Enrique Núñez, Carmen Aragón, and Beatriz López-Corcuera
The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.
Rescue of two trafficking-defective variants of the neuronal glycine transporter GlyT2 associated to hyperekplexia
Andrés de la Rocha-Muñoz, Elena Melgarejo, Carmen Aragón, Beatriz López-Corcuera
Hyperekplexia is a rare sensorimotor syndrome characterized by pathological startle reflex in response to unexpected trivial stimuli for which there is no specific treatment. Neonates suffer from hypertonia and are at high risk of sudden death due to apnea episodes. Mutations in the human SLC6A5 gene encoding the neuronal glycine transporter GlyT2 may disrupt the inhibitory glycinergic neurotransmission and cause a presynaptic form of the disease. The phenotype of missense mutations giving rise to protein misfolding but maintaining residual activity could be rescued by facilitating folding or intracellular trafficking. In this report, we characterized the trafficking properties of two mutants associated with hyperekplexia (A277T and Y707C, rat numbering). Transporter molecules were partially retained in the endoplasmic reticulum showing increased interaction with the endoplasmic reticulum chaperone calnexin. One transporter variant had export difficulties and increased ubiquitination levels, suggestive of enhanced endoplasmic reticulum-associated degradation. However, the two mutant transporters were amenable to correction by calnexin overexpression. Within the search for compounds capable of rescuing mutant phenotypes, we found that the arachidonic acid derivative N-arachidonoyl glycine can rescue the trafficking defects of the two variants in heterologous cells and rat brain cortical neurons. N-arachidonoyl glycine improves the endoplasmic reticulum output by reducing the interaction transporter/calnexin, increasing membrane expression and improving transport activity in a comparable way as the well-established chemical chaperone 4-phenyl-butyrate. This work identifies N-arachidonoyl glycine as a promising compound with potential for hyperekplexia therapy.
Reconstitution of GABA, Glycine and Glutamate Transporters
Niels Christian Danbolt, Beatriz López‑Corcuera, Yun Zhou
In contrast to water soluble enzymes which can be purified and studied while in solution, studies of solute carrier (transporter) proteins require both that the protein of interest is situated in a phospholipid membrane and that this membrane forms a closed compartment. An additional challenge to the study of transporter proteins has been that the transport depends on the transmembrane electrochemical gradients. Baruch I. Kanner understood this early on and first developed techniques for studying plasma membrane vesicles. This advanced the field in that the experimenter could control the electrochemical gradients. Kanner, however, did not stop there, but started to solubilize the membranes so that the transporter proteins were taken out of their natural environment. In order to study them, Kanner then had to find a way to reconstitute them (reinsert them into phospholipid membranes). The scope of the present review is both to describe the reconstitution method in full detail as that has never been done, and also to reveal the scientific impact that this method has had. Kanner’s later work is not reviewed here although that also deserves a review because it too has had a huge impact.
Calcium‑Dependent Regulation of the Neuronal Glycine Transporter GlyT2 by M2 Muscarinic Acetylcholine Receptors
Esperanza Jiménez, Amparo Fornés, Raquel Felipe, Enrique Núñez, Carmen Aragón, Beatriz López‑Corcuera
The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission and plays a key role in regulating nociceptive signal progression. The cholinergic system acting through muscarinic acetylcholine receptors (mAChRs) also mediates important regulations of nociceptive transmission being the M2 subtype the most abundantly expressed in the spinal cord. Here we studied the effect of M2 mAChRs stimulation on GlyT2 function co-expressed in a heterologous system with negligible levels of muscarinic receptor activity. We found GlyT2 is down-regulated by carbachol in a calcium-dependent manner. Different components involved in cell calcium homeostasis were analysed to establish a role in the mechanism of GlyT2 inhibition. GlyT2 down-regulation by carbachol was increased by thapsigargin and reduced by internal store depletion, although calcium release from endoplasmic reticulum or mitochondria had a minor role on GlyT2 inhibition. Our results are consistent with a GlyT2 sensitivity to intracellular calcium mobilized by M2 mAChRs in the subcortical area of the plasma membrane. A crucial role of the plasma membrane sodium calcium exchanger NCX is proposed.
Coordinating DNA Replication and Mitosis through Ubiquitin/SUMO and CDK1
Antonio Galarreta, Pablo Valledor, Oscar Fernandez-Capetillo and Emilio Lecona
Post-translational modification of the DNA replication machinery by ubiquitin and SUMOPost-translational modification of the DNA replication machinery by ubiquitin and SUMOplays key roles in the faithful duplication of the genetic information. Among other functions, ubiquitinationand SUMOylation serve as signals for the extraction of factors from chromatin by the AAAATPase VCP. In addition to the regulation of DNA replication initiation and elongation, we now knowthat ubiquitination mediates the disassembly of the replisome after DNA replication termination, aprocess that is essential to preserve genomic stability. Here, we review the recent evidence showinghow active DNA replication restricts replisome ubiquitination to prevent the premature disassemblyof the DNA replication machinery. Ubiquitination also mediates the removal of the replisome toallow DNA repair. Further, we discuss the interplay between ubiquitin-mediated replisome disassemblyand the activation of CDK1 that is required to set up the transition from the S phase tomitosis. We propose the existence of a ubiquitin–CDK1 relay, where the disassembly of terminatedreplisomes increases CDK1 activity that, in turn, favors the ubiquitination and disassembly of morereplisomes. This model has important implications for the mechanism of action of cancer therapiesthat induce the untimely activation of CDK1, thereby triggering premature replisome disassemblyand DNA damage.
Stretching of the retinal pigment epithelium contributes to zebrafish optic cup morphogenesis
Tania Moreno-Mármol, Mario Ledesma-Terrón, Noemi Tabanera, Maria Jesús Martin-Bermejo, Marcos J Cardozo, Florencia Cavodeassi, Paola Bovolenta
The vertebrate eye-primordium consists of a pseudostratified neuroepithelium, the optic vesicle (OV), in which cells acquire neural retina or retinal pigment epithelium (RPE) fates. As these fates arise, the OV assumes a cup-shape, influenced by mechanical forces generated within the neural retina. Whether the RPE passively adapts to retinal changes or actively contributes to OV morphogenesis remains unexplored. We generated a zebrafish Tg(E1-bhlhe40:GFP) line to track RPE morphogenesis and interrogate its participation in OV folding. We show that, in virtual absence of proliferation, RPE cells stretch and flatten, thereby matching the retinal curvature and promoting OV folding. Localized interference with the RPE cytoskeleton disrupts tissue stretching and OV folding. Thus, extreme RPE flattening and accelerated differentiation are efficient solutions adopted by fast-developing species to enable timely optic cup formation. This mechanism differs in amniotes, in which proliferation drives RPE expansion with a much-reduced need of cell flattening.
SFRP1 modulates astrocyte-to-microglia crosstalk in acute and chronic neuroinflammation
Javier Rueda-Carrasco, María Jesús Martin-Bermejo, Guadalupe Pereyra, María Inés Mateo, Aldo Borroto, Frederic Brosseron, Markus P Kummer, Stephanie Schwartz, José P López-Atalaya, Balbino Alarcon, Pilar Esteve, Michael T Heneka, Paola Bovolenta
Neuroinflammation is a common feature of many neurodegenerative diseases. It fosters a dysfunctional neuron-microglia-astrocyte crosstalk that, in turn, maintains microglial cells in a perniciously reactive state that often enhances neuronal damage. The molecular components that mediate this critical communication are not fully explored. Here, we show that secreted frizzled-related protein 1 (SFRP1), a multifunctional regulator of cell-to-cell communication, is part of the cellular crosstalk underlying neuroinflammation. In mouse models of acute and chronic neuroinflammation, SFRP1, largely astrocyte-derived, promotes and sustains microglial activation, and thus a chronic inflammatory state. SFRP1 promotes the upregulation of components of the hypoxia-induced factor-dependent inflammatory pathway and, to a lower extent, of those downstream of the nuclear factor-kappa B. We thus propose that SFRP1 acts as an astrocyte-to-microglia amplifier of neuroinflammation, representing a potential valuable therapeutic target for counteracting the harmful effect of chronic inflammation in several neurodegenerative diseases.
Viral targeting of glioblastoma stem cells with patient-specific genetic and post-translational p53 deregulations
Jon Gil-Ranedo, Carlos Gallego-García, Jose M. Almendral
Oncolytic viruses are being developed for cancer therapy, but their biosafety and specificity are major concerns. GilRanedo et al. describe that human glioblastoma stem cells (GSCs) with patient-specific p53 mutants and p53- Ser15 phosphorylation are selective targets for two strains of the mouse parvovirus minute virus of mice (MVM) that are non-pathogenic for humans. The MVM strains disrupt GSC-derived brain tumors in orthotopic rodent models, showing promise for biosafe personalized therapy against human cancers.
GRK2 regulates GLP-1R-mediated early phase insulin secretion in vivo
Alba C. Arcones, Rocío Vila-Bedmar, Mercedes Mirasierra, Marta Cruces-Sande, Mario Vallejo, Ben Jones, Alejandra Tomas, Federico Mayor Jr & Cristina Murga
Insulin secretion from the pancreatic β-cell is finely modulated by different signals to allow an adequate control of glucose homeostasis. Incretin hormones such as glucagon-like peptide-1 (GLP-1) act as key physiological potentiators of insulin release through binding to the G protein-coupled receptor GLP-1R. Another key regulator of insulin signaling is the Ser/Thr kinase G protein-coupled receptor kinase 2 (GRK2). However, whether GRK2 affects insulin secretion or if GRK2 can control incretin actions in vivo remains to be analyzed.
Analysis of gene network bifurcation during optic cup morphogenesis in zebraﬁsh
Lorena Buono, Jorge Corbacho, Silvia Naranjo, María Almuedo-Castillo,Tania Moreno-Marmol, Berta de la Cerda, Estefanía Sanbria-Reinoso, Rocío Polvillo, Francisco-Javier Díaz-Corrales, Ozren Bogdanovic, Paola Bovolenta, Juan-Ramón Martínez-Morales
Sight depends on the tight cooperation between photoreceptors and pigmented cells, which derive from common progenitors through the bifurcation of a single gene regulatory network into the neural retina (NR) and retinal-pigmented epithelium (RPE) programs. Although genetic studies have identiﬁed upstream nodes controlling these networks, their regulatory logic remains poorly investigated. Here, we characterize transcriptome dynamics and chromatin accessibility in segregating NR/RPE populations in zebraﬁsh. We analyze cis-regulatory modules and enriched transcription factor motives to show extensive network redundancy and context-dependent activity. We identify downstream targets, highlighting an early recruitment of desmosomal genes in the ﬂattening RPE and revealing Tead factors as upstream regulators. We investigate the RPE speciﬁcation network dynamics to uncover an unexpected sequence of transcription factors recruitment, which is conserved in humans. This systematic interrogation of the NR/RPE bifurcation should improve both genetic counseling for eye disorders and hiPSCs-to-RPE differentiation protocols for cell-replacement therapies in degenerative diseases.
Genome-wide association study of more than 40,000 bipolar disorder cases provides new insights into the underlying biology
Niamh Mullins, Andreas J. Forstner, Ole A. Andreassen
Bipolar disorder is a heritable mental illness with complex etiology. We performed a genome-wide association study of 41,917 bipolar disorder cases and 371,549 controls of European ancestry, which identified 64 associated genomic loci. Bipolar disorder risk alleles were enriched in genes in synaptic signaling pathways and brain-expressed genes, particularly those with high specificity of expression in neurons of the prefrontal cortex and hippocampus. Significant signal enrichment was found in genes encoding targets of antipsychotics, calcium channel blockers, antiepileptics and anesthetics. Integrating expression quantitative trait locus data implicated 15 genes robustly linked to bipolar disorder via gene expression, encoding druggable targets such as HTR6, MCHR1, DCLK3 and FURIN. Analyses of bipolar disorder subtypes indicated high but imperfect genetic correlation between bipolar disorder type I and II and identified additional associated loci. Together, these results advance our understanding of the biological etiology of bipolar disorder, identify novel therapeutic leads and prioritize genes for functional follow-up studies.
CD9 inhibition reveals a functional connection of extracellular vesicle secretion with mitophagy in melanoma cells
Henar Suárez, Zoraida Andreu, Carla Mazzeo, Víctor Toribio, Aldo Emmanuel Pérez-Rivera, Soraya López-Martín, Susana García-Silva, Begoña Hurtado, Esperanza Morato, Laura Peláez, Egoitz Astigarraga Arribas, Tarson Tolentino-Cortez, Gabriel Barreda-Gómez, Ana Isabel Marina, Héctor Peinado, María Yáñez-Mó
Tetraspanins are often used as Extracellular Vesicle (EV) detection markers because of their abundance on these secreted vesicles. However, data on their function on EV biogenesis are controversial and compensatory mechanisms often occur upon gene deletion. To overcome this handicap, we have compared the effects of tetraspanin CD9 gene deletion with those elicited by cytopermeable peptides with blocking properties against tetraspanin CD9. Both CD9 peptide or gene deletion reduced the number of early endosomes. CD9 peptide induced an increase in lysosome numbers, while CD9 deletion augmented the number of MVB and EV secretion, probably because of compensatory CD63 expression upregulation. In vivo, CD9 peptide delayed primary tumour cell growth and reduced metastasis size. These effects on cell proliferation were shown to be concomitant with an impairment in mitochondrial quality control. CD9 KO cells were able to compensate the mitochondrial malfunction by increasing total mitochondrial mass reducing mitophagy. Our data thus provide the first evidence for a functional connection of tetraspanin CD9 with mitophagy in melanoma cells.
A novel bipartite antitermination system widespread in conjugative elements of Gram-positive bacteria
Andrés Miguel-Arribas, Jorge Val-Calvo, César Gago-Córdoba, José M Izquierdo, David Abia, Ling Juan Wu, Jeff Errington, Wilfried J J Meijer
Transcriptional regulation allows adaptive and coordinated gene expression, and is essential for life. Processive antitermination systems alter the transcription elongation complex to allow the RNA polymerase to read through multiple terminators in an operon. Here, we describe the discovery of a novel bipartite antitermination system that is widespread among conjugative elements from Gram-positive bacteria, which we named conAn. This system is composed of a large RNA element that exerts antitermination, and a protein that functions as a processivity factor. Besides allowing coordinated expression of very long operons, we show that these systems allow differential expression of genes within an operon, and probably contribute to strict regulation of the conjugation genes by minimizing the effects of spurious transcription. Mechanistic features of the conAn system are likely to decisively influence its host range, with important implications for the spread of antibiotic resistance and virulence genes.
Generation of mitochondrial reactive oxygen species is controlled by ATPase inhibitory factor 1 and regulates cognition
Pau B. Esparza-Moltó,Inés Romero-Carramiñana,Cristina Núñez de Arenas,Marta P. Pereira,Noelia Blanco,Beatriz Pardo,Georgina R. Bates,Carla Sánchez-Castillo,Rafael Artuch,Michael P. Murphy,José A. Esteban,José M. Cuezva
The mitochondrial ATP synthase emerges as key hub of cellular functions controlling the production of ATP, cellular signaling, and fate. It is regulated by the ATPase inhibitory factor 1 (IF1), which is highly abundant in neurons. Herein, we ablated or overexpressed IF1 in mouse neurons to show that IF1 dose defines the fraction of active/inactive enzyme in vivo, thereby controlling mitochondrial function and the production of mitochondrial reactive oxygen species (mtROS). Transcriptomic, proteomic, and metabolomic analyses indicate that IF1 dose regulates mitochondrial metabolism, synaptic function, and cognition. Ablation of IF1 impairs memory, whereas synaptic transmission and learning are enhanced by IF1 overexpression. Mechanistically, quenching the IF1-mediated increase in mtROS production in mice overexpressing IF1 reduces the increased synaptic transmission and obliterates the learning advantage afforded by the higher IF1 content. Overall, IF1 plays a key role in neuronal function by regulating the fraction of ATP synthase responsible for mitohormetic mtROS signaling.
Translational control of gene expression by eIF2 modulates proteostasis and extends lifespan
Tamara Jiménez-Saucedo, Juan José Berlanga, Miguel Rodríguez-Gabriel
Although the stress response in eukaryotes depends on early events triggered in cells by environmental insults, long-term processes such as aging are also affected. The loss of cellular proteostasis greatly impacts aging, which is regulated by the balancing of protein synthesis and degradation systems. As translation is the input event in proteostasis, we decided to study the role of translational activity on cell lifespan. Our hypothesis was that a reduction on translational activity or specific changes in translation may increase cellular longevity.
Using mutant strains of Schizosaccharomyces pombe and various stress conditions, we showed that translational reduction caused by phosphorylation of eukaryotic translation initiation factor 2 (eIF2) during the exponential growth phase enhances chronological lifespan (CLS). Furthermore, through next-generation sequence analysis, we found eIF2α phosphorylation-dependent translational activation of some specific genes, especially those involved in autophagy. This fact, together with the observed regulation of autophagy, points to a conserved mechanism involving general and specific control of translation and autophagy as mediators of the role of eIF2α phosphorylation in aging.
Aortic disease in Marfan syndrome is caused by overactivation of sGC-PRKG signaling by NO
Andrea de la Fuente-Alonso, Marta Toral, Alvaro Alfayate, María Jesús Ruiz-Rodríguez, Elena Bonzón-Kulichenko, Gisela Teixido-Tura, Sara Martínez-Martínez, María José Méndez-Olivares, Dolores López-Maderuelo, Ileana González-Valdés, Eusebio Garcia-Izquierdo, Susana Mingo, Carlos E. Martín, Laura Muiño-Mosquera, Julie De Backer, J. Francisco Nistal, Alberto Forteza, Arturo Evangelista, Jesús Vázquez, Miguel R. Campanero & Juan Miguel Redondo
Thoracic aortic aneurysm, as occurs in Marfan syndrome, is generally asymptomatic untildissection or rupture, requiring surgical intervention as the only available treatment. Here, weshow that nitric oxide (NO) signaling dysregulates actin cytoskeleton dynamics in MarfanSyndrome smooth muscle cells and that NO-donors induce Marfan-like aortopathy in wild-type mice, indicating that a marked increase in NO suffices to induce aortopathy. Levels ofnitrated proteins are higher in plasma from Marfan patients and mice and in aortic tissuefrom Marfan mice than in control samples, indicating elevated circulating and tissue NO.Soluble guanylate cyclase and cGMP-dependent protein kinase are both activated in Marfanpatients and mice and in wild-type mice treated with NO-donors, as shown by increasedplasma cGMP and pVASP-S239 staining in aortic tissue. Marfan aortopathy in mice isreverted by pharmacological inhibition of soluble guanylate cyclase and cGMP-dependentprotein kinase and lentiviral-mediatedPrkg1silencing. Thesefindings identify potential bio-markers for monitoring Marfan Syndrome in patients and urge evaluation of cGMP-dependent protein kinase and soluble guanylate cyclase as therapeutic targets.
A new non-aggregative splicing isoform of human Tau is decreased in Alzheimer's disease
Vega García-Escudero, Daniel Ruiz-Gabarre , Ricardo Gargini, Mar Pérez, Esther García, Raquel Cuadros, Ivó H Hernández, Jorge R Cabrera, Ramón García-Escudero, José J Lucas, Félix Hernández, Jesús Ávila
Tauopathies, including Alzheimer's disease (AD) and frontotemporal lobar degeneration with Tau pathology (FTLD-tau), are a group of neurodegenerative disorders characterized by Tau hyperphosphorylation. Post-translational modifications of Tau such as phosphorylation and truncation have been demonstrated to be an essential step in the molecular pathogenesis of these tauopathies. In this work, we demonstrate the existence of a new, human-specific truncated form of Tau generated by intron 12 retention in human neuroblastoma cells and, to a higher extent, in human RNA brain samples, using qPCR and further confirming the results on a larger database of human RNA-seq samples. Diminished protein levels of this new Tau isoform are found by Westernblotting in Alzheimer's patients' brains (Braak I n = 3; Braak II n = 6, Braak III n = 3, Braak IV n = 1, and Braak V n = 10, Braak VI n = 8) with respect to non-demented control subjects (n = 9), suggesting that the lack of this truncated isoform may play an important role in the pathology. This new Tau isoform exhibits similar post-transcriptional modifications by phosphorylation and affinity for microtubule binding, but more interestingly, is less prone to aggregate than other Tau isoforms. Finally, we present evidence suggesting this new Tau isoform could be linked to the inhibition of GSK3β, which would mediate intron 12 retention by modulating the serine/arginine rich splicing factor 2 (SRSF2). Our results show the existence of an important new isoform of Tau and suggest that further research on this less aggregation-prone Tau may help to develop future therapies for Alzheimer's disease and other tauopathies.
Huntington’s disease-specific mis-splicing unveils key effector genes and altered splicing factors
José J Lucas
Correction of mis-splicing events is a growing therapeutic approach for neurological diseases such as spinal muscular atrophy or neuronal ceroid lipofuscinosis 7, which are caused by splicing-affecting mutations. Non-mutation harboring mis-spliced effector genes are also good candidate therapeutic targets in diseases with more complex etiologies such as cancer, autism, muscular dystrophies or neurodegenerative diseases. Next-generation RNA sequencing (RNA-seq) has boosted investigation of global mis-splicing in diseased tissue to identify such key pathogenic mis-spliced genes. Nevertheless, while analysis of tumour or dystrophic muscle biopsies can be informative on early stage pathogenic mis-splicing, for neurodegenerative diseases, these analyses are intrinsically hampered by neuronal loss and neuroinflammation in post-mortem brains. To infer splicing alterations relevant to Huntington’s disease (HD) pathogenesis, here we performed intersect-RNA-seq analyses of human post-mortem striatal tissue and of an early symptomatic mouse model in which neuronal loss and gliosis are not yet present. Together with a human/mouse parallel motif scan analysis, this approach allowed us to identify the shared mis-splicing signature triggered by the HD-causing mutation in both species and to infer upstream deregulated splicing factors. Moreover, we identified a plethora of downstream neurodegeneration-linked mis-spliced effector genes that -together with the deregulated splicing factors- become new possible therapeutic targets. In summary, here we report pathogenic global mis-splicing in HD striatum captured by our new intersect-RNA-seq approach that can be readily applied to other neurodegenerative diseases for which bona fide animal models are available.
Microglia in Alzheimer’s Disease in the Context of Tau Pathology
Juan Ramón Perea, Marta Bolós, Jesús Avila
Microglia are the cells that comprise the innate immune system in the brain. First described more than a century ago, these cells were initially assigned a secondary role in the central nervous system (CNS) with respect to the protagonists, neurons. However, the latest advances have revealed the complexity and importance of microglia in neurodegenerative conditions such as Alzheimer’s disease (AD), the most common form of dementia associated with aging. This pathology is characterized by the accumulation of amyloid-β peptide (Aβ), which forms senile plaques in the neocortex, as well as by the aggregation of hyperphosphorylated tau protein, a process that leads to the development of neurofibrillary tangles (NFTs). Over the past few years, efforts have been focused on studying the interaction between Aβ and microglia, together with the ability of the latter to decrease the levels of this peptide. Given that most clinical trials following this strategy have failed, current endeavors focus on deciphering the molecular mechanisms that trigger the tau-induced inflammatory response of microglia. In this review, we summarize the most recent studies on the physiological and pathological functions of tau protein and microglia. In addition, we analyze the impact of microglial AD-risk genes (APOE, TREM2, and CD33) in tau pathology, and we discuss the role of extracellular soluble tau in neuroinflammation.
Involvement of the 14-3-3 Gene Family in Autism Spectrum Disorder and Schizophrenia: Genetics, Transcriptomics and Functional Analyses
Bàrbara Torrico, Ester Antón-Galindo, Noèlia Fernàndez-Castillo, Eva Rojo-Francàs, Sadaf Ghorbani, Laura Pineda-Cirera, Amaia Hervás, Isabel Rueda, Estefanía Moreno, Janice M. Fullerton, Vicent Casadó, Jan K. Buitelaar, Nanda Rommelse, Barbara Franke, Andreas Reif, Andreas G. Chiocchetti, Christine Freitag, Rune Kleppe, Jan Haavik, Claudio Toma, Bru Cormand
The 14-3-3 protein family are molecular chaperones involved in several biological functions and neurological diseases. We previously pinpointed YWHAZ (encoding 14-3-3ζ) as a candidate gene for autism spectrum disorder (ASD) through a whole-exome sequencing study, which identified a frameshift variant within the gene (c.659-660insT, p.L220Ffs*18). Here, we explored the contribution of the seven human 14-3-3 family members in ASD and other psychiatric disorders by investigating the: (i) functional impact of the 14-3-3ζ mutation p.L220Ffs*18 by assessing solubility, target binding and dimerization; (ii) contribution of common risk variants in 14-3-3 genes to ASD and additional psychiatric disorders; (iii) burden of rare variants in ASD and schizophrenia; and iv) 14-3-3 gene expression using ASD and schizophrenia transcriptomic data. We found that the mutant 14-3-3ζ protein had decreased solubility and lost its ability to form heterodimers and bind to its target tyrosine hydroxylase. Gene-based analyses using publicly available datasets revealed that common variants in YWHAE contribute to schizophrenia (p = 6.6 × 10−7), whereas ultra-rare variants were found enriched in ASD across the 14-3-3 genes (p = 0.017) and in schizophrenia for YWHAZ (meta-p = 0.017). Furthermore, expression of 14-3-3 genes was altered in post-mortem brains of ASD and schizophrenia patients. Our study supports a role for the 14-3-3 family in ASD and schizophrenia.
Temporal groups of lineage-related neurons have different neuropeptidergic fates and related functions in the Drosophila melanogaster CNS
Laura Díaz-de-la-Peña, Leila Maestro-Paramio, Fernando J. Díaz-Benjumea, Pilar Herrero
The central nervous system (CNS) of Drosophila is comprised of the brain and the ventral nerve cord (VNC), which are the homologous structures of the vertebrate brain and the spinal cord, respectively. Neurons of the CNS arise from neural stem cells called neuroblasts (NBs). Each neuroblast gives rise to a specific repertory of cell types whose fate is unknown in most lineages. A combination of spatial and temporal genetic cues defines the fate of each neuron. We studied the origin and specification of a group of peptidergic neurons present in several abdominal segments of the larval VNC that are characterized by the expression of the neuropeptide GPB5, the GPB5-expressing neurons (GPB5-ENs). Our data reveal that the progenitor NB that generates the GPB5-ENs also generates the abdominal leucokinergic neurons (ABLKs) in two different temporal windows. We also show that these two set of neurons share the same axonal projections in larvae and in adults and, as previously suggested, may both function in hydrosaline regulation. Our genetic analysis of potential specification determinants reveals that Klumpfuss (klu) and huckebein (hkb) are involved in the specification of the GPB5 cell fate. Additionally, we show that GPB5-ENs have a role in starvation resistance and longevity; however, their role in desiccation and ionic stress resistance is not as clear. We hypothesize that the neurons arising from the same neuroblast lineage are both architecturally similar and functionally related.
Identifying the role of PrimPol in TDF-induced toxicity and implications of its loss of function mutation in an HIV+ patient
Vincent N. Duong, Lei Zhou, María I. Martínez-Jiménez, Linh He, Moises Cosme, Luis Blanco, Elijah Paintsil, Karen S. Anderson
A key component of antiretroviral therapy (ART) for HIV patients is the nucleoside reverse transcriptase inhibitor (NRTI) is tenofovir. Recent reports of tenofovir toxicity in patients taking ART for HIV cannot be explained solely on the basis of off-target inhibition of mitochondrial DNA polymerase gamma (Polγ). PrimPol was discovered as a primase-polymerase localized to the mitochondria with repriming and translesion synthesis capabilities and, therefore, a potential contributor to mitochondrial toxicity. We established a possible role of PrimPol in tenofovir-induced toxicity in vitro and show that tenofovir-diphosphate incorporation by PrimPol is dependent on the n-1 nucleotide. We identified and characterized a PrimPol mutation, D114N, in an HIV+ patient on tenofovir-based ART with mitochondrial toxicity. This mutant form of PrimPol, targeting a catalytic metal ligand, was unable to synthesize primers, likely due to protein instability and weakened DNA binding. We performed cellular respiration and toxicity assays using PrimPol overexpression and shRNA knockdown strains in renal proximal tubular epithelial cells. The PrimPol-knockdown strain was hypersensitive to tenofovir treatment, indicating that PrimPol protects against tenofovir-induced mitochondrial toxicity. We show that a major cellular role of PrimPol is protecting against toxicity caused by ART and individuals with inactivating mutations may be predisposed to these effects.
MiR‐9‐5p protects from kidney fibrosis by metabolic reprogramming
Marta Fierro‐Fernández, Verónica Miguel, Laura Márquez‐Expósito, Cristina Nuevo‐Tapioles, J. Ignacio Herrero, Eva Blanco‐Ruiz, Jessica Tituaña, Carolina Castillo, Pablo Cannata, María Monsalve, Marta Ruiz‐Ortega, Ricardo Ramos, Santiago Lamas
MicroRNAs (miRNAs) regulate gene expression posttranscriptionally and control biological processes (BPs), including fibrogenesis. Kidney fibrosis remains a clinical challenge and miRNAs may represent a valid therapeutic avenue. We show that miR‐9‐5p protected from renal fibrosis in the mouse model of unilateral ureteral obstruction (UUO). This was reflected in reduced expression of pro‐fibrotic markers, decreased number of infiltrating monocytes/macrophages, and diminished tubular epithelial cell injury and transforming growth factor‐beta 1 (TGF‐β1)‐dependent de‐differentiation in human kidney proximal tubular (HKC‐8) cells. RNA‐sequencing (RNA‐Seq) studies in the UUO model revealed that treatment with miR‐9‐5p prevented the downregulation of genes related to key metabolic pathways, including mitochondrial function, oxidative phosphorylation (OXPHOS), fatty acid oxidation (FAO), and glycolysis. Studies in human tubular epithelial cells demonstrated that miR‐9‐5p impeded TGF‐β1‐induced bioenergetics derangement. The expression of the FAO‐related axis peroxisome proliferator‐activated receptor gamma coactivator 1 alpha (PGC‐1α)‐peroxisome proliferator‐activated receptor alpha (PPARα) was reduced by UUO, although preserved by the administration of miR‐9‐5p. We found that in mice null for the mitochondrial master regulator PGC‐1α, miR‐9‐5p was unable to promote a protective effect in the UUO model. We propose that miR‐9‐5p elicits a protective response to chronic kidney injury and renal fibrosis by inducing reprogramming of the metabolic derangement and mitochondrial dysfunction affecting tubular epithelial cells.
Epigenetic Priming in Immunodeficiencies
Jorge Martínez-Cano, Elena Campos-Sánchez and César Cobaleda
Immunodeficiencies (IDs) are disorders of the immune system that increase susceptibility to infections and cancer, and are therefore associated with elevated morbidity and mortality. IDs can be primary (not caused by other condition or exposure) or secondary due to the exposure to different agents (infections, chemicals, aging, etc.). Most primary immunodeficiencies (PIDs) are of genetic origin, caused by mutations affecting genes with key roles in the development or function of the cells of the immune system. A large percentage of PIDs are associated with a defective development and/or function of lymphocytes and, especially, B cells, the ones in charge of generating the different types of antibodies. B-cell development is a tightly regulated process in which many different factors participate. Among the regulators of B-cell differentiation, a correct epigenetic control of cellular identity is essential for normal cell function. With the advent of next-generation sequencing (NGS) techniques, more and more alterations in different types of epigenetic regulators are being described at the root of PIDs, both in humans and in animal models. At the same time, it is becoming increasingly clear that epigenetic alterations triggered by the exposure to environmental agents have a key role in the development of secondary immunodeficiencies (SIDs). Due to their largely reversible nature, epigenetic modifications are quickly becoming key therapeutic targets in other diseases where their contribution has been known for more time, like cancer. Here, we establish a parallelism between IDs and the nowadays accepted role of epigenetics in cancer initiation and progression, and propose that epigenetics forms a “third axis” (together with genetics and external agents) to be considered in the etiology of IDs, and linking PIDs and SIDs at the molecular level. We therefore postulate that IDs arise due to a variable contribution of (i) genetic, (ii) environmental, and (iii) epigenetic causes, which in fact form a continuum landscape of all possible combinations of these factors. Additionally, this implies the possibility of a fully epigenetically triggered mechanism for some IDs. This concept would have important prophylactic and translational implications, and would also imply a more blurred frontier between primary and secondary immunodeficiencies.
Synergistic Lethal Mutagenesis of Hepatitis C Virus
Isabel Gallego, María Eugenia Soria, Josep Gregori, Ana I. de Ávila, Carlos García-Crespo, Elena Moreno, Ignacio Gadea, Jaime Esteban, Ricardo Fernández-Roblas, Juan Ignacio Esteban, Jordi Gómez, Josep Quer, Esteban Domingo, Celia Perales
Lethal mutagenesis is an antiviral approach that consists of extinguishing a virus by an excess of mutations acquired during replication in the presence of a mutagenic agent, often a nucleotide analogue. One of its advantages is its broad-spectrum nature, which renders the strategy potentially effective against emergent RNA viral infections. Here we describe the synergistic lethal mutagenesis of hepatitis C virus (HCV) by a combination of favipiravir (T-705) and ribavirin. Synergy has been documented over a broad range of analogue concentrations using the Chou-Talalay method implemented in CompuSyn graphics software, with the average dose reduction index (DRI) being above 1 (68.02 ± 101.6 for favipiravir and 5.83 ± 6.07 for ribavirin) and the average combination indices (CI) being below 1 (0.52 ± 0.28). Furthermore, analogue concentrations that individually did not extinguish high-fitness HCV in 10 serial infections extinguished high-fitness HCV in 1 to 2 passages when used in combination. Although both analogues displayed a preference for G → A and C → U transitions, deep sequencing analysis of mutant spectra indicated a different preference of the two analogues for the mutation sites, thus unveiling a new possible synergy mechanism in lethal mutagenesis. The prospects for synergy among mutagenic nucleotides as a strategy to confront emerging viral infections are discussed.
MEPSAnd: minimum energy path surface analysis over n-dimensional surfaces
Iñigo Marcos-Alcalde, Eduardo López-Viñas, Paulino Gómez-Puertas
n-dimensional energy surfaces are becoming computationally accessible, yet interpreting their information is not straightforward. We present minimum energy path surface analysis over n-dimensional surfaces (MEPSAnd), an open source GUI-based program that natively calculates minimum energy paths across energy surfaces of any number of dimensions. Among other features, MEPSAnd can compute the path through lowest barriers and automatically provide a set of alternative paths. MEPSAnd offers distinct plotting solutions as well as direct python scripting.
Esteban Domingo, Celia Perales
Viral quasispecies refers to a population structure that consists of extremely large numbers of variant genomes, termed mutant spectra, mutant swarms or mutant clouds. Fueled by high mutation rates, mutants arise continually, and they change in relative frequency as viral replication proceeds. The term quasispecies was adopted from a theory of the origin of life in which primitive replicons) consisted of mutant distributions, as found experimentally with present day RNA viruses. The theory provided a new definition of wild type, and a conceptual framework for the interpretation of the adaptive potential of RNA viruses that contrasted with classical studies based on consensus sequences. Standard clonal analyses and deep sequencing methodologies have confirmed the presence of myriads of mutant genomes in viral populations, and their participation in adaptive processes. The quasispecies concept applies to any biological entity, but its impact is more evident when the genome size is limited and the mutation rate is high. This is the case of the RNA viruses, ubiquitous in our biosphere, and that comprise many important pathogens. In virology, quasispecies are defined as complex distributions of closely related variant genomes subjected to genetic variation, competition and selection, and that may act as a unit of selection. Despite being an integral part of their replication, high mutation rates have an upper limit compatible with inheritable information. Crossing such a limit leads to RNA virus extinction, a transition that is the basis of an antiviral design termed lethal mutagenesis.
Genetic deficiency or pharmacological inhibition of miR-33 protects from kidney fibrosis
Nathan L. Price, Verónica Miguel, Wen Ding, Abhishek K. Singh, Shipra Malik, Noemi Rotllan, Anna Moshnikova, Jakub Toczek, Caroline Zeiss, Mehran M. Sadeghi, Noemi Arias, Ángel Baldán, Oleg A. Andreev, Diego Rodríguez-Puyol, Raman Bahal, Yana K. Reshetnyak, Yajaira Suárez, Carlos Fernández-Hernando, and Santiago Lamas
Previous work has reported the important links between cellular bioenergetics and the development of chronic kidney disease, highlighting the potential for targeting metabolic functions to regulate disease progression. More recently, it has been shown that alterations in fatty acid oxidation (FAO) can have an important impact on the progression of kidney disease. In this work, we demonstrate that loss of miR-33, an important regulator of lipid metabolism, can partially prevent the repression of FAO in fibrotic kidneys and reduce lipid accumulation. These changes were associated with a dramatic reduction in the extent of fibrosis induced in 2 mouse models of kidney disease. These effects were not related to changes in circulating leukocytes because bone marrow transplants from miR-33–deficient animals did not have a similar impact on disease progression. Most important, targeted delivery of miR-33 peptide nucleic acid inhibitors to the kidney and other acidic microenvironments was accomplished using pH low insertion peptides as a carrier. This was effective at both increasing the expression of factors involved in FAO and reducing the development of fibrosis. Together, these findings suggest that miR-33 may be an attractive therapeutic target for the treatment of chronic kidney disease.
Extracellular Tuning of Mitochondrial Respiration Leads to Aortic Aneurysm
Jorge Oller, Enrique Gabandé-Rodríguez, María Jesús Ruiz-Rodriguez, Gabriela Desdín-Micó, Juan Francisco Aranda, Raquel Rodrigues-Diez, Constanza Ballesteros-Martínez, Eva María Blanco, Raquel Roldan-Montero, Pedro Acuña, Alberto Forteza Gil, Carlos E. Martín-López, J. Francisco Nistal, Christian L. Lino Cardenas, Mark Evan Lindsay, José Luís Martín-Ventura, Ana M. Briones, Juan Miguel Redondo, and Maréa Mittelbrunn
Marfan syndrome (MFS) is an autosomal dominant disorder of the connective tissue caused by mutations in the FBN1 gene encoding a large glycoprotein in the extracellular matrix called fibrillin-1. The major complication of this connective disorder is the risk to develop thoracic aortic aneurysm (TAA). To date, no effective pharmacological therapies have been identified for the management of thoracic aortic disease and the only options capable of preventing aneurysm rupture are endovascular repair or open surgery. Here, we have studied the role of mitochondrial dysfunction in the progression of thoracic aortic aneurysm and mitochondrial boosting strategies as a potential treatment to managing aortic aneurysms