Representative publications

Covadonga Díaz-Díaz, Gabriel Baonza, Fernando Martín-Belmonte.

Epithelial tissues are made of highly specialized cells present in many organs and represent the first barrier of protection from the external environment. Essential for this critical role in protection is their capacity to polarize in the apicobasal axis. The integrity of the epithelium and its properties as a protective barrier is mostly regulated by dynamic intercellular junctions composed of multiprotein complexes. The functionality and dynamics of these junctions are tightly controlled by several signaling processes, including Rho GTPases. Here, we review the most recent data in the contribution of Rho GTPases and their functional regulators during the morphogenesis of epithelial tissues and to maintain the homeostasis in adults.

Daniel Pérez-Núñez , Eva Castillo-Rosa, Gonzalo Vigara-Astillero, Raquel García-Belmonte, Carmina Gallardo and Yolanda Revilla.

 No efficient vaccines exist against African swine fever virus (ASFV), which causes a serious disease in wild boars and domestic pigs that produces great industrial and ecological concerns worldwide. An extensive genetic characterization of the original ASFV stocks used to produce live attenuated vaccine (LAV) prototypes is needed for vaccine biosecurity and control. Here, we sequenced for the first time the Arm/07 stock which was obtained from an infected pig during the Armenia outbreak in 2007, using an improved viral dsDNA purification method together with high coverage analysis. There was unexpected viral heterogeneity within the stock, with two genetically distinct ASFV subpopulations. The first, represented by the Arm/07/CBM/c2 clone, displayed high sequence identity to the updated genotype II Georgia 2007/1, whereas the second (exemplified by clone Arm/07/CBM/c4) displayed a hemadsorbing phenotype and grouped within genotype I based on a central region conserved among all members of this group. Intriguingly, Arm/07/CBM/c4 contained a unique EP402R sequence, produced by a single mutation in the N-terminal region. Importantly, Arm/07/CBM/c4 showed in vitro features of attenuated strains regarding innate immune response pathway. Both Arm/07/CBM/c2 and c4 represent well-characterized viral clones, useful for different molecular and virus-host interaction studies, including virulence studies and vaccine development.

Bruno Hernáez, Graciela Alonso, Iliana Georgana, Misbah El-Jesr, Rocío Martin, Kathy H. Y. Shairst, Cornelius Fischer, Sascha Sauer, Carlos Maluquer de Motes, Antonio Alcami

Cells contain numerous immune sensors to detect virus infection. The cyclic GMP-AMP (CGAMP) synthase (CGAS) recognizes cytosolic DNA and activates innate immune responses via stimulator of interferon genes (STING), but the impact of DNA sensing pathways on host protective responses has not been fully defined. We demonstrate that CGAS/STING activation is required to resist lethal poxvirus infection. We identified viral Schlafen (vsifn) as the main STING inhibitor, and ectromelia virus was severely attenuated in the absence of vSlfn. Both vsifn-mediated virulence and STING inhibitory activity were mapped to the recently discovered poxin CGAMP nuclease domain. Animals were protected from subcutaneous, respiratory, and intravenous infection in the absence of vsifn, and interferon was the main antiviral protective mechanism controlled by the DNA sensing pathway. Our findings support the idea that manipulation of DNA sensing is an efficient therapeutic strategy in diseases triggered by viral infection or tissue damage-mediated release of self-DNA.

 

Adrián Bartoll, Ana Toledano-Zaragoza, Josefina Casas, Manuel Guzmán, Edward H Schuchman & María Dolores Ledesma.

Acid sphingomyelinase deficiency (ASMD) leads to cellular accumulation of sphingomyelin (SM), neurodegeneration, and early death.

Here, we describe the downregulation of the endocannabinoid(eCB) system in neurons of ASM knockout (ASM-KO) mice and a ASMD patient. High SM reduced expression of the eCB receptor CB1 in neuronal processes and induced its accumulation in lyso- somes. Activation of CB1 receptor signaling, through inhibition of the eCB-degrading enzyme fatty acid amide hydrolase (FAAH), reduced SM levels in ASM-KO neurons. Oral treatment of ASM-KO mice with a FAAH inhibitor prevented SM buildup; alleviated inflammation, neurodegeneration, and behavioral alterations; and extended lifespan. This treatment showed benefits even after a single administration at advanced disease stages. We also found CB1 receptor downregulation in neurons of a mouse model and a patient of another sphingolipid storage disorder, Niemann–Pick disease type C (NPC). We showed the efficacy of FAAH inhibition to reduce SM and cholesterol levels in NPC patient-derived cells and in the brain of a NPC mouse model. Our findings reveal a patho-physiological crosstalk between neuronal SM and the eCB system and offer a new treatment for ASMD and other sphingolipidoses.

David Blom-Dahl , Sergio Cordoba, Hugo Gabilondo, Pablo Carr-Baena,Fernando J. Díaz-Benjumea, Carlos Estella.

The Sp family of transcription factors plays important functions during development and disease. An evolutionary conserved role for some Sp family members is the control of limb development. The family is characterized by the presence of three C2H2-type zinc fingers and an adjacent 10 aa region with an unknown function called the Buttonhead (BTD) box. The presence of this BTD-box in all Sp family members identified from arthropods to vertebrates, suggests that it plays an essential role during development. However, despite its conservation, the in vivo function of the BTD-box has never been studied. In this work, we have generated specific BTD-box deletion alleles for the Drosophila Sp family members Sp1 and buttonhead (btd) using gene editing tools and analyzed its role during development. Unexpectedly, btd and Sp1 mutant alleles that lack the BTD-box are viable and have almost normal appendages. However, in a sensitized background the requirement of this domain to fully regulate some of Sp1 and Btd target genes is revealed. Furthermore, we have also identified a novel Sp1 role promoting leg vs antenna identity through the repression of spineless (ss) expression in the leg, a function that also depends on the Sp1 BTD-box.

MDA5 cleavage by the Leader protease of foot-and-mouth disease virus reveals its pleiotropic effect against the host antiviral response 

Miguel Rodríguez Pulido , Encarnación Martínez-Salas , Francisco Sobrino  and Margarita Sáiz

The RIG-I-like receptor (RLR) melanoma differentiation-associated gene 5 (MDA5) plays a key role in triggering innate antiviral response during infection by RNA viruses. MDA5 activation leads to transcription induction of type-I interferon (IFN) and proinflammatory cytokines. MDA5 has also been associated with autoimmune and autoinflammatory diseases by dysfunctional activation of innate immune response in the absence of infection. Here, we show how foot-and-mouth disease virus (FMDV) counteracts the specific antiviral effect exerted by MDA5 targeting the protein for cleavage by the viral Leader protease (Lpro). MDA5 overexpression had an inhibitory effect on FMDV infection in IFN-competent cells. Remarkably, immunostimulatory viral RNA co-immunoprecipitated with MDA5 in infected cells.

Moreover, specific cleavage of MDA5 by Lpro was detected in co-transfected cells, as well as during the course of FMDV infection. A significant reduction in IFN induction associated with MDA5 cleavage was detected by comparison with a non-cleavable MDA5 mutant protein with preserved antiviral activity. The Lpro cleavage site in MDA5 was identified as the RGRAR sequence in the conserved helicase motif VI, coinciding with that recently reported for Lpro in LGP2, another member of the RLRs family involved in antiviral defenses. Interestingly, specific mutations within the MDA5 Lpro target sequence have been associated with immune disease in mice and humans. Our results reveal a pleiotropic strategy for immune evasion based on a viral protease targeting phylogenetically conserved domains of immune sensors. Identification of viral strategies aimed to disrupt MDA5 functionality may also contribute to develop new treatment tools for MDA5-related disorders.

GSK3a, not GSK3b, drives hippocampal NMDAR-dependent LTD via tau-mediated spine anchoring 

Jonathan E Draffin, Carla Sánchez-Castillo, Alba Fernández-Rodrigo, Xavier Sánchez-Sáez, Jesús Ávila , Florence F Wagner  & José A Esteban.

Glycogen synthase kinase-3 (GSK3) is an important signalling protein in the brain and modulates different forms of synaptic
plasticity. Neuronal functions of GSK3 are typically attributed to one of its two isoforms, GSK3b, simply because of its prevalent expression in the brain. Consequently, the importance of isoform-specific functions of GSK3 in synaptic plasticity has not been fully explored. We now directly address this question for NMDA receptor-dependent long-term depression (LTD) in the hippocampus. Here, we specifically target the GSK3 isoforms with shRNA knock-down in mouse hippocampus and with novel isoform-selective drugs to dissect their roles in LTD. Using electrophysiological and live imaging approaches, we find that GSK3a, but not GSK3b, is required for LTD. The specific engagement of GSK3a occurs via its transient anchoring in dendritic spines during LTD induction. We find that the major GSK3 substrate, the microtubule-binding protein tau, is required for this spine anchoring of GSK3a and mediates GSK3a-induced LTD. These results link GSK3a and tau in a common mechanism for synaptic depression and rule out a major role for GSK3b in this process.

 
Irene Pérez-Liébana, María José Casarejos, Andrea Alcaide, Eduardo Herrada-Soler, Irene Llorente-Folch, Laura Contreras, Jorgina Satrústegui, and Beatriz Pardo.

Aralar/AGC1/Slc25a12, the mitochondrial aspartate-glutamate carrier expressed in neurons, is the regulatory component of the NADH malate-aspartate shuttle. AGC1 deficiency is a neuropediatric rare disease characterized by hypomyelination, hypotonia, developmental arrest, and epilepsy. We have investigated whether b-hydroxybutyrate (bOHB), the main ketone body (KB) produced in ketogenic diet (KD), is neuroprotective in aralar-knock-out (KO) neurons and mice. We report that bOHB efficiently recovers aralar-KO neurons from deficits in basal-stimulated and glutamate-stimulated respiration, effects requiring bOHB entry into the neuron, and pro-
tects from glutamate excitotoxicity. Aralar-deficient mice were fed a KD to investigate its therapeutic potential early in development, but this approach was unfeasible. Therefore, aralar-KO pups were treated without distinction of gender with daily intraperitoneal injections of bOHB during 5 d. This treatment resulted in a recovery of striatal markers of the dopaminergic system including dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC)/DA ratio, and vesicular monoamine transporter 2 (VMAT2) protein. Regarding postnatal myelination, myelin basic protein (MBP) and myelin-associated glycoprotein (MAG) myelin proteins were markedly increased in the cortices of bOHB-treated aralar-KO mice. Although brain Asp and NAA levels did not change by bOHB administration, a 4-d bOHB treatment to aralar-KO, but not to control, neurons led to a substantial increase in Asp (3-fold) and NAA (4-fold) levels. These results suggest that the lack of increase in brain Asp and NAA is possibly because of its active utilization by the aralar-KO brain and the likely involvement of neuronal NAA in postnatal myelination in these mice. The effectiveness of bOHB as a therapeutic treatment in AGC1 deficiency deserves further investigation.

Carlos D. Ordóñez, Ana Lechuga, Margarita Salas & Modesto Redrejo‐Rodríguez.

The development of whole genome amplifcation (WGA) and related methods, coupled with the dramatic growth of sequencing capacities, has changed the paradigm of genomic and genetic analyses.This has led to a continual requirement of improved DNA amplifcation protocols and the elaboration of new tailored methods.As key elements inWGA, identifcation and engineering of novel, faithful and processive DNA polymerases is a driving force in the feld.We have engineered the B-family DNA polymerase of virus Bam35 with a C-terminal fusion of DNA-binding motifs.The new protein, named B35-HhH, shows faithful DNA replication in the presence of magnesium or an optimised combination of magnesium and manganese divalent cofactors, which enhances the replication of damaged DNA substrates. Overall, the newly generated variant displays improved amplifcation performance, sensitivity, translesion synthesis and resistance to salt, which are of great interest for several applications of isothermal DNA amplifcation. Further, rolling-circle amplifcation of abasic site-containing minicircles provides a proof-of-concept for using B35-HhH for processive amplifcation of damaged DNA samples.

 

Leticia Labat‐de‐Hoz, Miguel A. Alonso.

Formins are a conserved family of proteins that primarily act to form linear polymers of actin. Despite their importance to the normal functioning of the cytoskeleton, for a long time, the only two formin genes known to be a genetic cause of human disorders were DIAPH1 and DIAPH3, whose mutation causes two distinct forms of hereditary deafness. In the last 10 years, however, the formin INF2 has emerged as an important target of mutations responsible for the appearance of focal segmental glomerulosclerosis, which are histological lesions associated with glomerulus degeneration that often leads to end-stage renal disease. In some rare cases, focal segmental glomerulosclerosis concurs with Charcot–Marie–Tooth disease, which is a degenerative neurological disorder afecting peripheral nerves. All known INF2 gene mutations causing disease map to the exons encoding the amino-terminal domain. In this review, we summarize the structure, biochemical features and functions
of INF2, conduct a systematic and comprehensive analysis of the pathogenic INF2 mutations, including a detailed study exon-by-exon of patient cases and mutations, address the impact of the pathogenic mutations on the structure, regulation and known functions of INF2, draw a series of conclusions that could be useful for INF2-related disease diagnosis, and suggest lines of research for future work on the molecular mechanisms by which INF2 causes disease.

Genetic Material Manipulation and Modification by Optical Trapping and Nanosurgery-A Perspective

Alfonso Blázquez-Castro, José Fernández-Piqueras and Javier Santos.

Light can be employed as a tool to alter and manipulate matter in many ways. An example has been the implementation of optical trapping, the so called optical tweezers,
in which light can hold and move small objects with 3D control. Of interest for the Life Sciences and Biotechnology is the fact that biological objects in the size range from
tens of nanometers to hundreds of microns can be precisely manipulated through this technology. In particular, it has been shown possible to optically trap and move genetic material (DNA and chromatin) using optical tweezers. Also, these biological entities can be severed, rearranged and reconstructed by the combined use of laser scissors and optical tweezers. In this review, the background, current state and future possibilities of optical tweezers and laser scissors to manipulate, rearrange and alter genetic material (DNA, chromatin and chromosomes) will be presented. Sources of undesirable effects by the optical procedure and measures to avoid them will be discussed. In addition, first tentative approaches at cellular-level genetic and organelle surgery, in which genetic material or DNA-carrying organelles are extracted out or introduced into cells, will be presented.

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