Editorial: Molecular and Cellular Crosstalk on Neuronal Functionality and Regulation, From Development to Pathology

EDITORIAL articleWe are grateful to all authors and reviewers that contributed to this Research Topic. IS acknowledges to the Max Planck Society for support. JE acknowledges IRBLLEIDA and the University of Lleida for support and the Ministerio de Economia y Competitividad of Spain for funding (PGC2018-101910-B-I00)

Neuronal survival induced by neurotrophins requires calmodulin

It has been reported that phosphoinositide 3-kinase (PI 3-kinase) and its downstream target, protein kinase B (PKB), play a central role in the signaling of cell survival triggered by neurotrophins (NTs). In this report, we have analyzed the involvement of Ca2+ and calmodulin (CaM) in the activation of the PKB induced by NTs. We have found that reduction of intracellular Ca2+ concentration or functional blockade of CaM abolished NGF-induced activation of PKB in PC12 cells. Similar results were obtained in cultures of chicken spinal cord motoneurons treated with brain-derived neurotrophic factor (BDNF). Moreover, CaM inhibition prevented the cell survival triggered by NGF or BDNF. This effect was counteracted by the transient expression of constitutive active forms of the PKB, indicating that CaM regulates NT-induced cell survival through the activation of the PKB. We have investigated the mechanisms whereby CaM regulates the activation of the PKB, and we have found that CaM was necessary for the proper generation and/or accumulation of the products of the PI 3-kinase in intact cells

Sprouty1 controls genitourinary development via its N-terminal tyrosine

Background: Congenital anomalies of the kidney and urinary tract (CAKUT) is a group of diseases that include a broad spectrum of developmental defects of the genitourinary system. Mouse models indicate that perturbations of the GDNF-Ret signaling pathway are a major genetic cause of CAKUT. Sprouty1 is an intracellular Ret inhibitor whose mutation results in supernumerary kidneys, megaureters, and hydronephrosis in mice. Both the molecular mechanisms and the structural domains critical for Sprouty function are a matter of controversy, partly because studies pursuing this objective rely on ectopic overexpression in cell lines. A conserved N-terminal tyrosine has been frequently, but not always, identified as critical for their function in vitro. Methods: We have generated Sprouty1 knockin mice bearing a tyrosine-to-alanine substitution in position 53, corresponding to the conserved N-terminal tyrosine of Sprouty1. We have characterized development of the genitourinary systems of these mice via different methods, including the use of reporter mice expressing EGFP form the Ret locus, and whole mount cytokeratin staining. Results: Mice lacking this tyrosine grow ectopic ureteric buds that ultimately will form supernumerary kidneys, a phenotype indistinguishable to that of Sprouty1 knockout mice. Sprouty1 knockin mice also present megaureters and vesicoureteral reflux, caused by failure of ureters to separate from Wolffian ducts and migrate to their definitive position. Conclusions: Tyrosine 53 is absolutely necessary to convey Sprouty1 function during genitourinary development.This work was supported by grants BFU2010-47175-P and BFU2017-83646-P (AEI/FEDER, UE) from MINECO to ME. MV was supported by a predoctoral fellowship from AGAUR. CA was supported by a predoctoral fellowship from Universitat de Lleida. SC was supported by a Cofund action from the Marie Curie program of the EU. We are grateful to Dr. Sanjay Jain (Washington University, St Louis) for sharing RetEGFP mice, and to Dr. Tung-Tien Sun (New York University) for Uroplakin antibody. We thank Anna Macià (IRB Lleida) for her contribution to the initial development of this manuscript, as well as Marta Hereu, Maria Santacana, Mónica Domingo and Maria Carrele for their excellent technical assistance

FLRT2 and FLRT3 Cooperate in Maintaining the Tangential Migratory Streams of Cortical Interneurons during Development

Neuron migration is a hallmark of nervous system development that allows gathering of neurons from different origins for assembling of functional neuronal circuits. Cortical inhibitory interneurons arise in the ventral telencephalon and migrate tangentially forming three transient migratory streams in the cortex before reaching the final laminar destination. Although migration defects lead to the disruption of inhibitory circuits and are linked to aspects of psychiatric disorders such as autism and schizophrenia, the molecular mechanisms controlling cortical interneuron development and final layer positioning are incompletely understood. Here, we show that mouse embryos with a double deletion of FLRT2 and FLRT3 genes encoding cell adhesion molecules exhibit an abnormal distribution of interneurons within the streams during development, which in turn, affect the layering of somatostatin+ interneurons postnatally. Mechanistically, FLRT2 and FLRT3 proteins act in a noncell-autonomous manner, possibly through a repulsive mechanism. In support of such a conclusion, double knockouts deficient in the repulsive receptors for FLRTs, Unc5B and Unc5D, also display interneuron defects during development, similar to the FLRT2/FLRT3 mutants. Moreover, FLRT proteins are chemorepellent ligands for developing interneurons in vitro, an effect that is in part dependent on FLRT-Unc5 interaction. Together, we propose that FLRTs act through Unc5 receptors to control cortical interneuron distribution in a mechanism that involves cell repulsion.This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2010-1805, BFU2013-48563-P, and PGC2018-101910-B-I00 to J.E. and BES-2014-067618 to P.M.-O.), FP7-PEOPLE-2011-CIG (PCIG9-GA-2011-293980 to J.E.), the Max-Planck Society (R.K.), and the Jade Plus Fellowship Program 2011–2014 (C.F.). We thank Tristan Rodríguez (Sox1-Cre line) and Anne Eichmann (Unc5Blx line) for the transgenic mice; Michèle Studer for the vasointestinal peptide probe; Eve Seuntjens and Veronique van den Berghe for scientific discussion; Inmaculada Segura for reading the manuscript; Serafí Cambray, Alex Espinós, Ma José Menal, Inma Montoliu, Montse Ortega, Noel Pérez, Sònia Rius, Marc Tarrés, and the University of Lleida Scientific and Technical Services for technical assistance, and the University of Lleida animal house staff facility for animal care

Caveolin-1 is required for TGF-β-induced transactivation of the EGF receptor pathway in hepatocytes through the activation of the metalloprotease TACE/ADAM17

Transforming growth factor-beta (TGF-β) plays a dual role in hepatocytes, inducing both pro- and anti-apoptotic responses, whose balance decides cell fate. Survival signals are mediated by the epidermal growth factor receptor (EGFR) pathway, which is activated by TGF-β in these cells. Caveolin-1 (Cav1) is a structural protein of caveolae linked to TGF-β receptors trafficking and signaling. Previous results have indicated that in hepatocytes, Cav1 is required for TGF-β-induced anti-apoptotic signals, but the molecular mechanism is not fully understood yet. In this work, we show that immortalized Cav1−/− hepatocytes were more sensitive to the pro-apoptotic effects induced by TGF-β, showing a higher activation of caspase-3, higher decrease in cell viability and prolonged increase through time of intracellular reactive oxygen species (ROS). These results were coincident with attenuation of TGF-β-induced survival signals in Cav1−/− hepatocytes, such as AKT and ERK1/2 phosphorylation and NFκ-B activation. Transactivation of the EGFR pathway by TGF-β was impaired in Cav1−/− hepatocytes, which correlated with lack of activation of TACE/ADAM17, the metalloprotease responsible for the shedding of EGFR ligands. Reconstitution of Cav1 in Cav1−/− hepatocytes rescued wild-type phenotype features, both in terms of EGFR transactivation and TACE/ADAM17 activation. TACE/ADAM17 was localized in detergent-resistant membrane (DRM) fractions in Cav1+/+ cells, which was not the case in Cav1−/− cells. Disorganization of lipid rafts after treatment with cholesterol-binding agents caused loss of TACE/ADAM17 activation after TGF-β treatment. In conclusion, in hepatocytes, Cav1 is required for TGF-β-mediated activation of the metalloprotease TACE/ADAM17 that is responsible for shedding of EGFR ligands and activation of the EGFR pathway, which counteracts the TGF-β pro-apoptotic effects. Therefore, Cav1 contributes to the pro-tumorigenic effects of TGF-β in liver cancer cells

A dominant negative mutation uncovers cooperative control of caudal Wolffian duct development by Sprouty genes

The Wolffian ducts (WD) are paired epithelial tubules central to the development of the mammalian genitourinary tract. Outgrowths from the WD known as the ureteric buds (UB) generate the collecting ducts of the kidney. Later during development, the caudal portion of the WD will form the vas deferens, epididymis and seminal vesicle in males, and will degenerate in females. While the genetic pathways controlling the development of the UB are firmly established, less is known about those governing development of WD portions caudal to the UB. Sprouty proteins are inhibitors of receptor tyrosine kinase (RTK) signaling in vivo. We have recently shown that homozygous mutation of a conserved tyrosine (Tyr53) of Spry1 results in UB defects indistinguishable from that of Spry1 null mice. Here, we show that heterozygosity for the Spry1 Y53A allele causes caudal WD developmental defects consisting of ectopically branched seminal vesicles in males and persistent WD in females, without affecting kidney development. Detailed analysis reveals that this phenotype also occurs in Spry1+/- mice but with a much lower penetrance, indicating that removal of tyrosine 53 generates a dominant negative mutation in vivo. Supporting this notion, concomitant deletion of one allele of Spry1 and Spry2 also recapitulates the genital phenotype of Spry1Y53A/+ mice with high penetrance. Mechanistically, we show that unlike the effects of Spry1 in kidney development, these caudal WD defects are independent of Ret signaling, but can be completely rescued by lowering the genetic dosage of Fgf10. In conclusion, mutation of tyrosine 53 of Spry1 generates a dominant negative allele that uncovers fine-tuning of caudal WD development by Sprouty genes.Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This work was supported by grants BFU2017-83646-P (MINECO) and PID2020-114947 GB-I00 (MCIU) (both supported by funds from AEI/FEDER, UE) to ME. MV was supported by a predoctoral fellowship from AGAUR. GA and CA and GA are supported by a fellowship from Universitat de Lleida. SC was supported by a Cofund action from the Marie Curie program of the E

The role of viral and host microRNAs in the Aujeszky's disease virus during the infection process

Porcine production is a primary market in the world economy. Controlling swine diseases in the farm is essential in order to achieve the sector necessities. Aujeszky's disease is a viral condition affecting pigs and is endemic in many countries of the world, causing important economic losses in the swine industry. microRNAs (miRNAs) are non-coding RNAs which modulates gene expression in animals, plants and viruses. With the aim of understanding miRNA roles during the Aujeszky's disease virus [ADV] (also known as suid herpesvirus type 1 [SuHV-1]) infection, the expression profiles of host and viral miRNAs were determined through deep sequencing in SuHV-1 infected porcine cell line (PK-15) and in an animal experimental SuHV-1 infection with virulent (NIA-3) and attenuated (Begonia) strains. In the in vivo approach miR-206, miR-133a, miR-133b and miR-378 presented differential expression between virus strains infection. In the in vitro approach, most miRNAs were down-regulated in infected groups. miR-92a and miR-92b-3p were up-regulated in Begonia infected samples. Functional analysis of all this over expressed miRNAs during the infection revealed their association in pathways related to viral infection processes and immune response. Furthermore, 8 viral miRNAs were detected by stem loop RT-qPCR in both in vitro and in vivo approaches, presenting a gene regulatory network affecting 59 viral genes. Most described viral miRNAs were related to Large Latency Transcript (LLT) and to viral transcription activators EP0 and IE180, and also to regulatory genes regarding their important roles in the host-pathogen interaction during viral infection

Identification of microRNAs in PCV2 subclinically infected pigs by high throughput sequencing

Porcine circovirus type 2 (PCV2) is the essential etiological infectious agent of PCV2-systemic disease and has been associated with other swine diseases, all of them collectively known as porcine circovirus diseases. MicroRNAs (miRNAs) are a new class of small non-coding RNAs that regulate gene expression post-transcriptionally. miRNAs play an increasing role in many biological processes. The study of miRNA-mediated host-pathogen interactions has emerged in the last decade due to the important role that miRNAs play in antiviral defense. The objective of this study was to identify the miRNA expression pattern in PCV2 subclinically infected and non-infected pigs. For this purpose an experimental PCV2 infection was carried out and small-RNA libraries were constructed from tonsil and mediastinal lymph node (MLN) of infected and non-infected pigs. High throughput sequencing determined differences in miRNA expression in MLN between infected and non-infected while, in tonsil, a very conserved pattern was observed. In MLN, miRNA 126-3p, miRNA 126-5p, let-7d-3p, mir-129a and mir-let-7b-3p were up-regulated whereas mir-193a-5p, mir-574-5p and mir-34a down-regulated. Prediction of functional analysis showed that these miRNAs can be involved in pathways related to immune system and in processes related to the pathogenesis of PCV2, although functional assays are needed to support these predictions. This is the first study on miRNA gene expression in pigs infected with PCV2 using a high throughput sequencing approach in which several host miRNAs were differentially expressed in response to PCV2 infection

Common Variation in the PIN1 Locus Increases the Genetic Risk to Suffer from Sertoli Cell-Only Syndrome

Funding Information: Funding: This work was supported by the Plan Andaluz de Investigación, Desarrollo e Innovación (PAIDI 2020) (ref. PY20_00212, P20_00583), and the Spanish Ministry of Economy and Competitiveness through the Spanish National Plan for Scientific and Technical Research and Innovation (ref. SAF2016–78722-R, PID2020–120157RB-I00) and the Proyectos I + D + i del Programa Operativo FEDER 2020 (ref. B-CTS-584-UGR20, B-CTS-260-UGR20). FDC was supported by the “Ramón y Cajal” program (ref. RYC-2014–16458), and LBC was supported by the Spanish Ministry of Economy and Competitiveness through the “Juan de la Cierva Incorporación” program (Grant ref. IJC2018– 038026-I, funded by MCIN/AEI/10.13039/501100011033), all of them including FEDER funds. AGJ was funded by MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro”(grant ref. FPU20/02926). SGM was funded by a previously mentioned project (ref. PY20_00212). IPATIMUP integrates the i3S Research Unit, which is partially supported by the Portuguese Foundation for Science and Technology (FCT), financed by the European Social Funds (COMPETE-FEDER) and National Funds (projects PEstC/SAU/LA0003/2013 and POCI-01–0145-FEDER-007274). AML is funded by the Portuguese Government through FCT (IF/01262/2014). PIM is supported by the FCT post-doctoral fellowship (SFRH/BPD/120777/2016), financed from the Portuguese State Budget of the Ministry for Science, Technology and High Education and from the European Social Fund, available through the Programa Operacional do Capital Humano. ToxOmics—Centre for Toxicogenomics and Human Health, Genetics, Oncology and Human Toxicology, Nova Medical School, Lisbon, is also partially supported by FCT (Projects: UID/BIM/00009/2013 and UIDB/UIDP/00009/2020). SLarriba received support from Instituto de Salud Carlos III (grant DTS18/00101], co-funded by FEDER funds/European Regional Development Fund (ERDF)—a way to build Europe), and from “Generalitat de Catalunya” (grant 2017SGR191). SLarriba is sponsored by the “Researchers Consolidation Program” from the SNS-Dpt. Salut Generalitat de Catalunya (Exp. CES09/020). This article is related to the Ph.D. Doctoral Thesis of Miriam Cerván-Martín (grant ref. BES-2017–081222 funded by MCIN/AEI/10.13039/501100011033 and FSE “El FSE invierte en tu futuro”). Publisher Copyright: © 2022 by the authors. Licensee MDPI, Basel, Switzerland.We aimed to analyze the role of the common genetic variants located in the PIN1 locus, a relevant prolyl isomerase required to control the proliferation of spermatogonial stem cells and the integrity of the blood–testis barrier, in the genetic risk of developing male infertility due to a severe spermatogenic failure (SPGF). Genotyping was performed using TaqMan genotyping assays for three PIN1 taggers (rs2287839, rs2233678 and rs62105751). The study cohort included 715 males diagnosed with SPGF and classified as suffering from non-obstructive azoospermia (NOA, n = 505) or severe oligospermia (SO, n = 210), and 1058 controls from the Iberian Peninsula. The allelic frequency differences between cases and controls were analyzed by the means of logistic regression models. A subtype specific genetic association with the subset of NOA patients classified as suffering from the Sertoli cell-only (SCO) syndrome was observed with the minor alleles showing strong risk effects for this subset (ORaddrs2287839 = 1.85 (1.17–2.93), ORaddrs2233678 = 1.62 (1.11–2.36), ORaddrs62105751 = 1.43 (1.06–1.93)). The causal variants were predicted to affect the binding of key transcription factors and to produce an altered PIN1 gene expression and isoform balance. In conclusion, common non-coding single-nucleotide polymorphisms located in PIN1 increase the genetic risk to develop SCO.publishersversionpublishe

Contribution of TEX15 genetic variants to the risk of developing severe non-obstructive oligozoospermia

Background: Severe spermatogenic failure (SPGF) represents one of the most relevant causes of male infertility. This pathological condition can lead to extreme abnormalities in the seminal sperm count, such as severe oligozoospermia (SO) or non-obstructive azoospermia (NOA). Most cases of SPGF have an unknown aetiology, and it is known that this idiopathic form of male infertility represents a complex condition. In this study, we aimed to evaluate whether common genetic variation in TEX15, which encodes a key player in spermatogenesis, is involved in the susceptibility to idiopathic SPGF.Materials and Methods: We designed a genetic association study comprising a total of 727 SPGF cases (including 527 NOA and 200 SO) and 1,058 unaffected men from the Iberian Peninsula. Following a tagging strategy, three tag single-nucleotide polymorphisms (SNPs) of TEX15 (rs1362912, rs323342, and rs323346) were selected for genotyping using TaqMan probes. Case-control association tests were then performed by logistic regression models. In silico analyses were also carried out to shed light into the putative functional implications of the studied variants.Results: A significant increase in TEX15-rs1362912 minor allele frequency (MAF) was observed in the group of SO patients (MAF = 0.0842) compared to either the control cohort (MAF = 0.0468, OR = 1.90, p = 7.47E-03) or the NOA group (MAF = 0.0472, OR = 1.83, p = 1.23E-02). The genotype distribution of the SO population was also different from those of both control (p = 1.14E-02) and NOA groups (p = 4.33-02). The analysis of functional annotations of the human genome suggested that the effect of the SO-associated TEX15 variants is likely exerted by alteration of the binding affinity of crucial transcription factors for spermatogenesis.Conclusion: Our results suggest that common variation in TEX15 is involved in the genetic predisposition to SO, thus supporting the notion of idiopathic SPGF as a complex trait