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[eng] The Central nervous system (CNS) originates from the coordinated events that result into neural specification and the morphogenic events that shape the neural tube (NT). Then, the embryonic NT should grow and generate all the cell diversity present in the healthy organ. The morphogenic events that shape the NT occur in two consecutive, radically different processes referred as primary and secondary neurulation, that can be followed at different anterio-posterior levels in the Spinal cord (SpC). The posterior NT is formed by secondary neurulation, in a process concomitant to body axis elongation and mediated by the specification of nueromesodermal progenitors (NMPs). Defects in this process lead to caudal neural tube defects (NTDs). In the first chapter of this thesis, a new human 3D in vitro model for posterior SpC is set up. In these human organoid models, the neural specification, the morphogenesis of the NT, and its grow can be followed. Here, it is characterized a human organoid model were human embryonic stem cells (hESC) are guided into NMPs, expressing SOX2 and BRA and then into neural progenitor cells (NPCs), which maintain the SOX2 and lose BRA expression as it happens in vivo. Moreover, the NPCs are organized as an epithelium surrounding a central lumen. NPCs locate the centrosome and cilia at the lumen surface, where the polarity complexes are organized mimicking the polarity features of NPCs characterized in vivo. Additionally, in parallel to this epithelialization, the cell rearranges that shape the hollow NT formation in vivo, like the cell intercalation driving lumen resolution, can be followed in this organoid model. In the second chapter of this thesis, a screening to identify new mature centrosome com- ponents that would be potentially control the NPCs proliferation/differentiation rates was done independently with an in vivo, using the chick embryo as model, and an in silico approach. Unfortunately, the in vivo approach faced technical limitations intrinsic of the chick embryo model that prevent any successful identification of new candidates. However, the in silico approach provided a list of candidates to start their functional analysis., [spa] El desarrollo del sistema nervioso central (SNC) depende de la especificación del tejido neural y una serie de eventos morfogénicos que lo organizan en un tubo neural (NT). A partir de ahí, ese NT deberá crecer y generar toda la diversidad celular que presenta el órgano sano y le confiere su funcionalidad. Los eventos morfogénicos que dan forma al NT ocurren en dos procesos consecutivos radicalmente diferentes denominados neurulación primaria y secundaria, que pueden seguirse a diferentes niveles anteroposteriores en la médula espinal (SpC). El NT posterior se forma por neurulación secundaria, en un proceso simultáneo a la elongación del embrión y mediado por la especificación de progenitores nueromesodérmicos (NMP). Los defectos en este proceso conducen a defectos del tubo neural caudal (NTDs). En el primer capítulo de esta tesis, se establece in vitro un nuevo modelo 3D de SpC posterior humana. En estos organoides, se puede seguir la especificación neural, los procesos morfogenéticos de la formación del NT y su crecimiento. Aquí, se caracteriza un modelo de organoides humanos en el que las células madre embrionarias humanas (hESC) se guían hacia NMP, expresando SOX2 y BRA, que a continuación se diferencian en células progenitoras neurales (NPC). Estas últimas mantienen la SOX2 y pierden la expresión de BRA como sucede in vivo. Además, los NPC organizan un epitelio que rodea un único lumen central. Los NPC presentan su centrosoma y cilio en la superficie del lumen, donde los complejos de polaridad se organizan mimetizando las características de polaridad apical observadas in vivo. Además, en estos organoides se pueden seguir los reordenamientos celulares de los procesos morfogénicos que eventualmente forman un tubo neural hueco. En el segundo capítulo de esta tesis, se realizó un screening para identificar nuevos componentes de maduración del centrosoma que potencialmente controlarían el equilibrio de proliferación/diferenciación de los NPC. Este screening se realizó de forma independiente in vivo, utilizando el embrión de pollo como modelo, e in silico. Desafortunadamente, el enfoque in vivo enfrentó limitaciones técnicas intrínsecas del modelo que impidieron la identificación de nuevos candidatos. Sin embargo, el enfoque in silico proporcionó una lista de candidatos sobre los que comenzar un análisis funcional.

The clinical spectrum of GRIN-related neurodevelopmental disorders (GRD) results from gene- and variant-dependent primary alterations of the NMDA receptor, disturbing glutamatergic neurotransmission. Despite GRIN gene variants’ functional annotations being dually critical for stratification and precision medicine design, genetically diagnosed pathogenic GRIN variants currently outnumber their relative functional annotations. Based on high-resolution crystal 3D models and topological domains conservation between GluN1, GluN2A, and GluN2B subunits of the NMDAR, we have generated GluN1-GluN2A-GluN2B subunits structural superimposition model to find equivalent positions between GluN subunits. We have developed a GRIN structural algorithm that predicts functional changes in the equivalent structural positions in other GluN subunits. GRIN structural algorithm was computationally evaluated to the full GRIN missense variants repertoire, consisting of 4,525 variants. The analysis of this structure-based model revealed an absolute predictive power for GluN1, GluN2A, and GluN2B subunits, both in terms of pathogenicity-association (benign vs. pathogenic variants) and functional impact (loss-of-function, benign, gain-of-function). Further, we validated this computational algorithm experimentally, using an in silico library of GluN2B-equivalent GluN2A artificial variants, designed from pathogenic GluN2B variants. Thus, the implementation of the GRIN structural algorithm allows to computationally predict the pathogenicity and functional annotations of GRIN variants, resulting in the duplication of pathogenic GRIN variants assignment, reduction by 30% of GRIN variants with uncertain significance, and increase by 70% of functionally annotated GRIN variants. Finally, GRIN structural algorithm has been implemented into GRIN variants Database (http://lmc.uab.es/grindb), providing a computational tool that accelerates GRIN missense variants stratification, contributing to clinical therapeutic decisions for this neurodevelopmental disorder.

[eng] Intrauterine growth restriction (IUGR) is defined as a significant reduction on fetal growth rate, resulting in a birth weight below the 10th percentile for the corresponding gestational age (Sharma et al., 2016). The prevalence accounts for 5-10% of all pregnancies, and amounts to approximately 600.000 cases in Europe, being a serious health problem worldwide (Kady and Gardosi, 2004). Placental insufficiency, the main cause of IUGR, chronically decreases the blood flow and nutrient supply to the developing fetus resulting in an adverse intrauterine environment with chronic hypoxia conditions and undernutrition. This situation results to a wide range of changes in brain development including grey (GM) and white matter (WM) injury (Esteban et al., 2010; Pla et al., 2020), which are associated with short- and long-term neurodevelopmental damage and cognitive dysfunctions (Mwaniki et al., 2012; Batalle et al., 2014; Eixarch et al., 2016). The most prevalent causes of brain damage of prenatal origin manifest as subtle neurological abnormalities. Indeed, IUGR has been proposed as the cause of one-quarter of special educational needs postnatally (Mackay et al., 2013). But currently, there is no efficient treatment which avoids deleterious consequences related to IUGR, especially in the neurodevelopmental field. To better understand, which basic cellular processes of brain development are altered due to IUGR, we established a novel in vitro model based on primary rabbit neuronal progenitor cells (NPCs) (Barenys et al., 2021). IUGR was surgically induced in one uterus horn in pregnant rabbits on gestational day (GD) 25. Neural progenitor cells (NPCs) growing as three-dimensional (3D) cell aggregates known as neurospheres were obtained from rabbit pups’ brains immediately after caesarean delivery at GD30. Neurospheres are able to mimic basic processes of brain development such as NPC proliferation, migration, differentiation into the brain effector cells neurons, oligodendrocytes and astrocytes, synaptogenesis, and network formation (Barenys et al., 2017; Breier et al., 2010; Gassmann et al., 2010a; Moors et al., 2009, 2007a; Schreiber et al., 2010). We revealed a significantly lower ability to form oligodendrocytes due to a slower differentiation rate in IUGR neurospheres. This result correlates very well with the clinical outcome of IUGR-generated white matter alterations. In addition, IUGR neurospheres presented an increased neurite length, which is consistent with previous in vivo studies (Pla et al., 2020). We have discovered for the first time that IUGR neurospheres respond differently than control to the exposure of the compound EGCG, which triggers migration alterations, and we have revealed the mechanism behind this difference: an overexpression of the adhesion molecule Integrin-β1 in IUGR. Because Integrin-β1 is implied in NPC migration but also in axonal growth and neuronal branching, this discovery gives new insights into the characterization of IUGR-induced neurodevelopmental alterations. The thesis addresses the medical necessity by assessing the safety and efficacy of the potential neuroprotective therapies docosahexaenoic acid (DHA), melatonin (MEL), 3,3',5-triiodothyronine (T3), zinc, lactoferrin (LF) and its main metabolite sialic acid (SA), epigallocatechin gallate (EGCG) and derivatives in two different approaches: (1) exposure in vitro and (2) prenatal administration in vivo, both followed by the evaluation in vitro. DHA, and MEL, were identified as the best therapeutical agents for preventing/reverting impaired oligodendrogenesis caused by IUGR. LF and its main metabolite SA were revealed to reduce IUGR-induced neurite length extension. Finally, we integrated the discovered results about IUGR-induced changes in neurodevelopment into an “adverse outcome pathway” (AOP) approach and developed the putative AOP “Disrupted laminin-β1-integrin interaction leading to developmental neurotoxicity”. Overall, the novel neurosphere model is well suited for characterizing so far unknown neurodevelopmental effects on the cellular level induced by chemicals or IUGR. This new method opens the door to testing possible neuroprotective therapies for IUGR easily and cost- efficiently., [spa] El retraso del crecimiento intrauterino (RCIU), un grave problema de salud en todo el mundo, se define como una reducción significativa de la tasa de crecimiento fetal que da lugar a un peso al nacer inferior al percentil 10 para la edad gestacional correspondiente. La insuficiencia placentaria, principal causa del RCIU, reduce el flujo sanguíneo y el aporte de nutrientes al bebé en desarrollo, lo que provoca daños cerebrales y deterioro cognitivo a corto y largo plazo. En este trabajo, desarrollamos por primera vez un modelo de neuroesfera de conejo in vitro basado en células progenitoras neurales (CPN) primarias que imita el desarrollo cerebral complicado por RCIU. Las neuroesferas son agregados celulares en 3D capaces de reproducir procesos básicos del neurodesarrollo como la proliferación, la migración, la diferenciación, la sinaptogénesis y la formación de redes de CPNs. Las neuroesferas del grupo RCIU mostraron una tasa significativamente reducida de diferenciación de oligodendrocitos, que se correlaciona muy bien con los resultados clínico que describen lesiones de la materia blanca. Además, las neuronas de las neuroesferas IUGR mostraron neuritas significativamente más largas. Encontramos que las neuroesferas IUGR responden de forma diferente a las neuroesferas control respecto al compuesto EGCG, que induce alteraciones en la migración. Esta diferencia se debe a la sobreexpresión de la molécula de adhesión integrina-β1, que está implicada en la migración de las CNP, así como en el crecimiento axonal y la ramificación neuronal. Mediante estudios toxicológicos y farmacológicos, evaluamos la seguridad y eficacia de potenciales terapias neuroprotectoras utilizando el novedoso modelo de neuroesfera de conejo in vitro. El DHA y la MEL se identificaron como terapias beneficiosas por sus efectos promotores de la diferenciación de los oligodendrocitos no sólo tras la exposición in vitro, sino también tras la administración prenatal durante la gestación en conejo. La LF y su principal metabolito SA redujeron la longitud de las neuritas debido a una posible interacción entre la integrina-β1 y la proteína de la matriz extracelular laminina. Finalmente, integramos los resultados obtenidos en una “adverse outcome pathway” (AOP) diseñando el AOP putativo "Disrupted laminin-β1-integrin interaction leading to developmental neurotoxicity". En general, el nuevo modelo de neuroesferas resulta muy adecuado para caracterizar de forma rápida y rentable las alteraciones desconocidas del neurodesarrollo causadas por sustancias químicas o por la RCI. En futuros estudios, también será importante medir los efectos y la seguridad de las terapias probadas no sólo en términos de neurodesarrollo, sino también en términos de parámetros generales de desarrollo.

Emerging evidence points to coordinated action of chemical and mechanical cues during brain development. At early stages of neocortical development, angiogenic factors and chemokines such as CXCL12, ephrins, and semaphorins assume crucial roles in orchestrating neuronal migration and axon elongation of postmitotic neurons. Here we explore the intrinsic mechanical properties of the developing marginal zone of the pallium in the migratory pathways and brain distribution of the pioneer Cajal-Retzius cells. These neurons are generated in several proliferative regions in the developing brain (e.g., the cortical hem and the pallial subpallial boundary) and migrate tangentially in the preplate/marginal zone covering the upper portion of the developing cortex. These cells play crucial roles in correct neocortical layer formation by secreting several molecules such as Reelin. Our results indicate that the motogenic properties of Cajal-Retzius cells and their perinatal distribution in the marginal zone are modulated by both chemical and mechanical factors, by the specific mechanical properties of Cajal-Retzius cells, and by the differential stiffness of the migratory routes. Indeed, cells originating in the cortical hem display higher migratory capacities than those generated in the pallial subpallial boundary which may be involved in the differential distribution of these cells in the dorsal-lateral axis in the developing marginal zone.

Changes in the transcription factor (TF) expression are critical for brain development, and they may also underlie neurodevelopmental disorders. Indeed, T-box brain1 (Tbr1) is a TF crucial for the formation of neocortical layer VI, and mutations and microdeletions in that gene are associated with malformations in the human cerebral cortex, alterations that accompany autism spectrum disorder (ASD). Interestingly, Tbr1 upregulation has also been related to the occurrence of ASD-like symptoms, although limited studies have addressed the effect of increased Tbr1 levels during neocortical development. Here, we analysed the impact of Tbr1 misexpression in mouse neural progenitor cells (NPCs) at embryonic day 14.5 (E14.5), when they mainly generate neuronal layers II–IV. By E18.5, cells accumulated in the intermediate zone and in the deep cortical layers, whereas they became less abundant in the upper cortical layers. In accordance with this, the proportion of Sox5+ cells in layers V–VI increased, while that of Cux1+ cells in layers II–IV decreased. On postnatal day 7, fewer defects in migration were evident, although a higher proportion of Sox5+ cells were seen in the upper and deep layers. The abnormal neuronal migration could be partially due to the altered multipolar-bipolar neuron morphologies induced by Tbr1 misexpression, which also reduced dendrite growth and branching, and disrupted the corpus callosum. Our results indicate that Tbr1 misexpression in cortical NPCs delays or disrupts neuronal migration, neuronal specification, dendrite development and the formation of the callosal tract. Hence, genetic changes that provoke ectopic Tbr1 upregulation during development could provoke cortical brain malformations.

Long noncoding RNAs (lncRNAs) are regulatory molecules which have been traditionally considered as “non-coding”. Strikingly, recent evidence has demonstrated that many non-coding regions, including lncRNAs, do in fact contain small-open reading frames that code for small proteins that have been called microproteins. Only a few of them have been characterized so far, but they display key functions in a wide variety of cellular processes. Here, we show that TUNAR lncRNA encodes an evolutionarily conserved microprotein expressed in the nervous system that we have named pTUNAR. pTUNAR deficiency in mouse embryonic stem cells improves their differentiation potential towards neural lineage both in vitro and in vivo. Conversely, pTUNAR overexpression impairs neuronal differentiation by reduced neurite formation in different model systems. At the subcellular level, pTUNAR is a transmembrane protein that localizes in the endoplasmic reticulum and interacts with the calcium transporter SERCA2. pTUNAR overexpression reduces cytoplasmatic calcium, consistent with a possible role of pTUNAR as an activator of SERCA2. Altogether, our results suggest that our newly discovered microprotein has an important role in neural differentiation and neurite formation through the regulation of intracellular calcium. From a more general point of view, our results provide a proof of concept of the role of lncRNAs-encoded microproteins in neural differentiation., Work in the Abad lab is supported by VHIO, Fero Foundation, La Caixa Foundation (HR18-00256), Asociación Española Contra el Cancer (AECC), Cellex Foundation, Mutua Madrileña Foundation and by grants from the Spanish Ministry of Science and Innovation (SAF2015-69413-R; RTI2018-102046-B-I00). M.A. was recipient of a Ramon y Cajal contract from the Spanish Ministry of Science and Innovation (RYC-2013-14747). E.S. was recipient of a AECC Postdoctoral Fellowship. L.H-M. also acknowledges funding from grants SAF2017-88019-C3-1R y PID2020-116927RB-C21 from the Spanish Government. MG is supported by the advanced ERC grant NeuroCentro and the German Research Foundation (SFB870; SPP2202; SPP2306; SYNERGY; TRR274). DT is supported by the Ramón y Cajal program (RYC-2017-23486/RTI2018-095580-A-I00). MMA acknowledges funding from the Spanish Ministry of Science and Innovation PGC2018-094091-B-I00 co-funded by FEDER.

Detailed neural encoding of voice pitch and formant structure plays a crucial role in speech perception, and is of key importance for an appropriate acquisition of the phonetic repertoire in infants since birth. However, the extent to what newborns are capable of extracting pitch and formant structure information from the temporal envelope and the temporal fine structure of speech sounds, respectively, remains unclear. Here, we recorded the frequency-following response (FFR) elicited by a novel two-vowel, rising-pitch-ending stimulus to simultaneously characterize voice pitch and formant structure encoding accuracy in a sample of neonates and adults. Data revealed that newborns tracked changes in voice pitch reliably and no differently than adults, but exhibited weaker signatures of formant structure encoding, particularly at higher formant frequency ranges. Thus, our results indicate a well-developed encoding of voice pitch at birth, while formant structure representation is maturing in a frequency-dependent manner. Furthermore, we demonstrate the feasibility to assess voice pitch and formant structure encoding within clinical evaluation times in a hospital setting, and suggest the possibility to use this novel stimulus as a tool for longitudinal developmental studies of the auditory system.

Treball de fi de grau en Biologia Humana Supervisora: Cristina Pujades The Nuclear factor I A (Nfia) is a transcription factor which seems to be a key element during late embryonic brain neurogenesis. Since most of the studies about the role of Nfia in the Central Nervous System (CNS) are mainly focused on later stages, this project aims to shed light on the role of Nfia at embryonic stages. Firstly, I review the Nfia factor and its functions in the developing CNS. As there are hints that Nfia activity crosstalks with Notch signalling, I also review the Notch pathway and its operating mechanisms. Finally, I discuss the potential interplay between Nfia and Notch signalling. The main conclusions are that nfia is expressed in progenitor cells of the developing CNS, where it inhibits neurogenesis by promoting the maintenance of an undifferentiated pool of cells. These can give rise to late-born neurons. This role of maintaining cells in their undifferentiated state might be promoted by the synergy between Notch signalling and Nfia. Further studies are required in order to completely understand how this synergic mechanism is carried out. To finish this project, I propose several experiments in the context of zebrafish hindbrain which could help to answer some of the remaining questions.

[eng] Neurodevelopmental disorders (NDDs) are a group of chronic diseases in which the development of the central nervous system is impaired, resulting in disability at the neuropsychiatric, motor and/or intellectual level. Some of these disorders are considered syndromic. For instance, intellectual disability (ID) may present comorbidity with other neurological conditions (such as seizures or behavioral problems), dysmorphic features and/or internal organ anomalies. The vast majority of syndromic NDDs have a genetic origin and are considered to be rare, affecting less than 1 in 2000 people. However, globally, these diseases represent a serious social and health issue. Even though the majority of them are monogenic, many of them remain with an unknown molecular basis. Next-generation sequencing technologies have played a critical role in the optimization of the diagnosis of NDDs during the last decades. In this thesis, we have used a combination of whole-exome sequencing (WES) and functional studies to establish the diagnosis of 9 cases tentatively diagnosed as Opitz C, a clinical entity that encompasses patients with very different molecular causes. We identified the genetic origin of the disorder in the 7 tested families, which happened to be different variants in different genes for each of them. We showed that WES is a powerful approach to identify the molecular basis of ultra-rare NDDs. A significantly higher diagnosis yield was reached compared with other studies, potentially explained by a deep analysis of the sequencing data using in silico predictors, followed by the performance of specific functional studies for each case. We identified four different variants putatively affecting splicing patterns of different genes (ASXL1, KAT6A, PIGT and FOXP1) and tested them directly using fibroblasts obtained from patients or indirectly using a mini-gene splicing assay. We assessed the effect of variants in DPH1 in protein function combining a biochemical technique with a protein structural model and we established a correlation between the results of the tests and the severity of the patients’ phenotype. We contributed to the delineation of a recently described syndrome caused by germline mutations in TRAF7 by gathering and describing a cohort of 45 patients. We also performed a transcriptomics analysis on fibroblasts from different patients carrying TRAF7 mutations, which showed alterations in the expression of different genes that might contribute to the phenotype. Aiming to characterize truncating mutations in MAGEL2, which are responsible for Schaaf-Yang syndrome (SYS), we performed different experiments that suggest a potential toxic effect of the produced truncated form of the protein, which lacks its most relevant functional domain. Finally, as a first step to establish a relevant in vitro model of SYS, we reprogrammed fibroblasts from different patients to induced pluripotent stem cells (iPSCs), which can be then differentiated to relevant neural cell types and brain organoids to further study the pathophysiological mechanisms underlying this disease.

[eng] The Tousled like kinases 1 and 2 (TLK1 and TLK2) are evolutionarily conserved Ser/Thr kinases that are required for genome stability and normal development in numerous organisms. Both kinases contribute to histone deposition by targeting the histone chaperone ASF1 and are regulated by the DNA damage response. Their expression is ubiquitous and often elevated in cancer cells with suppressed immune gene expression. In cultured cells, TLK depletion causes replication stress, DNA damage and altered chromatin maintenance, eliciting an innate immune response dependent on the Alternative-lengthening of telomeres pathway. This suggested that TLKs would be a potential target in cancer therapy. In addition, TLKs have been linked to a distinct neurodevelopmental disorder (NDD) known as Mental Retardation Autosomal Dominant (MRD57). How TLKs contribute to these pathologies remains largely unknown. The aims of this thesis are to explore the therapeutic potential of targeting TLKs in cancer and understand how TLK2 deficiency contributes to NDDs. To this end, we have generated conditional mouse models where we can modulate TLK2 expression in both embryonic development and in developing breast cancer. We have also mimicked TLK2 mutations from MRD57 patients in vitro and addressed how they alter the normal kinase activity, localization and interactome of TLK2. We showed that depletion of TLK2 in already formed tumors stalls tumor growth and reduces lung metastases, and observed an early immune infiltration that could potentially sensitize these tumors to immune checkpoint blockade. Novel small molecule inhibitors towards TLKs were identified, indicating that they are inherently druggable targets. Consistent with what we observed in cancer, deletion of TLK2 in the telencephalon of mouse embryos caused elevated transcription of inflammatory genes and signatures of microglia and astrocytes. We also described 6 new cases of MRD57 and showed that patient-derived cell lines exhibited defects in chromatin compaction. Separately we identified two hyperactive MRD57 mutations, suggesting misregulation of TLK2 levels, rather than decreased activity, is likely the predisposing factor leading to NDD. Lastly, we showed in cells that TLK2 is in close proximity with a number of known autism-susceptibility proteins and proteins enriched in replication forks, and that these proximal interactions are altered by MRD57 mutations. Together, these results further suggest that TLKs may be a viable target for cancer therapy, as their loss leads to innate immune activation that may also influence neurodevelopment in MRD57 patients., [cat] Les Tousled like kinases i and 2 (TLK1 i TLK2) són Ser-Thr quinases conservades evolutivament que són necessàries pel manteniment de l’estabilitat del genoma i pel desenvolupament normal de diversos organismes. Ambdues quinases contribueixen a la deposició d’histones mitjançant la regulació de la xaperona d’histones ASF1, i són regulades per les vies de resposta al dany de l’ADN. La seva expressió és ubiqua i sovint elevada en cèl·lules canceroses que presenten l’expressió de gens immunes suprimida. En cèl·lules cultivades, la depleció de les TLKs causa estrès replicatiu, dany en l’ADN i altera el manteniment de la cromatina, provocant una resposta immune innata depenent de la via d’allargament alternatiu dels telòmers. Això suggereix que les TLKs són una diana terapèutica potencial per al tractament del càncer. A més a més, les TLKs han estat vinculades a un trastorn del neurodesenvolupament (TND), anomenat Retard mental autosòmic dominant (MRD57). Com les TLKs contribueixen a aquestes patologies continua sent un misteri. Els objectius d’aquesta tesi són explorar el potencial terapèutic en càncer de les TLKs i entendre com el dèficit de TLK2 contribueix a TND. Per assolir aquests objectius, hem generat models condicionals de ratolí on podem modular l’expressió de TLK2 tant en desenvolupament embriònic com durant la progressió de tumors mamaris. També hem mimetitzat mutacions de pacients amb MRD57 in vitro i estudiat com alteren l’activitat quinasa, la localització i l’interactoma normal de TLK2. Hem demostrat que la depleció de TLK2 en tumors mamaris n’atura el creixement i redueix significativament les metàstasis als pulmons. També hem observat un augment en la infiltració de cèl·lules immunes als tumors que poden sensibilitzar-los a inhibidors dels punts de control immunitaris. Hem identificat nous inhibidors de molècules petites de les TLKs, reafirmant que són dianes drogables. Consistent amb el que hem vist en càncer, la supressió de TLK2 en el telencèfal d’embrions de ratolí causa un augment en la transcripció de gens inflamatoris i en signatures de micròglia i astròcits. Hem descrit 6 nous casos de MRD57 i demostrat que línies cel·lulars derivades d’aquests pacients presenten defectes en la compactació de la cromatina. Separadament hem identificat dues mutacions de MRD57 que són hiperactives, suggerint que la desregulació dels nivells de TLK2 predisposen al desenvolupament de TND. Per últim, hem demostrat en cèl·lules que TLK2 està a prop de diverses proteïnes que confereixen susceptibilitat a autisme, així com de proteïnes que localitzen a les forquilles de replicació. Aquestes interaccions es veuen alterades per les mutacions de MRD57. Conjuntament, aquests resultats ens indiquen que les TLK2 són una diana viable pel tractament del càncer, ja que la seva pèrdua activa una resposta innata immunitària, i aquesta mateixa resposta sembla que també influeix en el neurodesenvolupament de pacients amb MRD57.

During development, two coordinated events shape the morphology of the mammalian cerebral cortex, leading to the cortex's columnar and layered structure: the proliferation of neuronal progenitors and cortical migration. Pyramidal neurons originating from germinal zones migrate along radial glial fibers to their final position in the cortical plate by both radial migration and tangential dispersion. These processes rely on the delicate balance of intercellular adhesive and repulsive signaling that takes place between neurons interacting with different substrates and guidance cues. Here, we focus on the function of the cell adhesion molecules fibronectin leucine-rich repeat transmembrane proteins (FLRTs) in regulating both the radial migration of neurons, as well as their tangential spread, and the impact these processes have on cortex morphogenesis. In combining structural and functional analysis, recent studies have begun to reveal how FLRT-mediated responses are precisely tuned - from forming different protein complexes to modulate either cell adhesion or repulsion in neurons. These approaches provide a deeper understanding of the context-dependent interactions of FLRTs with multiple receptors involved in axon guidance and synapse formation that contribute to finely regulated neuronal migration.

Prenatal alcohol exposure is associated to different physical, behavioral, cognitive, and neurological impairments collectively known as fetal alcohol spectrum disorder. The underlying mechanisms of ethanol toxicity are not completely understood. Experimental studies during human pregnancy to identify new diagnostic biomarkers are difficult to carry out beyond genetic or epigenetic analyses in biological matrices. Therefore, animal models are a useful tool to study the teratogenic effects of alcohol on the central nervous system and analyze the benefits of promising therapies. Animal models of alcohol spectrum disorder allow the analysis of key variables such as amount, timing and frequency of ethanol consumption to describe the harmful effects of prenatal alcohol exposure. In this review, we aim to synthetize neurodevelopmental disabilities in rodent fetal alcohol spectrum disorder phenotypes, considering facial dysmorphology and fetal growth restriction. We examine the different neurodevelopmental stages based on the most consistently implicated epigenetic mechanisms, cell types and molecular pathways, and assess the advantages and disadvantages of murine models in the study of fetal alcohol spectrum disorder, the different routes of alcohol administration, and alcohol consumption patterns applied to rodents. Finally, we analyze a wide range of phenotypic features to identify fetal alcohol spectrum disorder phenotypes in murine models, exploring facial dysmorphology, neurodevelopmental deficits, and growth restriction, as well as the methodologies used to evaluate behavioral and anatomical alterations produced by prenatal alcohol exposure in rodents., This work was supported by Red de Salud Materno-Infantil y del Desarrollo (SAMID) (RD12/0026/0003 and RD16/0022/0002) from Instituto de Salud Carlos III and the PI15/01179 grant from Instituto de Salud Carlos III

17 páginas, 7 figuras., Although previous studies suggest that neural stem cells (NSCs) exist in the adult olfactory bulb (OB), their location, identity, and capacity to generate mature neurons in vivo has been little explored. Here, we injected enhanced green fluorescent protein (EGFP)-expressing retroviral particles into the OB core of adult mice to label dividing cells and to track the differentiation/maturation of any neurons they might generate. EGFP-labeled cells initially expressed adult NSC markers on days 1 to 3 postinjection (dpi), including Nestin, GLAST, Sox2, Prominin-1, and GFAP. EGFP+ -doublecortin (DCX) cells with a migratory morphology were also detected and their abundance increased over a 7-day period. Furthermore, EGFP-labeled cells progressively became NeuN+ neurons, they acquired neuronal morphologies, and they became immunoreactive for OB neuron subtype markers, the most abundant representing calretinin expressing interneurons. OB-NSCs also generated glial cells, suggesting they could be multipotent in vivo. Significantly, the newly generated neurons established and received synaptic contacts, and they expressed presynaptic proteins and the transcription factor pCREB. By contrast, when the retroviral particles were injected into the subventricular zone (SVZ), nearly all (98%) EGFP+ -cells were postmitotic when they reached the OB core, implying that the vast majority of proliferating cells present in the OB are not derived from the SVZ. Furthermore, we detected slowly dividing label-retaining cells in this region that could correspond to the population of resident NSCs. This is the first time NSCs located in the adult OB core have been shown to generate neurons that incorporate into OB circuits in vivo., This work was funded by grants from the Spanish “Ministerio de Economía y Competitividad and Ministerio de Ciencia, Innovacion y Universidades ” (MINECO and MICIU: SAF2013-4759R and SAF2016-80419-R, PID2019-109059RB-I00 to C.V., SAF2015-70433-R to H.M., BFU2016-80870-P to A.C., and CIBERNED CB06/05/0065 to C.V.), from the “Comunidad de Madrid” (S2011/BMD-2336) to C.V. and H.M., from the “Generalitat Valenciana” (PROMETEO/2018/055) to H.M., and from the CSIC Intramural program (Refs. 201220E098 and 201320E054) to C.V. Additional support was provided by the European Research Council (ERC) 2012-StG (311736-PD-HUMMODEL), the Instituto de Salud Carlos III - ISCIII/FEDER (Red de Terapia Celular - TerCel RD16/0011/0024 and PIE14/00061) to A.C. Ç.D. received a short-term fellowship from the Red Olfativa Española (ROE). M.M.-E, M.D.-M., A.H.-C., and V.N.-E. were supported by FPI Fellowships from the MICINN and MINECO.

Intraventricular hemorrhage is a common cause of morbidity and mortality in premature infants. The rupture of the germinal zone into the ventricles entails loss of neural stem cells and disturbs the normal cytoarchitecture of the region, compromising late neurogliogenesis. Here we demonstrate that neural stem cells can be easily and robustly isolated from the hemorrhagic cerebrospinal fluid obtained during therapeutic neuroendoscopic lavage in preterm infants with severe intraventricular hemorrhage. Our analyses demonstrate that these neural stem cells, although similar to human fetal cell lines, display distinctive hallmarks related to their regional and developmental origin in the germinal zone of the ventral forebrain, the ganglionic eminences that give rise to interneurons and oligodendrocytes. These cells can be expanded, cryopreserved, and differentiated in vitro and in vivo in the brain of nude mice and show no sign of tumoral transformation 6 months after transplantation. This novel class of neural stem cells poses no ethical concerns, as the fluid is usually discarded, and could be useful for the development of an autologous therapy for preterm infants, aiming to restore late neurogliogenesis and attenuate neurocognitive deficits. Furthermore, these cells represent a valuable tool for the study of the final stages of human brain development and germinal zone biology., Germinal zone neural stem cells (Gz‐NSC) are isolated from the hemorrhagic cerebrospinal fluid of preterm infants with severe intraventricular hemorrhage. These cells express ventral and posterior forebrain markers, can be differentiated and do not cause tumors. Gz‐NSC represent a valuable tool for the development of new cell therapies and the study of human Gz biology.

The aim of this study was to develop a rabbit neurosphere culture to characterize differences in basic processes of neurogenesis induced by intrauterine growth restriction (IUGR). A novel in vitro neurosphere culture has been established using fresh or frozen neural progenitor cells from newborn (PND0) rabbit brains. After surgical IUGR induction in pregnant rabbits and cesarean section 5 days later, neural progenitor cells from both control and IUGR groups were isolated and directly cultured or frozen at −80°C. These neural progenitor cells spontaneously formed neurospheres after 7 days in culture. The ability of control and IUGR neurospheres to migrate, proliferate, differentiate to neurons, astrocytes, or oligodendrocytes was compared and the possibility to modulate their responses was tested by exposure to several positive and negative controls. Neurospheres obtained from IUGR brains have a significant impairment in oligodendrocyte differentiation, whereas no significant differences are observed in other basic processes of neurogenesis. This impairment can be reverted by in vitro exposure of IUGR neurospheres to thyroid hormone, which is known to play an essential role in white matter maturation in vivo. Our new rabbit neurosphere model and the results of this study open the possibility to test several substances in vitro as neuroprotective candidates against IUGR induced neurodevelopmental damage while decreasing the number of animals and resources and allowing a more mechanistic approach at a cellular functional level., Rabbit “Neurosphere Assay.” PND0 rabbit brains are dissected and either frozen or directly cultured until neurosphere formation. “Neurosphere Assay” starts when neurospheres are plated in 8‐chamber slides and maintained under differentiating conditions to evaluate migration and differentiation into neurons, into astrocytes or into oligodendrocytes. Neurospheres were also plated in 96‐well plates under proliferating conditions and evaluated for diameter increase.

[eng] In mammals, the term "Adult Neurogenesis” (AN) defines the process through which, throughout adulthood, new neurons are produced from neural stem cells (NSC). These NSC are located in a specific niche, concretely, in the subventricular zone (SVZ), lining the lateral ventricles, and in the subgranular zone (SGZ) in the dentate gyrus (DG) of the hippocampus. Controversially, new data have questioned the existence of this AN in the human brain seeing how only populations of immature neurons (IN), broadly dispersed within SGZ, have been detected. Either way, neurogenic activity in the hippocampus has been correlated with learning, memory formation and behavioral responses to stress, just like with the pathophysiology of many brain diseases and mood disorders. Various extracellular and intracellular stimuli have been shown to modulate survival, proliferation, and differentiation of adult-born cells in the hippocampus, especially through conserved stimuli-response mechanisms like the JNKs. In the present review, the JNK pathway and their control of adult hippocampal neurogenesis are described, evidencing the critical role of isoform JNK1., [cat] En mamíferos, el término “Neurogenesis Adulta (NA)”, se define como el proceso a través del cual, en adultos, se producen nuevas neuronas granulares a partir de células madre neurales (CMN). Estas CMN estan ubicadas en microambientes específicos, en concreto en la zona subventicular (ZSV), recubriendo los ventriculos laterales, y en la zona subgranular (ZSG) del giro dentado del hipocampo (GD). Sin embargo, nuevas informaciones han cuestionado la existencia de este proceso de neurogenesis adulta en el cerebro humano, ya que solamente se han detectado poblaciones de neuronas inmaduras (NI) dispersas a lo largo de la ZSG. Independientemente, la existencia de una actividad neurogénica en el hipocampo adulto se ha correlacionado con el aprendizaje, la formación de memoria y en el comportamiento ante situaciones de estrés, así como en la patofisiologia de diferentes patologías del cerebro, incluso en casos de alteraciones del estado de ánimo. Se ha demostrado que diferentes estímulos extracelulares e intracelulares controlan la supervivencia, la proliferación y la diferenciación de las nuevas neuronas del hipocampo, especialmente a través de mecanismos conservados de respuesta a estímulos como las JNKs. En la presente revisión se describe las JNK y su control de la neurogénesis hipocampal adulta, evidenciando el papel crucial de la isoforma JNK1.

[cat] La neurotransmissió és el procés funcional bàsic de comunicació neuronal, i té lloc a través de l’alliberament dels neurotransmissors a l’espai sinàptic. Els defectes clàssics monogènics dels neurotransmissors inclouen els trastorns del metabolisme, degradació i transport de les monoamines (o el seu cofactor la tetrahidrobiopterina –BH4-), el GABA, la glicina i la serina, majoritàriament. Recentment també s’inclouen altres neurotransmissors com l’acetilcolina o l’ATP, i els trastorns astrocitaris o dels gliotransmissors. Les manifestacions clíniques es troben dins de l’espectre de les sinaptopaties, i es solen caracteritzar per discapacitat intel·lectual, epilèpsia, trastorns del moviment o símptomes neuropsiquiàtrics. Poden debutar en qualsevol edat de la vida, però característicament solen ser malalties neuropediàtriques que repercuteixen en el neurodesenvolupament, i que per tant, debuten en els primers anys de la vida. El treball que es presenta en aquesta tesi fa referència a: A) els defectes monogènics clàssics dels neurotransmissors, i B) els defectes de la fisologia sinàptica. A. En el primer treball es realitza una presentació del primer grup internacional dedicat a l’estudi de les malalties dels neurotransmissors, l’International Working Group on Neurotransmitter Related Disorders (iNTD), s’actualitza quina ha estat la seva activitat aquests darrers anys i el resultat principal en forma del primer registre internacional de pacients. En el següent treball, es presenta la segona guia de pràctica clínica d’un grup de defectes dels neurotransmissors: els defectes síntesi o reciclatge de la tetrahidrobiopterina (BH4). B. Els defectes de la fisiologia sinàptica implicarien totes aquelles malalties responsables d’alterar el procés de neurotransmissió i la biodisponibilitat/homeòstasi del neurotransmissor a l’espai sinàptic. En el següent treball es proposa una nova categoria de malaltia basada en el cicle biològic de la vesícula sinàptica, centrades en el terminal presinàptic de la neurona. Inclouria defectes en la formació de novo de la vesícula sinàptica al soma neuronal, el transport, maduració, exocitosi i reciclatge. Es proposa una categorització en quatre grups fenotípics: encefalopatia epilèptica, trastorns del moviment, discapacitat intel·lectual sindròmica i no-sindròmica, i trastorns neuromusculars. A propòsit de les malalties de la vesícula sinàptica, el següent treball presenta diferents pacients amb mutació en el gen DNAJC6, que codifica per l’auxilina 1 i participa en l’endocitosi de la vesícula sinàptica. El fenotip d’aquests pacients és en forma de discapacitat intel·lectual i distonia-parkinsonisme juvenil progressiva. S’estudia el líquid cefalorraquidi i fibroblasts, i s’analitzen l’auxilina 1, la ciclina-G associada a quinasa (GAK), i diferents proteïnes sinàptiques. Es conclou que hi ha una especial vulnerabiliat de l’homeòstasi dopaminèrgica, amb un augment probablement compensatori de la GAK. El següent treball descriu una pacient amb mutació en el gen DLP1, que també implica la neurotransmissió dopaminèrgica, tot codificant una proteïna que participa de l’endocitosi de la vesícula sinàptica i la fissió mitocondrial. El darrer treball amplia els defectes de la neurotransmissió centrant-se en el GABA. Es presenta una cohort de 85 pacients amb diferents malalties neuropediàtriques, i s’analitza la fracció de GABA lliure i monoamines en líquid cefalorraquidi. Es reporta una especial vulnerabilitat de la neurotransmissió GABAèrgica en tota la cohort en fins a un 44% dels casos (davant del 20% de defectes de les monoamines), i especialment en els errors congènits del metabolisme. Finalment, els annexos presenten la feina realitzada al llarg de tots aquests anys i amb la difusió d’aquests grups de malalties a través de les reunions amb famílies i amb professionals, i de la creació d’una pàgina web. D’aquesta manera, s’aconsegueix tancar el cercle que va des del pacient, a l’estudi de la seva malaltia de base i el defecte molecular, i el retorn al pacient a través del contacte estret amb les associacions de famílies i la comunitat científica., [eng] Neurotrasmission is the basic functional process devoted to neuronal communication, that takes place in the synaptic cleft. Classical neurotransmitter defects include disorders of the synthesis, metabolism or transport of monoamines (mainly dopamine and serotonin, but also adrenalin and noradrenalin), aminoacids (such as GABA and glutamate, but also serine and glycine, among others), or other molecules identified in the last decades as having a role in neurotransmission (i.e. ATP, lactate or acetylcholine). Main clinical symptoms manifest in the form of global developmental delay and intellectual disability, movement disorders, neuropsychiatric symptoms and epilepsy, all in the spectrum of synaptopathies. The current thesis encompasses: A) 2 publications related to the classical neurotransmitter defects; and B) 4 publications related to disorders of the synaptic physiology and neurotransmission (that influence the process of neurotransmission, its homeostasis and the biodisponibility of the neurotransmitter in the synaptic cleft, not directly related to synthesis, transport or metabolism defects), all of them focused in the presynaptic terminal. A. The former include: 1) the first publication of the International Working Group for Neurotransmitter Related Disorders (iNTD: http://intd-online.org/) and the initiation of the international registry for patients with neurotransmitter defects, as well as 2) the first clinical guidelines related to tetrahydrobiopterin synthesis and regeneration disorders. B. The latter include: 1) a proposal for a new category of presynaptic disorder, focused on the synaptic vesicle cycle; 2) a cohort of patients carrying DNAJC6 mutations, that encode the protein auxilin 1 (implicated in the endocytosis of the synaptic vesicle), and the secondary dopaminergic disruption in these patients, studied in cerebrospinal fluid (CSF) and fibroblasts; 3) a patient with DLP1 mutation, that encodes dynamin-like protein 1, implicated in the mitochondrial and peroxisomal fission, as well as the endocytosis of the synaptic vesicle; and finally, 4) a cohort of 85 patients with different neuropediatric disorders, in whom free GABA levels were studied in CSF, revealing a special vulnerability of GABA levels compared to monoamine levels, especially in the group of inborn errors of metabolism. Finally, the annexes of the thesis reveal the dissemination of the work among the scientific communities and the empowering of families.

IntroductionThe Tousled-like kinases 1 and 2 (TLK1 and TLK2) are involved in many fundamental processes, including DNA replication, cell cycle checkpoint recovery and chromatin remodelling. Mutations in TLK2 were recently associated with ‘Mental Retardation Autosomal Dominant 57’ (MRD57, MIM# 618050), a neurodevelopmental disorder characterised by a highly variable phenotype, including mild-to-moderate intellectual disability, behavioural abnormalities, facial dysmorphisms, microcephaly, epilepsy and skeletal anomalies.MethodsWe re-evaluate whole exome sequencing and array-CGH data from a large cohort of patients affected by neurodevelopmental disorders. Using spatial proteomics (BioID) and single-cell gel electrophoresis, we investigated the proximity interaction landscape of TLK2 and analysed the effects of p.(Asp551Gly) and a previously reported missense variant (c.1850C>T; p.(Ser617Leu)) on TLK2 interactions, localisation and activity.ResultsWe identified three new unrelated MRD57 families. Two were sporadic and caused by a missense change (c.1652A>G; p.(Asp551Gly)) or a 39 kb deletion encompassing TLK2, and one was familial with three affected siblings who inherited a nonsense change from an affected mother (c.1423G>T; p.(Glu475Ter)). The clinical phenotypes were consistent with those of previously reported cases. The tested mutations strongly impaired TLK2 kinase activity. Proximal interactions between TLK2 and other factors implicated in neurological disorders, including CHD7, CHD8, BRD4 and NACC1, were identified. Finally, we demonstrated a more relaxed chromatin state in lymphoblastoid cells harbouring the p.(Asp551Gly) variant compared with control cells, conferring susceptibility to DNA damage.ConclusionOur study identified novel TLK2 pathogenic variants, confirming and further expanding the MRD57-related phenotype. The molecular characterisation of missense variants increases our knowledge about TLK2 function and provides new insights into its role in neurodevelopmental disorders.

Background: GRIN-related disorders (GRD), the so-called grinpathies, is a group of rare encephalopathies caused by mutations affecting GRIN genes (mostly GRIN1, GRIN2A and GRIN2B genes), which encode for the GluN subunit of the N-methyl D-aspartate (NMDA) type ionotropic glutamate receptors. A growing number of functional studies indicate that GRIN-encoded GluN1 subunit disturbances can be dichotomically classified into gain- and loss-of-function, although intermediate complex scenarios are often present. Methods: In this study, we aimed to delineate the structural and functional alterations of GRIN1 disease-associated variants, and their correlations with clinical symptoms in a Spanish cohort of 15 paediatric encephalopathy patients harbouring these variants. Results: Patients harbouring GRIN1 disease-associated variants have been clinically deeply-phenotyped. Further, using computational and in vitro approaches, we identified different critical checkpoints affecting GluN1 biogenesis (protein stability, subunit assembly and surface trafficking) and/or NMDAR biophysical properties, and their association with GRD clinical symptoms. Conclusions: Our findings show a strong correlation between GRIN1 variants-associated structural and functional outcomes. This structural-functional stratification provides relevant insights of genotype-phenotype association, contributing to future precision medicine of GRIN1-related encephalopathies.

[cat] 1. INTRODUCCIÓ El baix rendiment escolar (RE) s’associa a dificultats en l’assoliment de capacitats, comportant importants costos econòmics i emocionals pels nens i famílies. La prematuritat és un dels factors de risc més prevalent d’alteracions del desenvolupament. Cal avaluar si les eines de cribratge de dèficit del desenvolupament (DD) són útils per detectar de manera precoç futures alteracions de l’aprenentatge i/o del comportament a l’edat escolar. 2. OBJECTIUS 1. Avaluar l’associació del cribratge de l’ASQ3 als 4 anys (ASQ3-48) amb els resultats acadèmics de la Prova d’Avaluació Diagnòstica de Primària (PADP) als 8-9 anys en una població de prematurs tardans (PT) i a terme (AT). 2. Analitzar la prevalença de baix RE als 8-9 anys. 3. Analitzar les variables associades amb baix RE. 4. Comparar la freqüència d’intervenció psicopedagògica/reforç escolar i d’ alteracions del comportament entre ambdues cohorts. 3. PACIENTS I MÈTODE Estudi prospectiu de cohorts de PT i AT, avaluats amb ASQ3-48 i, als 8-9 anys en les competències comunicativa i lingüística, i matemàtiques, mitjançant la PADP. Es registra la necessitat de suport psicopedagògic/reforç escolar, i d’ alteracions del comportament. Càlcul de la prevalença de baix RE i classificació en RE normal i baix. Es valoren les diferències entre PT i AT. Es comparen les mitjanes de l’ASQ3-48 entre els nens amb baix RE i RE normal mitjançant test t de Student amb correcció de Welch. Es determina l’associació de l’ASQ3-48 en relació al RE, descrivint les propietats psicomètriques, i corba ROC utilitzant la puntuació total de l’ASQ3- 48. Es descriu la freqüència i significació de rebre reforç escolar i dels trastorns de comportament, de forma global i entre PT i AT. Model de regressió logística univariant i multivariant definint les variables relacionades amb baix RE. 4. RESULTATS La població inclosa en l’estudi és de 133 nens, 75 PT i 58 AT. La taxa de risc de DD segons ASQ3-48 és del 14,3%. La prevalença de baix RE és del 12,8%. S’observa diferència significativa entre la puntuació total de l’ASQ3-48 en el grup amb RE normal (281,46 punts) respecte el de baix RE (267,35 punts), p 0,0054. Les propietats psicomètriques que determinen l’associació de l’ASQ3-48 en relació a baix RE són: sensibilitat 47,1%, especificitat 90,5%, VPP 42,1%, VPN 92,1%, odds pre-test 14,7%, LR+ 4,96%, LR- 0,58. L’AUC de la corba ROC generada utilitzant la puntuació total d’ASQ3-48 per a predir baix RE, és 0,73 (sensibilitat 0,54, especificitat 0,62), determinant un punt de tall òptim per discriminar baix RE de 275 punts. Les AUC dels PT i AT per separat mostren superior valor predictiu en els PT (0,75 vs 0,69). Sense diferències significatives entre ambdues cohorts, PT 0,75 (IC95% 0,59-0,92) i AT 0,69 (IC95% 0,44-0,94). La freqüència d’intervenció específica/reforç escolar és del 21%, significativament superior en els PT que en els AT (29,3% vs 10,3%, p 0,0014). La freqüència de trastorns del comportament és d’un 17,2% amb diferència significativa entre PT i AT (24% vs 8,6%, p 0,036). En el model de regressió logística univariant resulten significatives, en relació amb el baix RE: tenir un domini per sota el punt de tall en l’avaluació ASQ3-48 (OR 6,48 [IC95% 1,89-22,18], p 0,0024), i rebre algun tipus de reforç escolar (OR 4,2 [IC95% 1,43-12,67], p 0,007), que es mantenen significatives en el model multivariant (p 0,0021 i p 0,009 respectivament). 1.5. CONCLUSIONS - L’ASQ3 als 4 anys per identificar riscos de DD prediu de manera acceptable el baix RE a l’etapa dels 8-9 anys en el nostre medi. - No es troben diferències en el RE entre PT i AT, però els PT requereixen, de forma significativa, superior percentatge d’intervenció psicopedagògica. - Els majors requeriments de suport escolar o intervenció psicopedagògica dels PT suggereix que, probablement, la prematuritat tardana és, a llarg termini, una població de major risc que la població dels nascuts AT. - El gran nombre que representen els PT i el caràcter més subtil de les seves alteracions, obliga a dedicar una atenció especial a l’avaluació del seu desenvolupament., [eng] 1. INTRODUCTION Poor school performance (SP) is associated with difficulty in acquiring skills. One of the most prevalent risk factors is prematurity. There is a need to determine if the screening tools used to detect the risk of developmental delay (DD) are also useful for early detection of future learning difficulties and/or behavioral problems at school age. 2. OBJECTIVES 1. Evaluate the association of the ASQ3 questionnaire carried out at four years old (ASQ3-48) and SP at 8-9 years old in a late preterm (LP) and a term-born (Term) infants. 2. Analyze the prevalence of poor SP at 8-9 years of age. 3. Analyze the variables associated with poor SP. 4. Compare the frequency of psychopedagogical intervention/extra tutoring and behavioral disturbances in LP and Term. 3. PATIENTS AND METHODS Prospective study of LP and Term infants assessed by ASQ3-48 and a further assessment by SP at 8-9 years old. It was recorded of psychopedagogical support/extra tutoring, and behavioral problems. The prevalence of poor SP was calculated. Possible differences between the LP and Terms were considered by comparing by means of the Student t test with Welch correction. The association of the ASQ3-48 in relation to SP was determined. The frequency and significance of extra tutoring and behavioral problems were described. Univariate and a multivariate logistic regression models were constructed defining the variables related with poor SP. 4. RESULTS The population consisted of 133 children, 75 LP and 58 Term. The rate of risk for DD was 14,3%. The prevalence of poor SP was 12,8%. Significant differences were observed in the total score on the ASQ3-48 in the normal SP group (281,46 points) compared to the poor SP group (267,35 points), p 0,0054. The psychometric properties that determined the association between the ASQ3-48 and poor SP were: sensitivity 47,1%, specificity 90,5%, VPP 42,1%, VPN 92,1%, odds pre-test 14,7%, LR+ 4,96%, and LR- 0,58. A ROC curve using the total score on the ASQ3-48 to predict poor SP, obtains AUC of 0,73 (sensitivity 0,54, specificity 0,62), and the optimal cutoff point to discriminate poor SP were 275. The ROC curves producing LP and Term were examined separately, revealing greater predictive value for the LP (0,75 vs 0,69). The frequency of specific intervention/extra tutoring was 21%; this was significantly greater in LP vs Term (29,3% vs 10,3% , p 0,0014). The frequency of behavioral problems was 17,2%, with a significant difference between LP and Term (24% vs 8,6%, p 0,036). In the univariate logistic regression model the following were significant in relation to poor SP: having a domain below the cutoff point in the ASQ3-48 assessment (OR 6,48 [IC 95%, 1,89-22,18], p 0,0024) and receiving some kind of extra tutoring (OR 4,2 [IC 95%, 1,43-12,67], p 0,007). These maintained significance in the multivariate model (p 0,0021 and p 0,009, respectively). 1.5. CONCLUSIONS - The ASQ3-48 in order to identify the risk of DD predicts poor SP acceptably at 8-9 years of age in our setting. - No differences were found in SP between the LP and Term, but the LP infants required, a greater percentage of psychopedagogical intervention. It suggests that late prematurity continues to be a population of long-term risk in comparison to those born at term. - The large number of those who are LP and the subtlety of their alterations highlight the need for special attention being given to assessment of their development.

Axon pathfinding and synapse formation are essential processes for nervous system development and function. The assembly of myelinated fibres and nodes of Ranvier is mediated by a number of cell adhesion molecules of the immunoglobulin superfamily including neurofascin, encoded by the NFASC gene, and its alternative isoforms Nfasc186 and Nfasc140 (located in the axonal membrane at the node of Ranvier) and Nfasc155 (a glial component of the paranodal axoglial junction). We identified 10 individuals from six unrelated families, exhibiting a neurodevelopmental disorder characterized with a spectrum of central (intellectual disability, developmental delay, motor impairment, speech difficulties) and peripheral (early onset demyelinating neuropathy) neurological involvement, who were found by exome or genome sequencing to carry one frameshift and four different homozygous non-synonymous variants in NFASC. Expression studies using immunostaining-based techniques identified absent expression of the Nfasc155 isoform as a consequence of the frameshift variant and a significant reduction of expression was also observed in association with two non-synonymous variants affecting the fibronectin type III domain. Cell aggregation studies revealed a severely impaired Nfasc155-CNTN1/CASPR1 complex interaction as a result of the identified variants. Immunofluorescence staining of myelinated fibres from two affected individuals showed a severe loss of myelinated fibres and abnormalities in the paranodal junction morphology. Our results establish that recessive variants affecting the Nfasc155 isoform can affect the formation of paranodal axoglial junctions at the nodes of Ranvier. The genetic disease caused by biallelic NFASC variants includes neurodevelopmental impairment and a spectrum of central and peripheral demyelination as part of its core clinical phenotype. Our findings support possible overlapping molecular mechanisms of paranodal damage at peripheral nerves in both the immune-mediated and the genetic disease, but the observation of prominent central neurological involvement in NFASC biallelic variant carriers highlights the importance of this gene in human brain development and function.

[eng] During the process of corticogenesis, cortical radial glial cells (RGs) generate sequentially most of the distinct types of neurons and glial cells that will populate the neocortex. The generation of these cells at the proper time and adequate numbers is key for the establishment of fully functional cerebral circuits. During the phase of neuronal production, RGs experience important changes in morphology, division mode and fate of their neuronal progeny. To provide evidence of the genetic networks underlying these changes, in the first part of this work we have compared the expression of protein-coding genes at the onset of neurogenesis (embryonic day (E)11.5) and during middle-late neurogenesis (E15.5). We found that the expression levels of 25% of these genes are regulated during neurogenesis, and a quarter of them are targets of developmentally regulated miRNAs. Down-regulated genes at E15.5 are mainly associated to cell cycle regulation, while most of the up-regulated genes encode for ion-transport proteins that may be involved in modulating the membrane potential of RGs. Moreover, we found significant up-regulation of synapse-related genes, supporting published reports that claim for the existence of a transcriptional “pre-patterning” in RGs. Among the regulated genes, we found significant enrichment in TEAD2 targets. YAP-TEAD transcriptional activity is regulated by mechanical cues and we found that a great number of differentially expressed genes in RGs encode for structural extracellular matrix (ECM) proteins, suggesting a dynamic remodelling of the ECM along neurogenesis. This remodelling likely varies the physical properties of the ECM, and thereby YAP-TEAD transcriptional activity in RGs. The integrated analysis of the developmental regulated mRNAs and miRNAs suggests that let-7 miRNAs are involved in the regulation of collagen and other structural ECM genes. Lastly, our RNA-seq data shows that alternative splicing (AS) is also developmentally regulated in RGs and affects the expression of splicing events in 19% of the expressed protein-coding genes. Functional enrichment analysis of these genes indicates that AS in RGs may be involved in silencing synapse-related genes and the regulation of critical cellular functions such as the biogenesis of the cilium. In the second part of this work, we addressed the effects of DYRK1A haploinsufficiency in the generation and differentiation of cortical glial cells. DYRK1A is a kinase that has a conserved role in brain growth through evolution and regulates the generation, survival and differentiation of different neuron types. Mutations in DYRK1A cause a syndrome, named DYRK1A-haploinsufficiency syndrome, that is characterized by the presence of microcephaly and other clinical features including intellectual disability, autistic traits and epilepsy. Gliosis and hypomyelination have also been reported in affected children. Previous work of the laboratory has shown that the Dyrk1a+/- mouse mimics several of the phenotypic traits of the syndrome, including an increased number of astrocytes and hypomyelination of the corpus callosum (CC). Here, we show that the augmented number of astrocytes in Dyrk1a+/- neocortices results from an enhanced developmental astrogliogenesis. Moreover, we have observed a decreased generation of oligodendrocyte precursor cells (OPCs) in Dyrk1a+/- embryos that correlates with a delay in the appearance of oligodendrocytes in the CC. Focal demyelination experiments in the adult CC indicate that Dyrk1a+/- OPCs differentiate properly, suggesting that the oligodendroglial phenotype in Dyrk1a+/- mutants results from alterations in the generation of OPCs during development. In addition to hypomyelination, the CC of adult Dyrk1a+/- mice shows altered proportion of the myelin proteins PLP and MBP, shorter nodes of Ranvier and a deficit in the expression of the nodal sodium channel NAV1.6. Collectively, these results show that deficits in the generation and differentiation of glial cells may contribute to the neurological defects reported in individuals with DYRK1A-haploinsufficiency syndrome., [spa] La mayoría de las neuronas y células gliales de la corteza cerebral se generan secuencialmente durante el desarrollo a partir de progenitores pluripotentes denominados glía radial (GR). La correcta generación y diferenciación de estas células es crítica para el establecimiento de circuitos funcionales. En la primera parte de esta Tesis, hemos estudiado los cambios de expresión génica que tienen lugar en la GR durante la neurogénesis. Los resultados obtenidos muestran cambios de expresión en el 25% de los genes codificantes e indican que la expresión de una cuarta parte de éstos está regulada por micro-ARNs. Entre los genes expresados diferencialmente encontramos un enriquecimiento en targets del complejo YAP-TEAD, cuya actividad transcripcional responde a estímulos mecánicos. Además, encontramos enriquecimiento en genes que codifican componentes de la matriz extracelular (MEC), sugiriendo que cambios en las propiedades físicas de ésta podrían modular la actividad YAP-TEAD en la GR. Por último, nuestros resultados indican que la regulación por splicing alternativo durante la neurogénesis es muy extensa (afectando al 19% de los genes codificantes) y podría tener un papel en silenciar genes neuronales en la GR y en otras funciones como la biogénesis del cilio. En la segunda parte, hemos estudiado los efectos de la haploinsuficiencia de DYRK1A en la generación y diferenciación de células gliales. DYRK1A codifica una quinasa con funciones clave en el desarrollo cerebral. Mutaciones de novo en DYRK1A causan un síndrome de discapacidad intelectual y autismo. Los cerebros de niños afectados presentan gliosis e hipomielinización. El ratón Dyrk1a+/- recapitula varios de los fenotipos del síndrome, incluyendo un incremento de astrocitos cerebrales e hipomielinización del cuerpo calloso (CC). Nuestros estudios en este modelo indican que la generación de células gliales está alterada durante el desarrollo y correlaciona con un retraso en la aparición de oligodendrocitos en el CC. La corteza adulta del ratón Dyrk1a+/- muestra alteraciones en los nodos de Ranvier y en la composición de proteínas de la mielina. Este fenotipo glial podría contribuir a los problemas neurológicos observados en el modelo Dyrk1a+/- y abrir nuevas vías de terapia para los individuos con mutaciones en el gen DYRK1A.

Contains fulltext : 207577.pdf (Publisher’s version ) (Open Access) Brain imaging methods have contributed to shed light on the mechanisms of recovery after early brain insult. The assumption that the unaffected right hemisphere can take over language functions after left perinatal stroke is still under debate. Here, we report how patterns of brain structural and functional reorganization were associated with language outcomes in a group of four-year-old children with left perinatal arterial ischemic stroke (PAIS). Specifically, we gathered specific fine-grained developmental measures of receptive and productive aspects of language as well as standardized measures of cognitive development. We also collected structural neuroimaging data as well as functional activations during a passive listening story-telling fMRI task and a resting state session (rs-fMRI). Children with a left perinatal stroke showed larger lateralization indices of both structural and functional connectivity of the dorsal language pathway towards the right hemisphere that, in turn, were associated with better language outcomes. Importantly, the pattern of structural asymmetry was significantly more right-lateralized in children with a left perinatal brain insult than in a group of matched healthy controls. These results strongly suggest that early lesions of the left dorsal pathway and the associated perisylvian regions can induce the interhemispheric transfer of language functions to right homolog regions. This study provides combined evidence of structural and functional brain reorganization of language networks after early stroke with strong implications for neurobiological models of language development. 18 p.

[eng] The γ-­‐tubulin ring complex (γTuRC) is required to efficiently generate new microtubules (MT) in a process known as MT nucleation. In mitotic cells, MT nucleation by γTuRC occurs from the centrosome, in vicinity of the chromosomes and from the lattice of pre-­‐ existing MTs in a process additionally involving the augmin complex. In neurons, the centrosome loses its MT nucleation capacity, while the γTuRC can still promote nucleation elsewhere. Still, it remains unclear how the γTuRC is regulated in these cells and whether other non-­‐centrosomal sites function as microtubule-­‐organizing centre in neurons. The aim of this thesis is to understand how MT nucleation mediated by augmin and γTuRC contributes to brain development and how nucleation is regulated during this process. In this thesis we show that the augmin complex is required for both axonal and dendritic development in vitro. In the axon, augmin guarantees that MTs are nucleated with the correct orientation, ensuring uniform axonal MT polarity. On the other hand, augmin depletion leads to an overall decrease in MT density in dendrites with no major effect on their polarity. Surprisingly, despite our findings, analysis of augmin function by other groups in vivo in flies and zebrafish has not revealed any dramatic defects. Strikingly, we show that, in conditional KO mice with gene deletion in the augmin subunit Haus6 in neural progenitors, brain development is halted before embryonic day 13 and the animals die at birth. These major brain defects are caused by impaired mitosis and massive cell death in neuroprogenitors, indicating significant, species-­‐specific differences in the requirement for augmin function. In the last part of this thesis we identified KIF2A and CEP170 as γTuRC interactors in in vitro cultured mouse cortical neurons and we show that, in agreement with published data on KIF2A function, CEP170 seems to inhibit growth of axon collateral branches. We speculate that KIF2A and CEP170 may function as negative regulators of microtubules nucleation by γTuRC. Together these results establish augmin-­‐mediated nucleation as essential for mammalian brain development and provide first insight in the regulation of microtubule nucleation in neurons by γTuRC interactors., [spa] El complejo de anillo gamma-­‐tubulina (γTuRC) es necesario para generar microtúbulos de manera eficiente en un proceso conocido como nucleación de microtúbulos. En células mitóticas, la nucleación a partir del γTuRC tiene lugar en el centrosoma, en regiones próximas a los cromosomas y en la superficie de microtúbulos previamente formados, en un proceso que requiere adicionalmente la presencia del complejo de augmina. A pesar de que en neuronas el centrosoma pierde su capacidad nucleadora de microtúbulos, el γTuRC aún puede inducir la nucleación en otros lugares. Sin embargo, aún se desconoce cómo se regula el γTuRC en estas células y si, aparte del centrosoma, existen otros lugares que puedan funcionar como centros organizadores de microtúbulos. El objetivo de esta tesis doctoral es entender cómo la nucleación de microtúbulos dependiente de augmina y del γTuRC contribuyen al desarrollo cerebral y cómo se regula la nucleación de microtúbulos durante este proceso. En esta tesis hemos demostrado que el complejo de augmina es necesario para el desarrollo de axones y dendritas in vitro. En el axón, augmina garantiza que los microtúbulos se nucleen en una orientación adecuada, asegurando una polaridad microtubular uniforme a lo largo del axón. Por otro lado, la ausencia de augmina causa una disminución general en la densidad de microtúbulos de las dendritas, sin afectar a su polaridad. Sorprendentemente, a pesar de nuestros descubrimientos, otros grupos han analizado la función de augmina in vivo (en moscas y pez cebra) sin encontrar ningún defecto significativo. Curiosamente, hemos observado que, en ratones knockout condicionales en los que la subunidad Haus6 de augmina ha sido delecionada de progenitores neuronales, el desarrollo cerebral se interrumpe antes del decimotercer día del desarrollo embrionario. Adicionalmente, esta condición resulta letal para los animales, que mueren durante el nacimiento. Se ha encontrado que las causas de estos dramáticos defectos cerebrales son problemas en mitosis y una muerte masiva de neuroprogenitores. Estos resultados indican que existen diferencias en el requerimiento de augmina entre diferentes especies. En la última parte de esta tesis identificamos KIF2A y CEP170 como interactores del γTuRC en neuronas corticales de ratones cultivadas in vitro. También demostramos que, en concordancia con datos publicados sobre KIF2A, CEP170 parece inhibir el crecimiento de ramificaciones laterales en axones. Nuestra hipótesis es que tanto KIF2A como CEP170 podrían actuar como reguladores negativos de la nucleación de microtúbulos dependiente de γTuRC. En conclusión, estos resultados demuestran que la nucleación dependiente de augmina es un proceso esencial en el desarrollo cerebral humano, y proporcionan una primera visión de cómo interactores del γTuRC pueden regular la nucleación de microtúbulos en neuronas.

[eng] During neural development, signaling molecules and networks of expressed transcription factors cooperate to control cellular fate. Chromatin acting factors are essential players in cell proliferation and differentiation events. PHF2 is a histone demethylase, previously associated with cancer and autism spectrum disorder (ASD) but despite its clinical relevance its function during neural development remains unclear. PHF2 belongs to the KDM7 family of demethylases. It harbors an N-terminal plant homeodomain, (PHD) a domain that was shown to be associated with methylated lysine residues and an enzymatically active Jumonji-C (JmjC) domain responsible for their demethylase activity towards H3K9me and H3K9me2. PHF2 has been proposed to be a transcriptional coactivator, although, its genomic localization still remains unclear. To address the function of this protein during early neurogenesis constitutes the major goal of this Ph.D. thesis. To do so, we are using in vitro (cortical neural stem cells, NSC) and in vivo (the chick embryo neural tube) models. We demonstrated that this histone demethylase binds mainly promoter regions along the genome and more specifically cell cycle gene promoters facilitating their transcription in NSCs. PHF2 was shown to regulate genes involved in G1-S transition (E2f2/3/7/8, Cdc7, Cdc25a, Cdk4, Mcm3/4/8), DNA replication (Orc1/2/6, Pcna), mitosis (Cdk1, Smc2/3/4, Aurkb, Topo2a), as well as chromatin activity (Cenpa, Kdm1b, Hat1, Parp1, Prmt5). In addition, we demontrated that this demethylase colocalizes with H3K4me2/3 marks, partially colocalizes with E2F1 and E2F4 transcription factors and mediates H3K9 demethylation at global and promoter levels. Moreover, PHF2 binds to the centromeric and pericentromeric regions and requiring its catalytic activity suppresses unprogrammed transcription from satellite repeats. In that way, PHF2 allows neural stem cells proliferation and preserves heterochromatin integrity during progenitor expansion. We also showed that PHF2 interacts with heterochromatic components such as the histone methyltransferase SUV39H1 and the HP1-binding protein 3. Furthermore, PHF2 fine-tunes H3K9 methylation levels, ensuring genome stability and chromatin homeostasis, as we observed that its lack of function leads to γH2Ax and R-loops accumulation in NSCs. Interestingly, we demonstrated that PHF2 is essential for neural progenitor self- renewal in the chick embryo neural tube and for neural subpopulation specification. To conclude, this work helps to move forward our understanding of the multiple crosstalks between epigenetics and developmental programs., [spa] Durante el desarrollo neural, los factores reguladores de la actividad de la cromatina son esenciales en el control de la proliferación y diferenciación de las células. PHF2 es una desmetilasa de histonas, asociada a cáncer y a trastornos del espectro autista (ASD) cuya función en el desarrollo neural no ha sido estudiada. PHF2 pertenece a la familia de desmetilasas KDM7. Contiene un dominio PHD, capaz de unirse a residuos de lysinas metiladas y un dominio Jumonji-C (JmjC) enzimáticamente activo, responsable de la actividad desmetilasa hacía las marcas H3K9me y H3K9me2. PHF2 ha sido propuesta como un activador transcripcional pero su localización genómica no ha sido descrita. El objetivo principal de esta tesis doctoral es abordar la función de esta desmetilasa de histonas durante la neurogénesis temprana. Para ello, hemos usado modelos in vitro (progenitores neurales de córtex, NSCs) e in vivo (el tubo neural del embrión de pollo). Nuestros resultados han permitido identificar los lugares de unión de PHF2 en el genoma, esencialmente promotores y más específicamente promotores de genes de ciclo celular facilitando su transcripción. Además, esta desmetilasa colocaliza con las marcas H3K4me2/3, media desmetilación de las marcas H3K9 a nivel global y en promotores. Los experimentos realizados han demostrado que PHF2 se une a las regiones centromérica y pericentromérica y así mantiene niveles bajos de la transcripción de las secuencias repetidas. De esta forma preserva la integridad de la heterocromatina, asegurando la estabilidad genómica y la homeostasis de la cromatina. Estos resultados han sido validados en nuestro modelo in vivo; en el tubo neural, hemos demostrado que PHF2 es esencial para la proliferación y el mantenimiento de la pluripotencia de los progenitores, así como para la especificación de los diferentes tipos de las poblaciones neurales. En resumen, el trabajo presentado ayuda a avanzar en el conocimiento de las múltiples interacciones moleculares entre los programas de desarrollo y la epigenética.

[eng] Reelin is a large extracellular matrix glycoprotein with a crucial role both during brain development, where it is key for neuronal migration and for the formation of the layered structure of cerebral cortex and cerebellum, and in the adulthood, where it is involved in adult synaptic plasticity, including neurogenesis in the dentate gyrus and dendritogenesis amongst other processes. Reelin acts through the binding to its canonical receptors (apolipoprotein E receptor 2, ApoER2; and very low density lipoprotein receptor, VLDLR) which trigger a complex signaling cascade involving numerous kinases and the adaptor protein Dab1. At the embryonic stage, Reelin is expressed mainly by Cajal-Retzius cells on the developing brain whereas at perinatal stages its expression gradually disappears from Cajal-Retzius cells and starts to be expressed by GABAergic interneurons of the cortex and hippocampus. In the neocortex, postmitotic neurons migrate in an ordered sequence that determines the normal “inside-out” layer formation. The malpositioning of cortical neurons is a result of abnormal migration and could cause severe layering malformations with functional consequences related with neurodevelopmental diseases such as Schizophrenia, Autism and Epilepsy. In this context, one of the most studied models has been the reeler mouse which presents a characteristic phenotype caused by an autosomal mutation in the Rln gene. The reeler mouse presents several morphological defects including a failed pre-plate splitting that causes a roughly inverted neuronal layering in the cortex, mispositioning of pyramidal neurons as well as granular cells on the dentate gyrus and profound cerebellar hypoplasia. However the study of the effects of Reelin signaling in the adult brain is difficult in the reeler mouse model due to the failed migration and mispositioning during development. Thus, to unravel the function of Reelin at different developmental stages (from embryonic to adult) as well as to gain insight in the potential distinct contribution of Reelin from different cell-types, we have generated three Reelin deficient conditional transgenic lines which allow us to ubiquitously delete Reelin in a temporally-controlled manner (Cre fR/fR) or selectively remove Reelin from Cajal-Retzius cells (CR fR/fR) or GABAergic interneurons (Gad fR/fR). Analysis of the cortical organization using layer-specific markers reveals that, unlike the reeler mouse, none of our transgenic lines shows the characteristic inversion of cortical layers. Moreover, our data strongly indicates that Reelin from Cajal-Retzius cells is important for the typical inside-out laminar cortical development but seems to be dispensable for pre-plate splitting. Furthermore, our results suggest that the absence of Reelin during early postnatal and adult stages seems to impact on the well-defined laminar structure of the cortex, leading to an invasion of layer I by late-born neurons from layer II-III. Regarding the hippocampus, our results suggest, on the one hand, a differential contribution of Reelin expressed by Cajal-Retzius cells and by GABAergic interneurons in the formation of the laminar structures of the hippocampus. On the other hand, temporally-controlled removal of Reelin at postnatal stages demonstrates that it is essential for the correct formation of the hippocampus whereas in the adult seems to be key for several aspects of hippocampus neurogenesis, including neuronal positioning in the dentate gyrus and dendritic orientation at different maturation stages of adult new-born granule cells. Finally, our findings also support the importance of Reelin expression for proper Purkinje cell migration, but not for granule cell disposition in the cerebellum at early postnatal and adult stages. Taken altogether, our results suggest a causal relation between the absence of Reelin and structural alterations in the hippocampus, cortex and cerebellum, either at developments stages or adult stages., [spa] Reelina es una glicoproteína extracelular de matriz esencial para la regulación de los procesos de migración neuronal y posicionamiento de las neuronas corticales durante el desarrollo del encéfalo. Durante la embriogénesis, Reelina es producida por las células Cajal-Retzius de la superficie de la corteza en desarrollo. En este estadío, las neuronas postmitóticas migran de forma ordenada originando una estructura laminar en seis capas, en las cuáles las neuronas más jóvenes se sitúan en las capas más externas. La pérdida de Reelina durante el desarrollo comporta fallos en la migración de las neuronas, provocando a su vez grandes alteraciones en la estructuración de la corteza que contribuyen a la patogénesis de muchos trastornos neurológicos como el autismo, la epilepsia, la esquizofrenia, o el trastorno bipolar. En este contexto, uno de los fenotipos más estudiado es el del ratón mutante de Reelina, reeler, que presenta una estructura cortical alterada con las capas invertidas. Sin embargo, dado que la expresión de Reelina durante el desarrollo ocurre a edades embrionarias muy tempranas, es difícil estudiar el efecto de su pérdida en este tipo de mutantes a edades más tardías, en los que los primeros efectos de su pérdida son tan profundos. Todo ello evidencia la necesidad de desarrollar otro tipo de modelos en los que la pérdida de Reelina sea más gradual o selectiva. En estadíos perinatales y en el cerebro adulto Reelina es expresada principalmente por interneuronas gabaérgicas, donde presumiblemente Reelina controla funciones de formación de sinapsis y mantenimiento de la plasticidad sináptica de las neuronas del córtex y del hipocampo. Nuestros resultados muestran un fenotipo diferencial para cada uno de nuestros mutantes, sugiriendo un papel diferente de Reelina expresada por cada tipo celular, o en función del estadío en el cuál la deleccionamos. En concreto, hemos visto que la función de Reelina es imprescindible para la correcta laminación del córtex, para la formación del hipocampo, y para el correcto posicionamiento de las células Purkinje del cerebelo. Además la pérdida de Reelina en estadíos adultos comporta fallos en la neurogénesis de la zona subventricular del giro dentado.

Objectives To perform a comprehensive assessment of cortical development in fetuses with isolated nonsevere ventriculomegaly (INSVM) by neurosonography. Methods We prospectively included 40 fetuses with INSVM and 40 controls. INSVM was defined as atrial width between 10.0 and 14.9 mm without associated malformation, infection, or chromosomal abnormality. Cortical development was assessed by neurosonography at 26 and 30 weeks of gestation measuring depth of selected sulci and applying a maturation scale from 0 (no appearance) to 5 (maximally developed) of main sulci and areas. Results INSVM showed underdeveloped calcarine and parieto-occipital sulci. In addition, significant delayed maturation pattern was also observed in regions distant to ventricular system including Insula depth (controls 30.8 mm [SD 1.7] vs INSVM 31.7 mm [1.8]; P = .04), Sylvian fissure grading (>2 at 26 weeks: controls 87.5% vs INSVM 50%, P = .01), mesial area grading (>2 at 30 weeks: controls 95% vs INSVM 62.5%; P = .03), and cingulate sulcus grading (>2 at 30 weeks: controls 100% vs INSVM 80.5%; P = .01). Conclusions Fetuses with INSVM showed underdeveloped cortical maturation including also regions, where effect of ventricular dilatation is unlikely. These results suggest that in a proportion of fetuses with INSVM, ventricular dilation might be related with altered cortical architecture.

[eng] The chloride channel ClCN2 is expressed in glia in the mature nervous system of vertebrates and some physiological functions have been proposed. In addition, human patients with mutations in the gene or dysfunction of the protein, have, among other defects, learning disabilities and mental retardation. Since these later symptoms usually arise from errors in the assembly of neural circuits during development, it is possible that ClC-2 has a role in glia during development. Glia act as niche cells in the neurogenic niche of both vertebrates and Drosophila during development. The process of neurogenesis has to adapt to each developmental stage and has to react to systemic changes. In this work we studied the role of ClC-2 fly homologue ClC-a during CNS development. We showed that the ionic homeostasis of the niche microenvironment is also necessary for the correct regulation of stem cell proliferation. In Drosophila melanogaster, the chloride channel ClC-a is expressed in cortex glial cells that extend processes that ensheath both neuroepithelia and central brain neuroblasts and the loss of the channel in those cortex glial niche leads to a decreased neuroepithelial proliferation. Probably neuroblast proliferation is also decreased. Also, cortex glial cells wrap neuronal cell bodies and ClC-a is required for their survival. We provide evidence that defects in central brain neuroblast lineage distribution, a decreased proliferation and an increase in cell death conclude in a reduction of a specific population of migratory glia in the optic lobe, necessary for the correct axonal guidance of photoreceptors. Altogether, the results of this work contribute to the understanding of how a chloride channel can affect the development of a neural tissue, controlling the niche microenvironment during neurogenesis., [spa] El canal de cloro ClCN2 se expresa en glia en el sistema nervioso maduro de vertebrados y se le han atribuido algunas funcionas fisiológicas. Además, pacientes humanos con mutaciones en el gen o disfunciones en la proteína, tienen, entre otros defectos, dificultades de aprendizaje y retardo mental. Ya que estos síntomas normalmente son consecuencias de errores en el ensamblaje de circuitos neuronales durante el desarrollo, es posible que ClC-2 tenga una función en glia durante el desarrollo. Cabe destacar que la glia es parte del nicho neurogénico de vertebrados y Drosophila durante el desarrollo. En este trabajo, hemos estudiado la función de ClC-a, el homólogo de ClC-2 en la mosca, durante el desarrollo del sistema nervioso central. Hemos demostrado que la homeostasis iónica del microambiente del nicho es también necesaria para la correcta regulación de la proliferación de las células madre. En Drosophila melanogaster, el canal de cloro ClC-a se expresa en células de cortex glia que envuelven los neuroepitelios y los neuroblastos del cerebro central. La pérdida del canal en el nicho de cortex glia conlleva a una reducción de la proliferación de los neuroepitelios. Probablemente, la proliferación de los neuroblastos también se reduce. Las células de cortex glia también envuelven los somas neuronales y ClC-a es requerido para la supervivencia de estos últimos. Presentamos evidencias de que defectos en la distribución de linajes de neuroblastos en el cerebro central, combinado con una reducción de la proliferación y un aumento de muerte celular, conllevan a una reducción de una población específica de glia migratoria en el lóbulo óptico. Estas células son necesarias para la correcta guía axonal de los fotorreceptores. Resumiendo, los resultados de este trabajo contribuyen al entendimiento de como un canal de cloro puede regular el desarrollo del tejido neural, controlando el microambiente del nicho durante la neurogénesis.

[cat] La correcta migració, la polarització neuronal i el manteniment de l'estructura neuronal són claus per l'òptim funcionament del cervell. Existeix un reconeixement molecular que permet la formació de les connexions neuronals. El manteniment d'aquestes connexions són claus en cervell adult. A més a més, la pèrdua progressiva d'aquestes donen lloc a processos neurodegeneratius. Les molècules d'adhesió cel·lulars realitzen diferents funcions essencials durant el desenvolupament i en la plasticitat neuronal. Estan involucrades en la migració ,creixement neurític, sinaptogènesi i plasticitat. Aquesta tesi es focalitza en l'estudi de les funcions que realitza una d'aquestes molècules d'adhesió, la molècula d'adhesió cel·lular neuronal 2 (neural cell adhesion molecule 2, Ncam2). Ncam2 és una glicoproteïna de la família de les Ncam. En concret, el gen Ncam2 és paràleg amb Ncam1 i presenta el mateix domini extracel·lular que Ncam1. Ncam2 presenta dues isoformes degut a un empalmament alternatiu que genera la isoforma Ncam2.1 i la isoforma Ncam2.2. En mamífers, Ncam2 s'expressa en diferents teixits del cos i el teixit on és més abundant és el cervell. Ncam2 s'expressa en diferents regions del cervell però la seva funció ha estat extensament estudiada en el bulb olfactori, on s'ha vist que participa en la formació i el manteniments de les connexions en el glomèrul. En concret, participa en la fasciculació dels axons i el creixement neurític. A més a més, en cultius de neurones corticals Ncam2 participa en la formació de fil·lopodis i la ramificació de les neurites. Tot i l'expressió en hipocamp i còrtex, la funció de Ncam2 no és coneixia en aquests teixits. En aquest sentit, al llarg de la tesi hem modulat l'expressió en diferents experiments in vitro i in vivo per determinar la funció de Ncam2 en el desenvolupament, morfogènesi neuronal i plasticitat adulta. En desenvolupament hem observat que Ncam2 s'expressa en les neurones que migren radialment i la seva expressió és necessària pel correcte posicionament en el còrtex. A més a més, l'expressió de Ncam2 en hipocamp és necessària per la morfogènesi neuronal degut a que participa en els processos de polarització, creixement dendrític i ramificació axonal. En hipocamp adult, Ncam2 controla la plasticitat neuronal i la neurogènesi adulta. En concret, Ncam2 controla la densitat d'espines de les neurones piramidals de CA1 i les neurones granulars del gir dentat. A més a més, també modula la densitat de contactes que formen les fibres molsoses a la CA3. A nivell de neurogènesi adulta, els nostres resultats indiquen que Ncam2 controla la diferenciació neuronal de les neurones generades en el gir dentat. A nivell de senyalització, els nostres resultats indiquen que Ncam2 interacciona amb diferents molècules que controlen les dinàmiques del citoesquelet d'actina i microtúbuls, la senyalització de calci, la transcripció genètica i el tràfic cel·lular. Aquestes dades obren la via d'estudi dels mecanismes que vehiculen les funcions de Ncam2. Les nostres dades posen de manifest diferents funcions que realitza Ncam2 en el cervell. En cap sentit, les interaccions i funcions descrites semblen exclusives de Ncam2 sinó que són processos en que hi ha un solapament funcional de moltes proteïnes d'adhesió, fet que confereix robustesa als processos de formació, manteniment i remodelació de les estructures neuronals. En concret, Ncam2 formaria part del conjunt de molècules que permeten la identitat molecular de les neurones. Una identitat clau perquè permet la formació i el manteniment de circuits neuronals específics., [eng] The proper migration, neuronal polarity and long-term maintenance of neurons are crucial for correct brain functioning, with special relevance for the connectivity orchestration during development and learning processes. Furthermore, changes in these processes are one of the keys features in neurodegenerative diseases. Cell Adhesion Molecules (CAMs) play an important role during brain development and brain plasticity processes, being involved in neurite outgrowth, synaptogenesis and synaptic plasticity. Neural cell adhesion molecule 2 (Ncam2) is a CAM family member homologous to Ncam1 with a similar ectodomain. Ncam2 has two different isoforms generated by alternative splicing known as Ncam2.1 (transmembrane isoform) and Ncam2.2 (glycosylphosphatidylinositol (GPI) anchored isoform). In mammals, NCAM2 is expressed predominantly in brain, and its expression pattern demonstrates a putative important role of this protein in both embryonic and adult brain function. NCAM2 role has been extensively investigated in the olfactory system where it plays an important role in axonal fasciculation and neurite outgrowth. Recently, different studies have revealed that Ncam2 also participates in the formation of filopodia and in neurite branching of cortical mouse neurons. Nevertheless its function in hippocampal development and adult plasticity remains largely unknown. We used different cell biology and molecular approaches, including loos-of- and gain-of-function models, to determine the role of Ncam2 in the development of the brain and adult plasticity. Our data show that Ncam2 is expressed in migrating neurons and that it is necessary for their correct position in the cortex. Moreover, Ncam2 is also expressed in the hippocampus and it is essential for the appropriate neuronal polarity. Ncam2 plays a role in neurite outgrowth, axonal branching and dendrite formation by modulating cytoskeletal dynamics. In adult hippocampus, our data show that Ncam2 contributes in synaptic plasticity and adult neurogenesis. Ncam2 modulates the spine density of pyramidal neurons in CA1 and granule cells in dentate gyrus. Furthermore, Ncam2 controls the mossy fibber contacts in CA3. Regarding to the adult neurogenesis, gain-of function of Ncam2 changes the neuronal differentiation of the new neurons. Our experiments indicate that Ncam2 interacts with a diversity of proteins including cytoskeletal components associated to the microtubule and actin networks, thus pointing to hypothetical mechanistic insights to be explored. Taken together, our results indicate that Ncam2 is an important molecule for the development, specification and connectivity of brain formation and adult plasticity.

[eng] BACKGROUND: Chronic hypoxia due to placental insufficiency and prenatal undernutrition are probably the two major causes worldwide of an adverse intrauterine environment having an impact on neurodevelopment. Clinically, both situations manifest as an intrauterine growth restriction (IUGR), a situation defined as a significant reduction in fetal growth resulting in a birth weight below the 10th percentile. This situation is a well-recognized cause of neurobehavioral and cognitive impairments extending beyond childhood and early adulthood period. Although all these evidence, the structural ground of these functional impairments and the pathophysiological mechanisms are not fully characterized. An improvement in these two aspects would allow us to propose different therapeutic strategies aiming to ameliorate and even revert the long-lasting consequences of the IUGR. HYPOTHESIS: We hypothesized that IUGR produces subtle structural brain changes that underlie the long-term neurobehavioral and cognitive impairments. The severity of the neurodevelopmental consequences might be related to the severity of the prenatal insult (reduction in nutrients with or without a reduction in oxygen). High-resolution brain imaging along with specific histological techniques focused on neuronal connectivity could evidence these structural brain changes. Additionally, we hypothesized that an early postnatal stimulation might ameliorate the structural and functional impairments that persist at the long-term period after IUGR. METHODS: Two animal models of IUGR were used in this thesis: i. A cohort of pregnant rabbits was randomized to reproduce an undernutrition model based on maternal food reduction intake, ii. Another cohort of pregnant rabbits was randomized to the placental insufficiency model based on the surgical ligation of 40-50% of the uteroplacental vessels that irrigate each gestational sac. After the delivery in both models, IUGR and controls animals were followed up to the 70th postnatal days. At the 30th postnatal days, a subgroup of IUGR animals was randomized to an environmental enrichment strategy. In all the groups at the neonatal period, general motor skills, reflexes, and olfactory sensitivity were evaluated. Similarly, at the 70th postnatal days, anxiety, memory, and learning were evaluated. Afterward, animals were sacrificed and brains were fixed and diffusion MRI was then performed. In a subset of animals, changes at the microstructural level and differences in the number of fibers in two specific brain circuits (anxiety and memory circuits) were performed by using a Voxel-Based approach (VBA) and Tractography analysis, respectively. Moreover, brain networks were obtained and evaluated by means of a Connectomics. Finally, a subgroup of animals was also histologically evaluated by means of dendritic spine and perineural nets evaluation in the Hippocampus. RESULTS: IUGR animals showed poorer functional performance in both moments, especially in the model of placental insufficiency. At the long-term period, IUGR animals presented an altered brain network architecture, being again these differences more pronounced in the placental insufficiency model. Moreover, VBA analysis and Tractography analysis evidenced microstructural brain changes mostly affecting gray matter and a decreased in the number of fibers involved in the anxiety and memory circuits in the IUGR animals in comparison to controls. At the cellular level, IUGR animals presented abnormal neuronal connectivity with changes in the dendritic spine density and in the perineural nets. In contrast, stimulated IUGR animals presented a functional and structural improvement in comparison to non-stimulated IUGR animals over the long-term period. CONCLUSIONS: This thesis adds to the previous evidence new insights regarding the pathophysiological mechanisms underlying IUGR and gives strong evidence linking IUGR with altered brain connectivity as the basis for the neurological sequelae associated with IUGR. Additionally, it gives preliminary evidence suggesting that a strategy based on physical, sensory, cognitive as well as social stimulation applied during early postnatal life, where brain plasticity is higher, might ameliorate the neurodevelopmental consequences of IUGR.

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes. Funding was from Associació Síndrome Opitz C, Terrassa, Spain; Spanish Ministerio de Economía y Competitividad (SAF2014-56562-R; FECYT, crowdfunding PRECIPITA); Catalan Government (2014SGR932) and from CIBERER (U720). We acknowledge support of the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017'.

Ageing is the main risk factor for human neurological disorders. Among the diverse molecular pathways that govern ageing, epigenetics can guide age-associated decline in part by regulating gene expression and also through the modulation of genomic instability and high-order chromatin architecture. Epigenetic mechanisms are involved in the regulation of neural differentiation as well as in functional processes related to memory consolidation, learning or cognition during healthy lifespan. On the other side of the coin, many neurodegenerative diseases are associated with epigenetic dysregulation. The reversible nature of epigenetic factors and, especially, their role as mediators between the genome and the environment make them exciting candidates as therapeutic targets. Rather than providing a broad description of the pathways epigenetically deregulated in human neurological disorders, in this review, we have focused on the potential use of epigenetic enzymes as druggable targets to ameliorate neural decline during normal ageing and especially in neurological disorders. We will firstly discuss recent progress that supports a key role of epigenetic regulation during healthy ageing with an emphasis on the role of epigenetic regulation in adult neurogenesis. Then, we will focus on epigenetic alterations associated with ageing-related human disorders of the central nervous system. We will discuss examples in the context of psychiatric disorders, including schizophrenia and posttraumatic stress disorders, and also dementia or Alzheimer's disease as the most frequent neurodegenerative disease. Finally, methodological limitations and future perspectives are discussed. EU Joint Programme-Neurodegenerative Disease Research (JPND; EPI-AD Consortium) RecerCaixa Foundation, Federación Española de Enfermedades Raras (FEDER) Federación Española de Enfermedades Neuromusculares (ASEM) Fundación Isabel Gemio Asociación Española Contra el Cáncer (AECC) Ministerio de Sanidad, Servicios Sociales e Igualdad

[eng] Phosphoenolpyruvate carboxykinase (PEPCK) is an enzyme that catalyses decarboxylation of oxaloacetate to phosphoenolpyruvate and it is part of gluconeogenic/glyceroneogenic pathway. There are two known isoforms of PEPCK, the mitochondrial and the cytosolic isozyme that are catalysing chemically identical reactions, but they differ in regulation and expression pattern. Selective presence of mitochondrial isoform of this enzyme (PEPCK-M, PCK2) in all types of cancer examined and in cycling neuroprogenitors, suggests a functional relationship with the metabolic adaptations of these cells. This thesis has had as its main objectives the characterization of the role of PEPCK-M in tumour cells and in neuronal progenitor cells. Metabolism of cell in CNS is not completely elucidated yet. Here we demonstrate that Tbr2 positive neuronal progenitors are metabolically dependent on lactate, which is favouring maintenance of their undifferentiated state. Lactate as metabolite can feed anabolic pathways and sustain ATP production by its oxidation in the TCA cycle. However, essential pathways like PPP, glycerol synthesis or one carbon metabolism pathways require carbons to feed the glycolytic intermediate pool. PEPCK-M in this setting, with lactate as sole carbon source is the only known pathway to fulfil the above-mentioned anabolic requirements. By using inhibitor of PEPCK-M we were able to prove that Tbr2 positive neuronal progenitors are metabolically dependent on PEPCK-M activity and their number significantly decrease after inhibiting PEPCK-M in vitro and in vivo. PEPCK-M activity in tumour cells is necessary for survival and growth in 2D and in cultures on semi-solid agar (anchorage-independent growth), which suggests that this enzyme has a fundamental role in the survival program to cell stress. A Kaplan-Meier analysis from datasets available in the GEO database (> 5000 patients) shows that elevated PCK2 expression is significantly associated with a worse prognosis in patients with breast cancer. Despite its potential relevance for metabolic adaptations in cancer, the mechanisms responsible for its pro- survival activity are not known. Therefore, we have proposed to study these mechanisms through metabolomic analysis where we wanted to examine whether PEPCK-M feeds an alternative pathway to glucose using carbons from glutamine in an experimental model with reduced and overexpressed levels of PEPCK-M activity. We demonstrated the functionality of PEPCK-M driven cataplerosis in MCF7 cells grown under glucose deprivation by showing synthesis of serine and glycine from glutamine by observing contribution of 13C-labeled carbons from [U-13C] glutamine into these metabolites. In the absence of nutritional stress (high abundance of glucose and amino acids), the silencing of PEPCK-M induces oxidative stress and the accumulation of succinate, with the consequent induction of p21 and deficiencies in cell growth. Glutamine cataplerosis is not affected by alterations in PEPCK-M activity. However, a higher enrichment of all carbons with 13C in intermediates of the Krebs cycle (TCA cycle) suggests a reduction in flux through this pathway. Together, these data increase our understanding of metabolic adaptations in tumours and the role of PEPCK in providing alternative carbon fluxes to deal with nutritional stress. Finally, these studies allow us to propose PEPCK-M as a new target for the treatment of tumorigenic processes that will need to be validated in the future., [spa] El fosfoenolpiruvato carboxiquinasa mitocondrial (PEPCK-M; PCK2) se regula transcripcionalmente por limitación de aminoácidos y por ER-estrés, de una manera dependiente de ATF4, aumentando así la supervivencia de la célula. La presencia selectiva de esta isoenzima en todos los tipos de cáncer examinado y en células neuroprogenitoras, sugiere una relación funcional con las adaptaciones metabólicas de estas células. Esta tesis ha tenido como objetivos fundamentales la caracterización del rol de la PEPCK-M en célula tumoral y en célula neuroprogenitora En cultivos neuronales, los neuroprogenitores Tbr2 positivos requieren lactato como sustrato metabólico para el mantenimiento de su fenotipo y su metabolismo. La PEPCK-M se expresa a niveles altos en este tipo celular y su actividad es necesaria para mantener la viabilidad de estos progenitores y cumplir con los requerimientos anabólicos a partir de carbonos provenientes del lactato. La actividad PEPCK-M en célula tumoral es necesaria para la supervivencia y crecimiento. A pesar de su potencial relevancia para las adaptaciones metabólicas en cáncer, no se conocen los mecanismos responsables de su actividad pro-supervivencia. Por ello, nos hemos propuesto estudiar estos mecanismos mediante análisis de metabolómica con los que hemos querido examinar si la PEPCK-M alimenta una vía alternativa a la glucosa utilizando carbonos provenientes de glutamina en un modelo experimental con niveles de actividad PEPCK-M reducidos y sobreexpresados. La contribución de carbonos marcados con 13C a partir de [U- 13C] glutamina en los productos de ramificación de glicolisis como serina y glicina, esta correlacionando directamente con los niveles de actividad PEPCK-M en condiciones de estrés nutricional (baja glucosa). La cataplerosis de glutamina no se ve afectada por alteraciones en la actividad de PEPCK-M. Sin embargo, un mayor enriquecimiento de 13C en intermediarios del ciclo de Krebs sugieren una reducción del flujo a través de esta vía. En conjunto, estos datos aumentan nuestra comprensión de las adaptaciones metabólicas en los tumores y el papel de la PEPCK en la provisión de flujos de carbono alternativas para lidiar con el estrés nutricional. Finalmente, estos estudios nos permiten proponer a la PEPCK-M como una nueva diana para el tratamiento de procesos tumorogénicos que necesitará ser validada en el futuro.

The study of psychiatric disorders usually focuses on emotional symptoms assessment. However, cognitive deficiencies frequently constitute the core symptoms, are often poorly controlled and handicap individual's quality of life. Adenosine receptors, through the control of both dopamine and glutamate systems, have been implicated in the pathophysiology of several psychiatric disorders such as schizophrenia and attention deficit/hyperactivity disorder. Indeed, clinical data indicate that poorly responsive schizophrenia patients treated with adenosine adjuvants show improved treatment outcomes. The A2A adenosine receptor subtype (A2AR) is highly expressed in brain areas controlling cognition and motivational responses including the striatum, hippocampus and cerebral cortex. Accordingly, we study the role of A2AR in the regulation of cognitive processes based on a complete cognitive behavioural analysis coupled with the assessment of neurogenesis and sub-synaptic protein expression in adult and middle-aged A2AR constitutional knockout mice and wild-type littermates. Our results show overall cognitive impairments in A2AR knockout mice associated with a decrease in new-born hippocampal neuron proliferation and concomitant changes in synaptic protein expression, in both the prefrontal cortex and the hippocampus. These results suggest a deficient adenosine signalling in cognitive processes, thus providing new opportunities for the therapeutic management of cognitive deficits associated with psychiatric disorders. This study was supported by MINECO (SAF 2013-41761-RFEDER, SAF 2014-55700-P, SAF 2016-75966-R, FEDER, PCIN-2013-019-C03-03), Ministry of Health (ReticeISCIIIeRD/12/0028/0024-FEDER; RD16/017/010-FEDER, PIE14/00034), Plan Nacional sobre Drogas (2014/020), IWT (SBO-140028), Fundacio la Marato de TV3 (Grant 20152031), UE Medbioinformatic project (grant number 634143) and Generalitat de Catalunya (2014 SGR 34, 2014 SGR 1251). IG-R was funded by an FPI fellowship, BES-2011-046655, associated with SAF 2010-15793.

DOI: 10.1007/s00204-016-1709-8 URL: http://link.springer.com/article/10.1007%2Fs00204-016-1709-8 Filiació URV: SI Memòria Food supplements based on herbal products are widely used during pregnancy as part of a self-care approach. The idea that such supplements are safe and healthy is deeply seated in the general population, although they do not underlie the same strict safety regulations than medical drugs. We aimed to characterize the neurodevelopmental effects of the green tea catechin epigallocatechin gallate (EGCG), which is now commercialized as high-dose food supplement. We used the “Neurosphere Assay” to study the effects and unravel underlying molecular mechanisms of EGCG treatment on human and rat neural progenitor cells (NPCs) development in vitro. EGCG alters human and rat NPC development in vitro. It disturbs migration distance, migration pattern, and nuclear density of NPCs growing as neurospheres. These functional impairments are initiated by EGCG binding to the extracellular matrix glycoprotein laminin, preventing its binding to β1-integrin subunits, thereby prohibiting cell adhesion and resulting in altered glia alignment and decreased number of migrating young neurons. Our data raise a concern on the intake of high-dose EGCG food supplements during pregnancy and highlight the need of an in vivo characterization of the effects of high-dose EGCG exposure during neurodevelopment.

Supported by the Spanish Institute of Health Carlos III (Co-funded by European Regional Development Fund “A way to make Europe”) (CP14/00108, PI16/00261, MS14/00108 [to J.J.]), the Agència de Gestió d’Ajuts Universitaris i de Recerca, Generalitat de Catalunya - Fons Social Europeu (2017 FI_B 00636 [to N.V-T.]), the Spanish Ministry of Science and Innovation (RYC-2012-10995 [to P.D.] and RYC-2011-08796 [to D.R.]), Obra Social Cajastur/Fundación Liberbank, Universidad de Oviedo, CIBERESP, Department of Health of the Basque Government, the Provincial Government of Gipuzkoa, the municipalities of the study area (Zumarraga, Urretxu, Legazpi, Azkoitia y Azpeitia y Beasain), Generalitat Valenciana, Generalitat de Catalunya, Fundació La marató de TV3, and the EU Commission. ISGlobal is a member of the CERCA Programme, Generalitat de Catalunya., López-Vicente, M., Ribas Fitó, N., Vilor-Tejedor, N., Garcia-Esteban, R., Fernández-Barrés, S., Dadvand, P., Murcia, M., Rebagliato, M., Ibarluzea, J., Lertxundi, A., Fernández-Somoano, A., Tardón, A., López-Sabater, M.C., Romaguera, D., Vrijheid, M., Sunyer, J., Julvez, J.

Adverse conditions during fetal life have been associated to both structural and functional changes in neurodevelopment from the neonatal period to adolescence. In this study, connectomics was used to assess the evolution of brain networks from infancy to early adolescence. Brain network reorganization over time in subjects who had suffered adverse perinatal conditions is characterized and related to neurodevelopment and cognition. Three cohorts of prematurely born infants and children (between 28 and 35 weeks of gestational age), including individuals with a birth weight appropriated for gestational age and with intrauterine growth restriction (IUGR), were evaluated at 1, 6, and 10 years of age, respectively. A common developmental trajectory of brain networks was identified in both control and IUGR groups: network efficiencies of the fractional anisotropy (FA)-weighted and normalized connectomes increase with age, which can be related to maturation and myelination of fiber connections while the number of connections decreases, which can be associated to an axonal pruning process and reorganization. Comparing subjects with or without IUGR, a similar pattern of network differences between groups was observed in the three developmental stages, mainly characterized by IUGR group having reduced brain network efficiencies in binary and FA-weighted connectomes and increased efficiencies in the connectome normalized by its total connection strength (FA). Associations between brain networks and neurobehavioral impairments were also evaluated showing a relationship between different network metrics and specific social cognition-related scores, as well as a higher risk of inattention/hyperactivity and/or executive functional disorders in IUGR children.

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Barcelona.--2016-11-11.-- Sobresaliente "cum laude", [EN] Development of the spinal cord involves coordination between exposure to localized extracellular signals and controlled activation of intracellular signaling pathways. This way, neuroepithelial cells firstly proliferate apically to increase the progenitor pool and, later on, initiate neurogenic divisions giving rise to a variety of neuronal cell types. Class IA PI3Ks are heterodimeric enzymes (catalytic+regulatory subunits) activated by receptors tyrosine kinase (RTKs) or G protein coupled receptors (GPCRs) that, upon extracellular stimuli, modulate diverse target proteins through local production of PtdIns(3,4,5)P3 lipids. Vertebrates express three Class IA catalytic subunits (p110-alpha, p110-beta, and p110-delta), all important for the development of the central nervous system. However, it is unclear to what extent these p110-alpha isoforms have overlapping or distinct biological roles, and what exact functions they hold in neural development. Analysis of PI3Kalpha (p110-alpha-alpha+regulatory subunit) expression in the embryonic spinal cord revealed abundant mRNA and protein levels in cycling progenitors followed by restriction of PI3K-alpha exclusively to differentiating neurons. To examine the role of PI3K-alpha in progenitors and neurons, we interfered with normal PI3K-alpha regulation by expressing active mutants or knocking down of p110-alpha-alpha in the chicken neural tube. Loss of p110-alpha resulted in high apoptotic rates in both progenitors and neurons, sustaining a role for PI3K-alpha in neural survival as seen in other studies. Instead, uncontrolled upregulation of PI3K-alpha activity resulted in severely disrupted neural tubes, with abnormal cell masses in the luminal face of the neuroepithelium and ectopic mitosis. Additionally, we observed alterations in the neural lamination characterized by basement membrane breaches followed by enhanced neural migration and misoriented axonal growth. A thorough analysis of the tissue unveiled loss of polarity as the main cellular mechanism driving the luminal structural aberrations, suggesting a major role of PI3K-alpha in neuroepithelial apico basal polarity. Moreover, the rescue of the depolarization phenotype with a dominant negative form of RhoA proposes local regulation of the Rho family of small GTPases as the molecular mechanism responsible for the PIP3 dependent regulation of adherens junction dynamics. Alternatively, we found the neural overmigration caused by excess of PI3K-alpha activity explained by increased basal accumulation of PIP3, leading to actin based membrane protrusions and basement membrane breaches. Coherently, when we assessed the neural positioning after p110-alpha knock down, we detected neurons inserted in the proliferative layer and reduction of the neuronal cytoskeletal component beta III tubulin, suggesting that PI3K-alpha also modulates morphological maturation and apico basal positioning of differentiating neurons. Interestingly, PIP3 induced overmigration seemed to be carried out through local activation of other two members of the Rho GTPases, Cdc42 and Rac1. These results shed some light upon the PI3K-alpha/PIP3 specific roles during early neural tube development, stressing out its function in cell polarity. Furthermore, we propose a mechanism that may partially explain how the PI3K-alpha /PIP3 signaling is able to control different types of polarity corresponding to different developmental moments. This could help to understand the initial events leading to some neurodevelopmental disorders caused by hyperactivation of PI3K signaling., [ES] El desarrollo de la médula espinal requiere una fina coordinación entre señales extracelulares y la activación de vías intracelulares específicas. De este modo se da una primera fase de proliferación de las células neuroepiteliales en la zona apical para aumentar el número de progenitores y una segunda fase de neurogénesis, a partir de la cual se originan diferentes tipos de neuronas. La clase IA de las PI3Ks se encuentra implicada en la transducción de señales a través de receptores tirosina quinasa (RTKs) o receptores acoplados a proteínas G (GPCRs). En respuesta a estímulos extracelulares, controlan la actividad de distintas proteínas diana a través de la producción local de lípidos PtdIns(3,4,5)P3. La clase IA de las PI3Ks, formada por enzimas heterodiméricas, consta de tres tipos de subunidades catalíticas (p110-alfa, p110-beta y p110-delta). Todas ellas son importantes para el desarrollo del sistema nervioso, sin embargo no están claras las funciones específicas de cada isoforma. El análisis de la expresión de PI3K a nivel de RNAm y proteína en la médula espinal embrionaria reveló una expresión diferencial según el estadío, siendo alta en progenitores antes de la neurogénesis y restringida a neuronas en estadíos más tardíos. Para estudiar su función en progenitores y neuronas, transfectamos formas activas de PI3K-alfa o suprimimos transitoriamente la p110-alfa en el tubo neural de embriones de pollo. La pérdida de p110-alfa provocó una alta tasa de apoptosis en ambas poblaciones, revelando su importancia en supervivencia. La sobreexpresión de la PI3K-alfa activa, en cambio, generó disrupciones muy severas del tejido neural caracterizadas por la presencia de masas celulares en la pared ventricular y mitosis ectópicas. En el lado basal, se observaron alteraciones en la laminación neuronal con células atravesando la lámina basal y crecimiento axonal aberrante. Nuestros resultados apuntan hacia la pérdida de polaridad como la principal causa de las aberraciones estructurales apicales, indicando que la PI3K-alfa tiene una función en la regulación de la polaridad apico basal. Asimismo, la PI3K-alfa parece implicada en la maduración del citoesqueleto neural y en el posicionamiento de las neuronas en el eje apico basal, funciones parcialmente mediadas por miembros de las Rho GTPasas.

[eng] Development of the spinal cord involves coordination between exposure to localized extracellular signals and controlled activation of intracellular signaling pathways. This way, neuroepithelial cells firstly proliferate apically to increase the progenitor pool and, later on, initiate neurogenic divisions giving rise to a variety of neuronal cell types. Class IA PI3Ks are heterodimeric enzymes (catalytic+regulatory subunits) activated by receptors tyrosine kinase (RTKs) or G protein coupled receptors (GPCRs) that, upon extracellular stimuli, modulate diverse target proteins through local production of PtdIns(3,4,5)P3 lipids. Vertebrates express three Class IA catalytic subunits (p110-alpha, p110-beta, and p110-delta), all important for the development of the central nervous system. However, it is unclear to what extent these p110-alpha isoforms have overlapping or distinct biological roles, and what exact functions they hold in neural development. Analysis of PI3Kalpha (p110-alpha-alpha+regulatory subunit) expression in the embryonic spinal cord revealed abundant mRNA and protein levels in cycling progenitors followed by restriction of PI3K-alpha exclusively to differentiating neurons. To examine the role of PI3K-alpha in progenitors and neurons, we interfered with normal PI3K-alpha regulation by expressing active mutants or knocking down of p110-alpha-alpha in the chicken neural tube. Loss of p110-alpha resulted in high apoptotic rates in both progenitors and neurons, sustaining a role for PI3K-alpha in neural survival as seen in other studies. Instead, uncontrolled upregulation of PI3K-alpha activity resulted in severely disrupted neural tubes, with abnormal cell masses in the luminal face of the neuroepithelium and ectopic mitosis. Additionally, we observed alterations in the neural lamination characterized by basement membrane breaches followed by enhanced neural migration and misoriented axonal growth. A thorough analysis of the tissue unveiled loss of polarity as the main cellular mechanism driving the luminal structural aberrations, suggesting a major role of PI3K-alpha in neuroepithelial apico basal polarity. Moreover, the rescue of the depolarization phenotype with a dominant negative form of RhoA proposes local regulation of the Rho family of small GTPases as the molecular mechanism responsible for the PIP3 dependent regulation of adherens junction dynamics. Alternatively, we found the neural overmigration caused by excess of PI3K-alpha activity explained by increased basal accumulation of PIP3, leading to actin based membrane protrusions and basement membrane breaches. Coherently, when we assessed the neural positioning after p110-alpha knock down, we detected neurons inserted in the proliferative layer and reduction of the neuronal cytoskeletal component beta III tubulin, suggesting that PI3K-alpha also modulates morphological maturation and apico basal positioning of differentiating neurons. Interestingly, PIP3 induced overmigration seemed to be carried out through local activation of other two members of the Rho GTPases, Cdc42 and Rac1. These results shed some light upon the PI3K-alpha/PIP3 specific roles during early neural tube development, stressing out its function in cell polarity. Furthermore, we propose a mechanism that may partially explain how the PI3K-alpha /PIP3 signaling is able to control different types of polarity corresponding to different developmental moments. This could help to understand the initial events leading to some neurodevelopmental disorders caused by hyperactivation of PI3K signaling., [spa] El desarrollo de la médula espinal requiere una fina coordinación entre señales extracelulares y la activación de vías intracelulares específicas. De este modo se da una primera fase de proliferación de las células neuroepiteliales en la zona apical para aumentar el número de progenitores y una segunda fase de neurogénesis, a partir de la cual se originan diferentes tipos de neuronas. La clase IA de las PI3Ks se encuentra implicada en la transducción de señales a través de receptores tirosina quinasa (RTKs) o receptores acoplados a proteínas G (GPCRs). En respuesta a estímulos extracelulares, controlan la actividad de distintas proteínas diana a través de la producción local de lípidos PtdIns(3,4,5)P3. La clase IA de las PI3Ks, formada por enzimas heterodiméricas, consta de tres tipos de subunidades catalíticas (p110-alfa, p110-beta y p110-delta). Todas ellas son importantes para el desarrollo del sistema nervioso, sin embargo no están claras las funciones específicas de cada isoforma. El análisis de la expresión de PI3K a nivel de RNAm y proteína en la médula espinal embrionaria reveló una expresión diferencial según el estadío, siendo alta en progenitores antes de la neurogénesis y restringida a neuronas en estadíos más tardíos. Para estudiar su función en progenitores y neuronas, transfectamos formas activas de PI3K-alfa o suprimimos transitoriamente la p110-alfa en el tubo neural de embriones de pollo. La pérdida de p110-alfa provocó una alta tasa de apoptosis en ambas poblaciones, revelando su importancia en supervivencia. La sobreexpresión de la PI3K-alfa activa, en cambio, generó disrupciones muy severas del tejido neural caracterizadas por la presencia de masas celulares en la pared ventricular y mitosis ectópicas. En el lado basal, se observaron alteraciones en la laminación neuronal con células atravesando la lámina basal y crecimiento axonal aberrante. Nuestros resultados apuntan hacia la pérdida de polaridad como la principal causa de las aberraciones estructurales apicales, indicando que la PI3K-alfa tiene una función en la regulación de la polaridad apico basal. Asimismo, la PI3K-alfa parece implicada en la maduración del citoesqueleto neural y en el posicionamiento de las neuronas en el eje apico basal, funciones parcialmente mediadas por miembros de las Rho GTPasas.

Endocannabinoids participate in the control of neurogenesis, neural cell death and gliosis. The pharmacological effect of the fatty acid amide hydrolase (FAAH) inhibitor URB597, which limits the endocannabinoid degradation, was investigated in the present study. Cell proliferation (phospho-H3(+) or BrdU(+) cells) of the main adult neurogenic zones as well as apoptosis (cleaved caspase-3(+)), astroglia (GFAP(+)), and microglia (Iba1(+) cells) were analyzed in the hippocampus, hypothalamus and striatum of rats intraperitoneally treated with URB597 (0.3 mg/kg/day) at one dose/4-days resting or 5 doses (1 dose/day). Repeated URB597 treatment increased the plasma levels of the N-acylethanolamines oleoylethanolamide, palmitoylethanolamide and arachidonoylethanolamine, reduced the plasma levels of glucose, triglycerides and cholesterol, and induced a transitory body weight decrease. The hippocampi of repeated URB597-treated rats showed a reduced number of phospho-H3(+) and BrdU(+) subgranular cells as well as GFAP(+), Iba1(+) and cleaved caspase-3(+) cells, which was accompanied with decreased hippocampal expression of the cannabinoid CB1 receptor gene Cnr1 and Faah. In the hypothalami of these rats, the number of phospho-H3(+), GFAP(+) and 3-weeks-old BrdU(+) cells was specifically decreased. The reduced striatal expression of CB1 receptor in repeated URB597-treated rats was only associated with a reduced apoptosis. In contrast, the striatum of acute URB597-treated rats showed an increased number of subventricular proliferative, astroglial and apoptotic cells, which was accompanied with increased Faah expression. Main results indicated that FAAH inhibitor URB597 decreased neural proliferation, glia and apoptosis in a brain region-dependent manner, which were coupled to local changes in Faah and/or Cnr1 expression and a negative energy context. Grant sponsor: 7th Framework Programme of European Union; Grant number: HEALTH-F2-2008-223713, REPROBESITY to FR and BL; Grant sponsor: Ministerio de Ciencia e Innovación; Grant numbers: SAF2010-19087, SAF 2010-20521; Grant sponsor: Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, UE-ERDF; Grant number: CP12/03109; Grant sponsor: Red de Trastornos Adictivos; Grant numbers: RD12/0028/0001, RD12/0028/0009; Grant sponsor: CIBERobn; Grant sponsor: Plan Nacional Sobre Drogas, Ministerio de Sanidad y Consumo; Grant number: PNSD2010/143; Grant sponsor: Consejería de Economía, Innovación y Ciencia, Junta de Andalucía, UE/ERDF; Grant numbers: CTS-433, P-11-CVI-07637; Grant sponsor: Consejería de Salud, Junta de Andalucía; Grant numbers: PI0232/2008, PI0029/2008, SAS111224; Grant sponsor: Fundació La Marató de TV3; Grant number: 386/C/2011; Grant sponsor: German Research Foundation DFG; Grant number: FOR629, project CP2 to BL. JS, FP, and AS hold “Miguel Servet” research contracts from the National System of Health, ISCIII (Grant numbers: CP12/03109, CP14/00212, CP14/00173 respectively).

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia, Universitat de Barcelona, 2015. Tutora: Elena Escubedo Rafa., [cat] Fins fa unes dècades predominava el dogma universal sobre la incapacitat del sistema nerviós central de regenerar-se després del naixement, establint així, la no-formació de noves neurones durant l’etapa adulta. Recentment, s’ha demostrat el contrari, el cervell adult és capaç de regenerar-se i crear noves neurones madures funcionals, i ho fa al llarg de tota la vida. L’objectiu principal del present treball és, per tant, descriure i caracteritzar el procés de neurogènesi del cervell d’un mamífer adult a partir de la recerca de bibliografia científica que ens permeti abordar i resoldre els aspectes més importants del procés: què és, on i com es dóna, quina és la seva funció, com es troba regulat i quina implicació i relació té amb les malalties neurodegeneratives i el consum de drogues d’abús. Hi ha un gran nombre d’estudis que palesen la inhibició del procés de neurogènesi degut al consum de substàncies com ara l’alcohol, la cocaïna o els opioides. En el present treball s’ha volgut determinar també l’efecte de la mefedrona (sola i en combinació amb etanol) sobre la neurogènesi en l’hipocamp de ratolins adolescents. Per avaluar-ho, s’ha administrat als animals una solució de BrdU com a marcardor de cèl·lules noves i, 28 dies després, s’han sacrificat i s’ha fet una immunohistoquímica en portaobjectes per BrdU i NeuN amb els talls coronals de cervell. Amb tot, s’ha pogut observar un descens en la taxa de neurogènesi en els animals tractats amb mefedrona, i encara més, en els tractats concomitantment amb alcohol. Aquest fet, per tant, podria relacionar-se amb una alteració de la funcionalitat del sistema nerviós central., [eng] Until a few decades ago a universal dogma existed with regards to the inability of the central nervous system to regenerate after birth, impeding the formation of new neurons during adulthood. Recently, the opposite has been demonstrated. The adult brain is able to regenerate and create new functional neurons; this occurs during the whole lifespan. The main aim of the present work is to describe and characterize the process of neurogenesis in the adult mammalian brain through scientific literature search allowing us to address the most important aspects of the process: what it is, where and how it occurs, what its function is, how it is regulated and what involvement it has in neurodegenerative diseases and the use of drugs of abuse. There are a lot of studies that show the inhibition of the process of neurogenesis due to the intake of drugs of abuse like ethanol, cocaine or opioids. In this project we also wanted to determine the effect of mephedrone (alone and in combination with ethanol) on hippocampal neurogenesis in adolescent mice. To assess this, we administered animals with a solution of BrdU as a new cell marker and, 28 days later, the animals were sacrificed and an immunohistochemistry on slides for BrdU and NeuN was performed on coronal brain slices. A decrease in the neurogenesis rate of animals treated with mephedrone was observed; this effect was further enhanced in the mephedrone plus alcohol group. This phenomenon may be explained by an altered functionality of the central nervous system.

Alternative splicing (AS) generates extensive transcriptomic and proteomic complexity. However, the functions of species- and lineage-specific splice variants are largely unknown. Here we show that mammalian-specific skipping of polypyrimidine tract-binding protein 1 (PTBP1) exon 9 alters the splicing regulatory activities of PTBP1 and affects the inclusion levels of numerous exons. During neurogenesis, skipping of exon 9 reduces PTBP1 repressive activity so as to facilitate activation of a brain-specific AS program. Engineered skipping of the orthologous exon in chicken cells induces a large number of mammalian-like AS changes in PTBP1 target exons. These results thus reveal that a single exon-skipping event in an RNA binding regulator directs numerous AS changes between species. Our results further suggest that these changes contributed to evolutionary differences in the formation of vertebrate nervous systems SG is supported by an NSERC Alexander Graham Bell Studentship, TGP is supported by fellowships from EMBO and OSCI, and MI was supported by a LTF from the Human Frontiers Science Program Organization. This research was supported by grants from the CIHR to BJB and A-C.G. BJB holds the Banbury Chair of Medical Research at the University of Toronto. Data presented in this manuscript is archived in the gene Gene Expression Omnibus (GEO) under accession number GSE69656.

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 ( glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

[spa] El tráfico de membrana en el cono crecimiento axonal es clave durante el desarrollo del sistema nervioso. La familia de proteínas SNARE son una familia de moléculas cuya función principal es mediar la fusión entre membranas. Este hecho hace que las proteínas SNARE tengan potencialmente tengan un papel en el desarrollo axonal y otros procesos que impliquen una gran actividad en los procesos de tráfico de membrana: un ejemplo de casos como así es la progresión de tumores con un alto grado de recambio de membrana como el glioblastoma, que es el caso más común y mortal de astrocitoma de grado IV. Las proteínas SNAREs se clasifican en dos grupos t-SNARE (localizadas en las membranas objetivo) y v-SNARE (localizadas en las membranas vesiculares) y juntas median la fusión entre membranas a través de la formación del complejo SNARE. El complejo SNARE mejor caracterizado es el tetrámero formado por Sintaxina1 (Stx1), dos SNAP-25 y VAMP2. En esta tesis doctoral estudiamos en primer lugar el papel de las proteínas SNARE en los procesos de guía axonal. Para ello utilizamos como modelo el desarrollo del sistema nervioso embrionario en Drosophila melanogaster y obtuvimos como resultado que problemas en las proteínas SNARE, especialmente en Sintaxina1A (Syx1A), generan problemas de guía axonal en el sistema nervioso. Si nos centramos en los estudios del desarrollo del sistema nervioso en mutantes nulos para Syx1A, todas las estructuras nerviosas desde el sistema nervioso central, los nervios motores y el sistema periférico desarrollaron problemas de guía axonal. Además, estudios de interacción génica y una detalla caracterización del fenotipo sugieren que la alteración de las proteínas SNARE, especialmente Syx1A, genera problemas en la vía de guía axonal Slit/Robo. En segundo lugar, estudiamos el rol de las proteínas SNARE en la progresión tumoral. Pare ello analizamos los efectos in vitro e in vivo que tiene el bloqueo de la función de Stx1 en líneas celulares y modelos murínicos del glioblastoma Como resultado, detectamos una pérdida de la proliferación in vitro e in vivo al bloquear Stx1. Además, el bloqueo de Stx1 disminuye la capacidad invasiva in vitro de las células de glioblastoma., [eng] The membrane delivery/traffic in the growth cone is a key element during axons development. SNARE family proteins may be important players in this process since they mediate the fusion of membrane vesicles with their targets. Moreover, since membrane trafficking is a key element during the progression of several tumors, SNAREs may be important also during tumoral progression. Two groups form the SNARE family: t-SNARE (Syntaxins, SNAPs…) located in the target membranes, and, v-SNARE (VAMPs), located in the vesicles. Complexes between specific t- and v- SNAREs are formed which ultimately leads to the fusion of vesicles with their target membranes. The best-characterized SNARE complex is the tetramer formed by syntaxin1 (Stx1), two SNAP-25 and VAMP2, which mediates the fusion of synaptic vesicles during the exocytosis of neurotransmitters. In order to study the role of SNARE in axon guidance we used as a model Drosophila melanogaster embryonic nerve system development. Embryos mutant for SNARE proteins developed axon guidance defects. From all the stocks, Syx1A had a severe phenotype in comparison with the other mutants. Syx1A null embryos developed axon guidance problems in all the nerve system structures: ventral nerve cord, motor axons or peripheral nerves. In addition, a deep analysis of the phenotype and genetic interaction studies suggest that Syx1A mutants have problems in Slit/Robo axon guidance pathway. In order to study the role of SNARE protein in tumoral progression we studied in vitro and in vivo the effect of Stx1 inactivation in glioblastoma models (glioblastomas are the most common and deadliest astrocitomas). Our results show that the loss of function in Stx1 decreases in vitro and in vivo glioblastoma progression and it blocks in vitro cellular invasion.

N-methyl-D-aspartate glutamate receptors (NMDARs) play a pivotal role in neural development and synaptic plasticity, as well as in neurological disease. Since NMDARs exert their function at the cell surface, their density in the plasma membrane is finely tuned by a plethora of molecules that regulate their production, trafficking, docking and internalization in response to external stimuli. In addition to transcriptional regulation, the density of NMDARs is also influenced by post-translational mechanisms like phosphorylation, a modification that also affects their biophysical properties. We previously described the increased surface expression of GluN1/GluN2A receptors in transgenic mice overexpressing the Dual specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A), suggesting that DYRK1A regulates NMDARs. Here we have further investigated whether the density and activity of NMDARs were modulated by DYRK1A phosphorylation. Accordingly, we show that endogenous DYRK1A is recruited to GluN2A-containing NMDARs in the adult mouse brain, and we identify a DYRK1A phosphorylation site at Ser1048 of GluN2A, within its intracellular C-terminal domain. Mechanistically, the DYRK1A-dependent phosphorylation of GluN2A at Ser1048 hinders the internalization of GluN1/GluN2A, causing an increase of surface GluN1/GluN2A in heterologous systems, as well as in primary cortical neurons. Furthermore, GluN2A phosphorylation at Ser1048 increases the current density and potentiates the gating of GluN1/GluN2A receptors. We conclude that DYRK1A is a direct regulator of NMDA receptors and we propose a novel mechanism for the control of NMDAR activity in neurons. This work was supported by grants from the Spanish Ministry of Health (PS10/00548,PS13/00135 to Xavier Altafaj), the Jérôme Le jeune Foundation (to Xavier Altafaj), the Red HERACLES (RD12/0042/0014 to José M. Fernández-Fernández), the Spanish Ministry o fEconomy and Competitiveness (SAF2012-31089 to José M. Fernández-Fernándezand BFU2010-15347 to Susana de la Luna), FEDER Funds and the Generalitat de Catalunya (SGR14-297 to Xavier Altafaj and Carlos Sindreu, and SGR14-674 toSusana de la Luna). Xavier Altafaj and Cristina Grauare financed by a contract from the Instituto de Salud Carlos III (MS10/00548 andPS10/00548)

This PhD thesis is focused in the application of brain network models obtained from different modalities of magnetic resonance imaging (MRI) to characterize anomalies in neurodevelopment of a prenatal origin, using intrauterine growth restriction (IUGR) as a clinical model. Importantly, IUGR due to placental insufficiency affects 5-10% of all pregnancies and is a leading cause of fetal morbidity and mortality. The thesis is presented as a compendium of four studies published in international journals. Each of the studies is focused in the characterization of IUGR using brain networks obtained from a specific MRI modality (structural, diffusion and functional MRI) in a specific pediatric stage in the life of subjects with IUGR (neonatal, early infancy and pre-adolescence age). The first study focuses in the characterization of brain reorganization produced by IUGR at one year of age using brain networks based on a tractography obtain from diffusion MRI, using diffusion tensor imaging (DTI) approach. In this study it is demonstrated that brain network features of IUGR infants have alterations associated with an altered neurodevelopment later in life. The second study assess the viability to use a novel methodology to obtain structural brain networks based on simple anatomical MRI based on the similitude of gray matter (GM) patterns among different areas of the brain. We demonstrated alterations in infants with IUGR using this technique, and that the alterations found are also associated with neurodevelopmental problems found later in life. In the third study we used a rabbit model of IUGR to explore if the alterations in the structural brain network persist at long-term, during preadolescence. We demonstrated that indeed, there are alterations in the structural brain network organization that persist at long-term and that this alterations are associated with neurobehavioral outcomes. Finally, using normalization approaches, we observed a peculiar compensatory effect in the subjects with IUGR. In the forth study, we assessed functional brain networks of neonates with IUGR, demonstrating that this condition produces a reorganization of functional brain connectivity since such an early age, characterized by a pattern of increased co-activation and synchronization of brain regions together with a suboptimal organization when assessing normalized networks. In addition, functional brain network features were also associated to neurobehavioral alterations. Overall, our main conclusion is that IUGR condition produces structural and functional brain reorganization since early life that persists postnatally up to pre-adolescence. We hypothesize that the observed functional and structural reorganization could be a potential substrate of high risk of altered neurodevelopment in infants with IUGR, and postulate this condition as a possible brain network disorder. Importantly, the association of network features with neurobehavior and neurodevelopment since an early age opens the opportunity to further develop early image biomarkers of altered neurodevelopment, a clinical chance to improve the management of a condition that affects up to 10% of deliveries., Aquesta tesis doctoral està centrada en l'aplicació de models de xarxa del cervell obtinguts a partir de diferents modalitats de ressonància magnètica (RM) per caracteritzar anomalies en el desenvolupament d'origen prenatal utilitzant la restricció de creixement intrauterí (RCIU) com a model clínic. La tesi està presentada com a compendi de quatre estudis publicats en revistes internacionals de primer quartil. Cada un dels estudis està centrat en la caracterització de la RCIU mitjançant xarxes cerebrals obtingudes a partir d'una modalitat de RM determinada en una etapa pediàtrica diferent, en la vida de subjectes amb RCIU. Així doncs, el primer estudi es centra en la caracterització de la reorganització cerebral produïda per RCIU a l'any de vida mitjançant xarxes cerebrals estructurals basades en RM per difusió. En aquest estudi es demostra que les característiques de xarxa en els subjectes amb RCIU presenten una sèrie d'alteracions relacionades amb un neuro-desenvolupament futur anormal. El segon projecte analitza la utilització de xarxes estructurals cerebrals basades en RM anatòmica convencional per caracteritzar alteracions en nens d'un any amb RCIU. Es demostra que efectivament amb aquesta tècnica també es troben alteracions en els infants amb IUGR, i que aquestes alteracions estan també relacionades amb problemes en el neuro-desenvolupament posterior. En el tercer projecte s'utilitza un model animal de conill amb RCIU per explorar les alteracions en la xarxa cerebral estructural que persisteix a llarg termini. Es demostra que efectivament existeixen alteracions en la organització estructural del cervell persistents a llarg termini i s'observa un efecte compensatori en els subjectes amb RCIU. En el quart projecte s'analitzen les xarxes cerebrals funcional en neonats amb RCIU, demostrant que aquesta condició prenatal genera una reorganització en la connectivitat cerebral que té un substrat funcional, que es pot observar des d'etapes molt precoces de la vida i que està relacionada amb resultats de neuro-comportament.

Santiago Ramón y Cajal developed a great body of scientific research during the last decade of 19th century, mainly between 1888 and 1892, when he published more than 30 manuscripts. The neuronal theory, the structure of dendrites and spines, and fine microscopic descriptions of numerous neural circuits are among these studies. In addition, numerous cell types (neuronal and glial) were described by Ramón y Cajal during this time using this “reazione nera” or Golgi method. Among these neurons were the special cells of the molecular layer of the neocortex. These cells were also termed Cajal cells or Retzius cells by other colleagues. Today these cells are known as Cajal–Retzius cells. From the earliest description, several biological aspects of these fascinating cells have been analyzed (e.g., cell morphology, physiological properties, origin and cellular fate, putative function during cortical development, etc). In this review we will summarize in a temporal basis the emerging knowledge concerning this cell population with specific attention the pioneer studies of Santiago Ramón y Cajal.

For both the intricate morphogenetic layout of the sensory cells in the ear and the elegantly radial arrangement of the sensory neurons in the nose, numerous signaling molecules and genetic determinants are required in concert to generate these specialized neuronal populations that help connect us to our environment. In this review, we outline many of the proteins and pathways that play essential roles in the differentiation of otic and olfactory neurons and their integration into their non-neuronal support structures. In both cases, well-known signaling pathways together with region-specific factors transform thickened ectodermal placodes into complex sense organs containing numerous, diverse neuronal subtypes. Olfactory and otic placodes, in combination with migratory neural crest stem cells, generate highly specialized subtypes of neuronal cells that sense sound, position and movement in space, odors and pheromones throughout our lives. ECM was funded by a Marie Curie Intra European Fellowship(275978). This work was supported by the following grants: BFU2011-27006 to BA, NIH grants DE16459 and DC011577 to MEB, and a BBSRC project grant BB/J003050 to TTW

[spa] En el cerebro postnatal se produce regularmente neurogénesis endógena en áreas concretas; estas áreas comprenden mayoritariamente la zona subventricular (SVZ) y el giro dentado (DG). En el cerebro adulto, los astrocitos de la SVZ tienen características de célula madre neural, por su poder de auto-renovación, proliferación y diferenciación tanto hacia el linaje neural como hacia el linaje glial. Las células madre neurales se regulan por multitud de señales moleculares y celulares del nicho neurogénico, sin embargo, se conoce muy poco acerca de los mecanismos moleculares que regulan la neurogénesis endógena tanto en condiciones fisiológicas como patológicas. El conocimiento básico de los fenómenos que regulan la proliferación, diferenciación y funcionalidad de las neuronas generadas de novo de manera endógena en el cerebro postnatal es necesario para la futura aplicabilidad de la neurogénesis endógena como terapia celular. El objetivo principal de la presente tesis doctoral ha sido estudiar el efecto de dos citocinas sobre la neurogénesis endógena en condiciones fisiológicas. El IFNγ y la IL-10 son citocinas proinflamatorias y antinflamatorias respectivamente que se expresan en condiciones fisiológicas en muy bajas concentraciones, incrementando considerablemente sus niveles en situaciones patológicas asociadas a procesos inflamatorios. Empleando una metodología in vitro y estudios in vivo pudimos obtener resultados a cerca del efecto que ejercen las citocinas en estudio sobre el comportamiento de las células madre neurales en el nicho neurogénico de la SVZ y sobre la neurogénesis adulta en condiciones fisiológicas., [eng] In the postnatal brain endogenous neurogenesis occurs regularly in specific areas; these areas mostly comprise the subventricular zone (SVZ) and the dentate gyrus (DG). In the adult brain, astrocytes located in SVZ have characteristics of neural stem cell, by its power of self-renewal, proliferation and differentiation into neural and glial lineage. Neural stem cells are regulated by a multitude of molecular and cellular signals in the neurogenic niche, however, very little are known about the molecular mechanisms that regulate endogenous neurogenesis in both physiological and pathological conditions. Basic knowledge of the phenomena that regulate the proliferation, differentiation and function of neurons generated de novo endogenously in the postnatal brain is necessary for future applicability of endogenous neurogenesis and cellular therapy. The main objective of this thesis was to study the effect of two cytokines on endogenous neurogenesis in physiological conditions. IFNy and IL-10 are respectively proinflammatory and cytokines anti-inflammatory expressed under physiological conditions in very low concentrations, greatly increasing its levels in pathological conditions associated with inflammatory processes. Using in vitro and in vivo methodology we obtained results about IFNy and IL-10 effect on the behavior of neural stem cells in the SVZ and on adult neurogenesis under physiological conditions.

Background: The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings: In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion: Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. This study was supported by grants from Fondo de Investigaciones Sanitarias (FIS) (PI10/02593; PI13/01135) and Red de Salud Materno-Infantil y del Desarrollo (SAMID) (RD12/0026/0003) from the Instituto Carlos III (Spain), a grant from Mutua Madrileña (AP150572014), intramural funding of the Neuroscience Program at IMIM (Institut Hospital del Mar ' Investigacions Mèdiques), partially supported by Generalitat de Catalunya (Spain) (2009SGR1388; 2014SGR584) and from Spanish Ministry of Economy and Competitiveness (BFU2012-31994).