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A hydroxycinnamic acid derivative, namely ferulic acid (FA) has been successfully encapsulated in polymeric nanoparticles (NPs) based on poly(lactic-co-glycolic acid) (PLGA). FA-loaded polymeric NPs were prepared from O/W nano-emulsion templates using the phase inversion composition (PIC) low-energy emulsification method. The obtained PLGA NPs exhibited high colloidal stability, good drug-loading capacity, and particle hydrodynamic diameters in the range of 74 to 117 nm, depending on the FA concentration used. In vitro drug release studies confirmed a diffusion-controlled mechanism through which the amount of released FA reached a plateau at 60% after 6 hours-incubation. Five kinetic models were used to fit the FA release data as a function of time. The Weibull distribution and Korsmeyer-Peppas equation models provided the best fit to our experimental data and suggested quasi-Fickian diffusion behaviour. Moderate dose-response antioxidant and radical scavenging activities of FA-loaded PLGA NPs were demonstrated using the DPPH˙ assay achieving inhibition activities close to 60 and 40%, respectively. Cell culture studies confirmed that FA-loaded NPs were not toxic according to the MTT colorimetric assay, were able to internalise efficiently SH-SY5Y neuronal cells and supressed the intracellular ROS-level induced by H2O2 leading to 52% and 24.7% of cellular viability at 0.082 and 0.041 mg mL-1, respectively. The permeability of the NPs through the blood brain barrier was tested with an in vitro organ-on-a-chip model to evaluate the ability of the FA-loaded PLGA and non-loaded PLGA NPs to penetrate to the brain. NPs were able to penetrate the barrier, but permeability decreased when FA was loaded. These results are promising for the use of loaded PLGA NPs for the management of neurological diseases., The authors acknowledge the Agencia Estatal de Investigación for funding (Projects CTQ2017-84998-P and PID2021122187NB-C31) and the Instituto de Salud Carlos III (ISCIII) (CB06/01/1058 and CB06/01/0055). CIBER BBN is an initiative funded by the VI National R + D + I Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER actions and financed by ISCIII with assistance from the European Regional Development Fund. The authors would like to thank Gemma Fabriàs’ research group for assistance and access to the cytometer instrument. The authors want to thank NANBIOSIS ICTS and the Nanostructured Liquid Characterization Unit (U12) for DLS and ζ-potential measurements, hyperspectral microscopy and dark-field imaging. The authors thank the Electron Microscopy Service from the National Centre of Biotechnology (CNB-CSIC), especially Beatriz Martín for sample preparation and TEM observations. The authors also acknowledge the “Grupo de Nanotecnología Farmacéutica” from the University of Barcelona in the Faculty of Pharmacy and Food Sciences which forms a R&D Associate Unit to CSIC.

[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.

Strabismus is not a condition in itself but the consequence of an underlying problem. Eye misalignment can be caused by disease, injury, and/or abnormalities in any of the structures and processes involved in visual perception and oculomotor control, from the extraocular muscles and their innervations to the oculomotor and visual processing areas in the brain. A small percentage of all strabismus cases are the consequence of well-described genetic syndromes, acquired insult, or disease affecting the extraocular muscles (EOMs) or their innervations. We will refer to them as strabismus of peripheral origin since their etiology lies in the peripheral nervous system. However, in most strabismus cases, that is comitant, non-restrictive, non-paralytic strabismus, the EOMs and their innervations function properly. These cases are not related to specific syndromes and their precise causes remain poorly understood. They are generally believed to be caused by deficits in the central neural pathways involved in visual perception and oculomotor control. Therefore, we will refer to them as central strabismus. The goal of this narrative review is to discuss the possible causes behind this particular type of eye misalignment and to raise awareness among eyecare professionals about the important role the central nervous system plays in strabismus etiology, and the subsequent implications regarding its treatment. A non-systematic search was conducted using PubMed, Medline, Cochrane, and Google Scholar databases with the keywords “origins,” “causes,” and “etiology” combined with “strabismus.” A snowball approach was also used to find relevant references. In the following article, we will first describe EOM integrity in central strabismus; next, we will address numerous reasons that support the idea of central nervous system (CNS) involvement in the origin of the deviation, followed by listing several possible central causes of the ocular misalignment. Finally, we will discuss the implications CNS etiology has on strabismus treatment. Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature.

This study describes the clinical characteristics, diagnostic results, treatment regimens, and clinical course of a cohort of patients with Susac syndrome (SS). It is a retrospective observational study of all patients with the diagnosis of SS evaluated at the Hospital Clinic (Barcelona, Spain) between March 2006 and November 2020. Nine patients were diagnosed with SS. The median time from the onset of the symptoms to diagnosis was five months (IQR 9.0), and the median follow-up time was 44 months (IQR 63.5). There was no clear predominance of sex, and mean age of symptoms onset was 36 years (range 19–59). Six patients (67%) presented with incomplete classical clinical triad, but this eventually developed in six patients during the disease course. Encephalopathy, focal neurological signs, visual disturbances, and hearing loss were the most frequent manifestations. Brain magnetic resonance imaging showed callosal lesions in all patients. Most were in remission within two years. Only four patients met the proposed criteria for definite SS. When SS is suspected, a detailed diagnostic workup should be performed and repeated over time to identify the clinical manifestations that will lead to a definite diagnosis.

Luminance; Chromatic channels Luminancia; Canales cromáticos Lluminància; Canals cromàtics Color induction is the phenomenon where the physical and the perceived colors of an object differ owing to the color distribution and the spatial configuration of the surrounding objects. Previous works studying this phenomenon on the lsY MacLeod–Boynton color space, show that color assimilation is present only when the magnocellular pathway (i.e., the Y axis) is activated (i.e., when there are luminance differences). Concretely, the authors showed that the effect is mainly induced by the koniocellular pathway (s axis), but not by the parvocellular pathway (l axis), suggesting that when magnocellular pathway is activated it inhibits the koniocellular pathway. In the present work, we study whether parvo-, konio-, and magnocellular pathways may influence on each other through the color induction effect. Our results show that color assimilation does not depend on a chromatic–chromatic interaction, and that chromatic assimilation is driven by the interaction between luminance and chromatic channels (mainly the magno- and the koniocellular pathways). Our results also show that chromatic induction is greatly decreased when all three visual pathways are simultaneously activated, and that chromatic pathways could influence each other through the magnocellular (luminance) pathway. In addition, we observe that chromatic channels can influence the luminance channel, hence inducing a small brightness induction. All these results show that color induction is a highly complex process where interactions between the several visual pathways are yet unknown and should be studied in greater detail. Supported through the research projects: Grant PID2020-115734RB-C21 funded by MCIN/AEI/10.13039/501100011033 and Grant DPI2017-89867-C2-1-R funded by MCIN/AEI/10.13039/501100011033/ and FEDER Una manera de hacer Europa, by the Secretaria d'Universitats i Recerca del Departament d'Empresa i Coneixement de la Generalitat de Catalunya through 2017-SGR-649, and CERCA Programme/Generalitat de Catalunya.

Objective: To assess the central nervous system (CNS) impact of a kick&kill HIV cure strategy using therapeutic vaccine MVA.HIVconsv and the histone deacetylase inhibitor (HDACi) romidepsin (RMD) as latency-reversing agent. Design: Neurological observational substudy of the BCN02 trial (NCT02616874), a proof-of-concept, open-label, single-arm, phase I clinical trial testing the safety and immunogenicity of the MVA.HIVconsv vaccine and RMD in early-treated HIV-1-infected individuals. A monitored antiretroviral pause (MAP) was performed, with cART resumption after 2 pVL more than 2000 copies/ml. Reinitiated participants were followed for 24 weeks. Methods: Substudy participation was offered to all BCN02 participants (N = 15). Evaluations covered cognitive, functional, and brain imaging outcomes, performed before RMD administration (pre-RMD), after three RMD infusions (post-RMD), and at the end of the study (EoS). A group of early-treated HIV-1-infected individuals with matched clinical characteristics was additionally recruited (n = 10). Primary endpoint was change in a global cognitive score (NPZ-6). Results: Eleven participants from BCN02 trial were enrolled. No significant changes were observed in cognitive, functional, or brain imaging outcomes from pre-RMD to post-RMD. No relevant alterations were detected from pre-RMD to EoS either. Scores at EoS were similar in participants off cART for 32 weeks (n = 3) and those who resumed therapy for 24 weeks (n = 7). Controls showed comparable punctuations in NPZ-6 across all timepoints. Conclusion: No detrimental effects on cognitive status, functional outcomes, or brain imaging parameters were observed after using the HDACi RMD as latency-reversing agent with the MVA.HIVconsv vaccine in early-treated HIV-1-infected individuals. CNS safety was also confirmed after completion of the MAP.

We have here assessed, using Δ9-tetrahydrocannabinol (Δ9-THC) for comparison, the effect of Δ9-tetrahydrocannabinolic acid (Δ9-THCA) and of Δ9-tetrahydrocannabivarin (Δ9-THCV) that is mediated by human versions of CB1, CB2, and CB1-CB2 receptor functional units, expressed in a heterologous system. Binding to the CB1 and CB2 receptors was addressed in living cells by means of a homogeneous assay. A biphasic competition curve for the binding to the CB2 receptor, was obtained for Δ9-THCV in cells expressing the two receptors. Signaling studies included cAMP level determination, activation of the mitogen-activated protein kinase pathway and ß-arrestin recruitment were performed. The signaling triggered by Δ9-THCA and Δ9-THCV via individual receptors or receptor heteromers disclosed differential bias, i.e. the bias observed using a given phytocannabinoid depended on the receptor (CB1, CB2 or CB1-CB2) and on the compound used as reference to calculate the bias factor (Δ9-THC, a selective agonist or a non-selective agonist). These results are consistent with different binding modes leading to differential functional selectivity depending on the agonist structure, and the state (monomeric or heteromeric) of the cannabinoid receptor. In addition, on studying Gi-coupling we showed that Δ9-THCV and Δ9-THCA and Δ9-THCV were able to revert the effect of a selective CB2 receptor agonist, but only Δ9-THCV, and not Δ9-THCA, reverted the effect of arachidonyl-2'-chloroethylamide (ACEA 100 nM) a selective agonist of the CB1 receptor. Overall, these results indicate that cannabinoids may have a variety of binding modes that results in qualitatively different effects depending on the signaling pathway that is engaged upon cannabinoid receptor activation.

Although the biological components of the endocannabinoid system (ECS) are well known and have been explored in detail over many decades, its significance seems to enlarge with every new experimental study. The ECS employs a huge network of molecules (receptors, ligands, and enzymatic machinery molecules) whose interactions with other cellular networks have still not been fully elucidated. It has become evident that its historical role in pain alleviation is just the tip of an enormous iceberg of translationally significant information that can be derived from the so-called endocannabinoidome. The ECS is involved in the modulation of a large amount of cognitive and physiological processes involved in the homeostatic regulation of the body. The role and mechanism by which the ECS is involved in the regulation of metabolism is not fully known, but its action is in large part through cyclic AMP/receptor activation-related pathways activated by cannabinoid ligands [1,2]. Endogenous cannabinoids are molecules with the primary function of control of multiple metabolic pathways. They are pre-synthesized and stored in cellular vesicles and released upon endogenous and exogenous stimuli to regulate internal homeostasis. Their targets include classical cannabinoid receptors that belong to the G-protein coupled receptor (GPCR) family as well as their various heteromers (see below), contributing to the complexity of the ECS [3]. Cannabinoid ligands also act through various non-canonical pathways [4], employing secondary messenger systems (changes in intracellular Ca2+ levels, activation of protein kinases) thus preferentially triggering alternative outcomes depending on the initial stimuli. This work aims to contribute to the growing burden of evidence that the ECS might be significantly more used as a pharmacological target for various metabolic disorders, despite carrying a historical label of being legally and ethically compromised.

[spa] INTRODUCCIÓN: El uso de la ML en NRC está aún muy limitado y enfocado principalmente a cirugías de corta duración o específicas como la cirugía “awake”, la estimulación cerebral profunda y la implantación de una derivación ventriculoperitoneal (DVP). Además, es de elección en procedimientos cortos que no precisan de relajación muscular, favoreciendo el despertar precoz y sin relajación muscular residual, lo que nos permitiría la valoración neurológica temprana en pacientes neuroquirúrgicos. El manejo de la vía aérea con Dispositivos supragloticos (DSG) en pacientes sometidos a diversos procedimientos neuroquirúrgicos es una alternativa eficaz y segura con respecto al tratamiento convencional mediante intubación orotraqueal. MÉTODOS: Realizamos tres estudios en diferentes poblaciones neuroquirurgicas, en cirugia lumbar en posicion prono valorando la utilidad de la ML en pacientes que se posicionan ellos mismo en posicion genupectoral. Se reporto tambien nuestra experiencia de 9 años en el uso de los DSG en pacientes propuestos para embolización de aneurismas cerebrales no rotos y por ultimo comparamos el efecto a nivel hemodinamico sistemico y cerebral, la incidencia de tos y fistula de liquido cefalorraquideo comparando la extubacion vs el intercambio del tubo orotraqueal por la Mascarilla laringea durante la educción. CONCLUSIONES: - Los DSG, insertados en decúbito prono o posición genupectoral modificada, permiten un manejo adecuado de la vía aérea en pacientes bien seleccionados intervenidos de cirugía lumbar programada mínimamente invasiva bajo anestesia general. - El uso de DSG de segunda generación en cirugía lumbar programada bajo anestesia general en decúbito prono se asoció a una muy baja incidencia de complicaciones, requiriendo un cambio a IOT en un 2,8% de los casos, pudieron resolverse los incidentes sin comprometer la seguridad del paciente. - Los DSG permiten el manejo de la vía aérea en la embolización de aneurismas cerebrales no rotos con una incidencia de eventos adversos similar a la IOT y constituyen un avance en el abordaje progresivamente menos invasivo de estos procedimientos. - El despertar de la anestesia general en cirugía hipofisaria después de reemplazar el TOT por ML se asocia a un mejor perfil hemodinámico sistémico y cerebral. El intercambio del TOT por ML una vez finalizada la cirugía y previamente a la educción anestésica tras la cirugía de hipófisis se asocia a una menor incidencia tos. La incidencia mayor de tos en el grupo “tubo” podría relacionarse con la aparición de fístula de líquido cefalorraquídeo, [eng] INTRODUCTION: The use of LMA in CRN is still very limited and mainly focused on short or specific surgeries such as awake surgery, deep brain stimulation and ventriculoperitoneal shunt (VPD) implantation. In addition, it is of choice in short procedures that do not require muscle relaxation, favoring early awakening and without residual muscle relaxation, which would allow early neurological assessment in neurosurgical patients. Airway management with supraglottic devices (SDG) in patients undergoing various neurosurgical procedures is an effective and safe alternative to conventional treatment with orotracheal intubation. METHODS: We conducted three studies in different neurosurgical populations, in lumbar surgery in prone position assessing the usefulness of the LMA in patients who position themselves in genupectoral position. We also reported our experience of 9 years in the use of DSG in patients proposed for embolization of unruptured cerebral aneurysms and finally we compared the effect on systemic and cerebral hemodynamic level, the incidence of cough and cerebrospinal fluid fistula comparing extubation vs the exchange of the orotracheal tube by the laryngeal mask during education. CONCLUSIONS: - DSGs, inserted in prone or modified genupectoral position, allow adequate airway management in well-selected patients undergoing minimally invasive scheduled lumbar surgery under general anesthesia. - The use of second generation DSGs in programmed lumbar surgery under general anesthesia in prone decubitus was associated with a very low incidence of complications, requiring a change to IOT in 2.8% of the cases, and the incidents could be resolved without compromising patient safety. - DSGs allow airway management in the embolization of unruptured cerebral aneurysms with an incidence of adverse events similar to IOT and constitute an advance in the progressively less invasive approach to these procedures. - Awakening from general anesthesia in pituitary surgery after replacing TOT with ML is associated with a better systemic and cerebral hemodynamic profile. The exchange of TOT for LMA after surgery and prior to anesthetic education after pituitary surgery is associated with a lower incidence of cough. The higher incidence of cough in the "tube" group could be related to the appearance of cerebrospinal fluid fistula.

The combination of the scaffolds of the cholinesterase inhibitor huprine Y and the antioxidant capsaicin results in compounds with nanomolar potencies toward human acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) that retain or improve the antioxidant properties of capsaicin. Crystal structures of their complexes with AChE and BChE revealed the molecular basis for their high potency. Brain penetration was confirmed by biodistribution studies in C57BL6 mice, with one compound (5i) displaying better brain/plasma ratio than donepezil. Chronic treatment of 10 month-old APP/PS1 mice with 5i (2 mg/kg, i.p., 3 times per week, 4 weeks) rescued learning and memory impairments, as measured by three different behavioral tests, delayed the Alzheimer-like pathology progression, as suggested by a significantly reduced Aβ42/Aβ40 ratio in the hippocampus, improved basal synaptic efficacy, and significantly reduced hippocampal oxidative stress and neuroinflammation. Compound 5i emerges as an interesting anti-Alzheimer lead with beneficial effects on cognitive symptoms and on some underlying disease mechanisms.

La infiltració de el sistema nerviós central (SNC) en els pacients amb síndromes limfoproliferatives és una troballa poc freqüent però associat amb mal pronòstic. En el limfoma cerebral primari el diagnòstic és complicat en moltes ocasions a causa de la dificultat per a realitzar una biòpsia cerebral i els riscos associats a l’procés quirúrgic. D’altra banda, les recaigudes en el SNC en els pacients amb limfoma sistèmic són en general poc freqüents, encara que determinats subtipus histològics i la presència de certs factors clínics i biològics augmenten el risc forma significativa. A més, la majoria de les recaigudes en el SNC ocorren de manera precoç el que suggereix que les cèl·lules malignes podrien estar ja presents en el líquid cefaloraquidi (LCR) i no ser detectades pels estudis convencionals. A causa de el mal pronòstic associat amb les recaigudes neuromeníngeas, es recomana realitzar profilaxi de l’SNC en els pacients d’alt risc, encara que la seva eficàcia és controvertida i tant l’esquema com la via d’administració no estan d’el tot establertes. En el primer treball, vam demostrar que l’anàlisi de DNA circulant tumoral (ctDNA) al LCR permet detectar la presència de malaltia i monitoritzar la resposta a l’tractament en els pacients amb limfoma cerebral. A més, també observem que l’anàlisi de ctDNA era capaç de detectar la presència de malaltia en el SNC de forma precoç en pacients amb limfoma sistèmic i limfoma cerebral. En el segon treball, observem que la profilaxi de l’SNC amb quimioteràpia intratecal o dosis altes de metotrexat endovenós no sembla aportar un benefici clar en la prevenció de la recaiguda neuromeníngea en els pacients amb limfoma difús de cèl·lules grans B d’alt risc. Aquests resultats suggereixen que l’anàlisi de ctDNA en el LCR permetria detectar de forma més precisa la presència de limfoma en el SNC. A més, podria ser útil per predir la recaiguda neuromeníngea i identificar millor els pacients que es beneficiarien de realitzar tractaments dirigits a l’SNC. Finalment, posen de manifest la necessitat d’investigar esquemes de profilaxi més eficaços. La infiltración del sistema nervioso central (SNC) en los pacientes con síndromes linfoproliferativos es un hallazgo poco frecuente pero asociado con mal pronóstico. En el caso del linfoma cerebral primario, el diagnóstico es complicado en muchas ocasiones debido a la dificultad para realizar la biopsia cerebral y los riesgos asociados al proceso quirúrgico. Por otro lado, las recaídas en el SNC en los pacientes con linfoma sistémico son en general poco frecuentes, aunque determinados subtipos histológicos y la presencia de ciertos factores clínicos y biológicos aumentan el riesgo forma significativa. Además, la mayoría de las recaídas en el SNC ocurren de forma precoz lo que sugiere que las células malignas podrían estar ya presentes en el líquido cefalorraquídeo (LCR) y no ser detectadas por los estudios convencionales. Debido al mal pronóstico asociado con las recaídas neuromeníngeas, se recomienda realizar profilaxis del SNC en los pacientes de alto riesgo, aunque su eficacia es controvertida y tanto el esquema como la vía de administración no están del todo establecidas. En el primer trabajo, observamos que el análisis de DNA circulante tumoral (ctDNA) en el LCR permite detectar enfermedad y monitorizar la respuesta al tratamiento en los pacientes con linfoma cerebral. Además, el análisis de ctDNA fue capaz de detectar la presencia de enfermedad en el SNC de forma precoz en un paciente con linfoma sistémico y otro con linfoma cerebral. En el segundo trabajo, observamos que la profilaxis del SNC con quimioterapia intratecal no parece aportar un beneficio claro en la prevención de la recaída neuromeníngea en los pacientes con linfoma difuso de células grandes B de alto riesgo. Además, nuestros resultados tampoco demuestran que la profilaxis con dosis altas de metotrexato endovenoso sea más eficaz que la profilaxis IT. Estos resultados sugieren que el análisis de ctDNA en el LCR permitiría detectar y caracterizar mejor la presencia de linfoma en el SNC, además podría ayudar a predecir la recaída neuromeníngea. Además, ponen de manifiesto la necesidad de investigar esquemas de profilaxis más eficaces. Central nervous system involvement (CNS) in patients with non-Hodgkin lymphoma is an uncommon event associated with dismal prognosis. The diagnosis of CNS lymphoma can be challenging due to the difficulty in accessing the tumor and the inherent risks associated with cranial surgery. Although CNS relapse is an uncommon complication in patients with systemic lymphoma, the presence of certain risk factors might increase the risk of CNS relapse significantly. Furthermore, CNS relapse is usually diagnosed during frontline chemotherapy shortly after the completion of treatment, suggesting the tumor cells were undetected in the cerebrospinal fluid (CSF) by standard techniques. In high-risk patients, CNS prophylaxis is usually recommended, although the optimal regimen remains unclear. In the first work, we showed that cell free circulating tumor DNA (ctDNA) in the CSF could complement de diagnosis, identify somatic mutations, monitor tumor burden and response to treatment in patients with CNS lymphoma. In addition, we observed that CSF ctDNA facilitated early detection of CNS relapse in one patient with high-risk systemic lymphoma and one patient with CNS lymphoma. In the second work, we did not observe a clear benefit of intrathecal (IT) prophylaxis on preventing CNS relapse. Furthermore, we did not demonstrate clinical advantage for using intravenous high-dose methotrexate (MTX) over traditional IT. Overall, our results suggest that CSF ctDNA analysis could be useful to detect and characterize CNS lymphoma, as well as facilitate early detection of CNS relapse. Moreover, they highlight the need for developing more effective CNS prophylaxis regimens than MTX. Universitat Autònoma de Barcelona. Programa de Doctorat en Medicina

Limfoma no Hodgkin agressiu; Limfoma del SNC Linfoma no Hodgkin agresivo; Linfoma del SNC Aggressive Non-Hodgkin's Lymphoma; CNS lymphoma The levels of cell free circulating tumor DNA (ctDNA) in plasma correlated with treatment response and outcome in systemic lymphomas. Notably, in brain tumors, the levels of ctDNA in the cerebrospinal fluid (CSF) are higher than in plasma. Nevertheless, their role in central nervous system (CNS) lymphomas remains elusive. We evaluated the CSF and plasma from 19 patients: 6 restricted CNS lymphomas, 1 systemic and CNS lymphoma, and 12 systemic lymphomas. We performed whole exome sequencing or targeted sequencing to identify somatic mutations of the primary tumor, then variant-specific droplet digital PCR was designed for each mutation. At time of enrolment, we found ctDNA in the CSF of all patients with restricted CNS lymphoma but not in patients with systemic lymphoma without CNS involvement. Conversely, plasma ctDNA was detected in only 2/6 patients with restricted CNS lymphoma with lower variant allele frequencies than CSF ctDNA. Moreover, we detected CSF ctDNA in 1 patient with CNS lymphoma in complete remission and in 1 patient with systemic lymphoma, 3 and 8 months before CNS relapse was confirmed; indicating CSF ctDNA might detect CNS relapse earlier than conventional methods. Finally, in 2 cases with CNS lymphoma, CSF ctDNA was still detected after treatment even though a complete decrease in CSF tumor cells was observed by flow cytometry (FC), indicating CSF ctDNA better detected residual disease than FC. In conclusion, CSF ctDNA can better detect CNS lesions than plasma ctDNA and FC. In addition, CSF ctDNA predicted CNS relapse in CNS and systemic lymphomas. This work was supported by research funding from Fundación Asociación Española contra el Cáncer (AECC) (to JS, MC and PA); FERO (to JS), laCaixa (to JS), BBVA (CAIMI) (to JS), the Instituto de Salud Carlos III, Fondo de Investigaciones Sanitarias (PI16/01278 to JS; PI17/00950 to MC; PI17/00943 to FB) cofinanced by the European Regional Development Fund (ERDF) and Gilead Fellowships (GLD16/00144, GLD18/00047, to FB). MC holds a contract from Ministerio de Ciencia, Innovación y Universidades (RYC-2012-12018). SB received funding from Fundación Alfonso Martin Escudero. LE received funding from the Juan de la Cierva fellowship. We thank CERCA Programme / Generalitat de Catalunya for institutional support.

The current obesity epidemic is a major worldwide health and economic burden. In the modern environment, an increase in the intake of high-fat and high-sugar foods plays a crucial role in the development of obesity by disrupting the mechanisms governing food intake and energy balance. Food intake and whole-body energy balance are regulated by the central nervous system through a sophisticated neuronal network located mostly in the hypothalamus. In particular, the hypothalamic arcuate nucleus (ARC) is a fundamental center that senses hormonal and nutrient-related signals informing about the energy state of the organism. The ARC contains two small, defined populations of neurons with opposite functions: anorexigenic proopiomelanocortin (POMC)-expressing neurons and orexigenic Agouti-related protein (AgRP)-expressing neurons. AgRP neurons, which also co-produce neuropeptide Y (NPY) and γ-Aminobutyric acid (GABA), are involved in an increase in hunger and a decrease in energy expenditure. In this review, we summarize the key findings from the most common animal models targeting AgRP neurons and the tools used to discern the role of this specific neuronal population in the control of peripheral metabolism, appetite, feeding-related behavior, and other complex behaviors. We also discuss how knowledge gained from these studies has revealed new pathways and key proteins that could be potential therapeutic targets to reduce appetite and food addictions in obesity and other diseases.

We investigated the frequency, predictors, and evolution of acute lymphoblastic leukemia (ALL) in patients with CNS relapse and introduced a novel method for studying BCR-ABL1 protein variants in cDNA from bone marrow (BM) and cerebrospinal fluid (CSF) blast cells. A total of 128 patients were analyzed in two PETHEMA clinical trials. All achieved complete remission after imatinib treatment. Of these, 30 (23%) experienced a relapse after achieving complete remission, and 13 (10%) had an isolated CNS relapse or combined CNS and BM relapses. We compared the characteristics of patients with and without CNS relapse and further analyzed CSF and BM samples from two of the 13 patients with CNS relapse. In both patients, classical sequencing analysis of the kinase domain of BCR-ABL1 from the cDNA of CSF blasts revealed the pathogenic variant p.L387M. We also performed ultra-deep next-generation sequencing (NGS) in three samples from one of the relapsed patients. We did not find the mutation in the BM sample, but we did find it in CSF blasts with 45% of reads at the time of relapse. These data demonstrate the feasibility of detecting BCR-ABL1 mutations in CSF blasts by NGS and highlight the importance of monitoring clonal evolution over time., This work was supported by the Fundación CRIS and Red Temática de Investigación Cooperativa en Cáncer (RTICC), Instituto de Salud Carlos III (Ref.: RD12/0036/0061 to JML).

Per poder funcionar de manera coordinada, requereix que el nostre sistema nerviós té un òrgan anomenat cos callós. Aquest òrgan s’encarrega de connectar la informació de l’hemisferi dret amb l’hemisferi esquerre fent possible moltes de les accions que duem a terme dia a dia, com caminar i escriure sense dificultat, gràcies a les fibres nervioses que van a zones específiques i traslladen la informació. En aquest treball prioritzo al sistema visual. És de vital importància un correcte desenvolupament del cos callós per tenir una bona visió, ja que aquest està íntimament lligat amb la coordinació dels dos ulls i amb als moviments oculars, per fer possible una bona binocularitat. També té relació amb la percepció visual. El cos callós és un dels principals protagonistes de la lateralitat: permet que la part esquerre i la dreta del cos no funcionin de manera independent i fa possible els moviments coordinats i contra laterals. També explico una algunes de les lesions i deficiències del cos callós. Per exemplificar hi ha dos casos de teràpia visual complerts fets a la consulta on he realitzat les pràctiques i un annex on explico diferents eines i jocs emprats en la teràpia visual

In Huntington’s disease (HD), striatal medium spiny neurons (MSNs) are particularly sensitive to the presence of a CAG repeat in the huntingtin (HTT) gene. However, there are many evidences that cells from the peripheral immune system and central nervous system (CNS) immune cells, namely microglia, play an important role in the etiology and the progression of HD. However, it remains unclear whether MSNs neurodegeneration is mediated by a non-cell autonomous mechanism. The homeostasis in the healthy CNS is maintained by several mechanisms of interaction between all brain cells. Neurons can control microglia activation through several inhibitory mechanisms, such as the CD200–CD200R1 interaction. Due to the complete lack of knowledge about the CD200–CD200R1 system in HD, we determined the temporal patterns of CD200 and CD200R1 expression in the neocortex, hippocampus and striatum in the HD mouse models R6/1 and HdhQ111/7 from pre-symptomatic to manifest stages. In order to explore any alteration in the peripheral immune system, we also studied the levels of expression of CD200 and CD200R1 in whole blood. Although CD200R1 expression was not altered, we observed and increase in CD200 gene expression and protein levels in the brain parenchyma of all the regions we examined, along with HD pathogenesis in R6/1 mice. Interestingly, the expression of CD200 mRNA was also up-regulated in blood following a similar temporal pattern. These results suggest that canonical neuronal–microglial communication through CD200–CD200R1 interaction is not compromised, and CD200 up-regulation in R6/1 brain parenchyma could represent a neurotrophic signal to sustain or extend neuronal function in the latest stages of HD as pro-survival mechanism., This study was supported by grants from the Ministerio de Ciencia, Innovación y Universidades (Spain), (RTI2018-099001-B-I00 to JMC); Instituto de Salud Carlos III, Ministerio de Ciencia, Innovacio´n y Universidades and European Regional Development Fund (ERDF) [CIBERNED and RETICS (Red de Terapia Celular, RD16/0011/ 0012 to JMC)], Spain; Generalitat de Catalunya (2017SGR-1408 to J. M. C.), Spain; the CHDI Foundation (A12076 to JMC), USA; and ADVANCE (CAT) with the support of ACCIO´ (Catalonia Trade & Investment; Generalitat de Catalunya) and the European Community under the Catalonian ERDF operational program 2014-2020], Spain. T

The dissemination in the central nervous system (CNS) is an uncommon but fatal complication occurring in patients with diffuse large B-cell lymphoma (DLBCL). Standard prophylaxis has been demonstrated to reduce CNS relapse and improve survival rates. Intrathecal (IT) liposomal cytarabine allows maintaining elevated drug levels in the cerebrospinal fluid for an extended period of time. Data on the efficacy and safety of liposomal cytarabine as CNS prophylaxis in patients with DLBCL are still insufficient. The objective of the present study was to evaluate the effectiveness and safety of the prophylaxis with IT liposomal cytarabine in prevention of CNS relapse in high-risk patients with DLBCL who were included in a trial of first line systemic therapy with 6 cycles of dose-dense R-CHOP every 14 days. Twenty-four (18.6 %) out of 129 patients were identified to have risk factors for CNS involvement, defined as follows: >30 % bone marrow infiltration, testes infiltration, retroperitoneal mass ≥10 cm, Waldeyer ring, or bulky cervical nodes involvement. Liposomal cytarabine (50 mg) was administered by lumbar puncture the first day of the 1st, 2nd, and 6th cycle of R-CHOP14 scheme. Among 70 IT infusions, grade 3-4 adverse events reported were headache (one patient) and nausea/vomiting (one patient). With a median follow-up of 40.1 months, no CNS involvement by DLBCL was observed in any patient. In conclusion, IT liposomal cytarabine is safe, feasible, and effective for CNS prophylaxis, causing few associated risks and little discomfort to patients with DLBCL. The study was conducted with the help of a grant from BRedes Temáticas de Investigación Cooperativa en Cáncer (RTICC).

ObjectiveTo report the presence of a new neuronal surface antibody against the metabotropic glutamate receptor 2 antibody (mGluR2-Ab) in 2 patients with paraneoplastic cerebellar ataxia.MethodsmGluR2-Abs were initially characterized by immunohistochemistry on the rat brain and confirmed by immunofluorescence on HEK293 cells transfected with mGluR2. Additional studies included analysis of potential cross-reactivity with other mGluRs, expression of mGluR2 in patients' tumors, and the effects of mGluR2-Abs on cultures of rat hippocampal neurons.ResultsPatient 1 was a 78-year-old woman with progressive cerebellar ataxia with an initial relapsing-remitting course who developed a small-cell tumor of unknown origin. Patient 2 was a 3-year-old girl who presented a steroid-responsive acute cerebellitis preceding the diagnosis of an alveolar rhabdomyosarcoma. Patients' serum and CSF showed a characteristic immunostaining of the hippocampus and cerebellum in rat brain sections and immunolabeled the cell surface of live rat hippocampal neurons. HEK293 cells transfected with mGluR1, 2, 3, and 5 confirmed that patients' antibodies only recognized mGluR2. mGluR2-Abs were not detected in 160 controls, 120 with paraneoplastic, autoimmune, or degenerative ataxias, and 40 with autoimmune encephalitis and antibodies against mGluR5 or unknown antigens. Expression of mGluR2 in tumors was confirmed by immunohistochemistry using a commercial mGluR2-Ab. Incubation of live rat hippocampal neurons with CSF of patient 2 did not modify the density of surface mGluR2 clusters.ConclusionsmGluR2-Abs are a novel biomarker of paraneoplastic cerebellar ataxia. The potential pathogenic effect of the antibodies is not mediated by downregulation or internalization of neuronal surface mGluR2.

Treball Final de Grau de Podologia, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, curs: 2017-2018, Tutor: J. Ignasi Beltrán Ruiz, El constructo teórico de cadenas musculares es empleado habitualmente por los profesionales de múltiples disciplinas, a veces desconociendo su definición e identidad. Por ello, este trabajo pretende clarificar dicho concepto, así como su origen y contextualización, realizando una descripción de los diferentes modelos de cadenas musculares desde su origen con Françoise Mézières y algunos derivados y modificados de este método cadenista. Analizaremos el modelo de G.D.S., donde se describen las cadenas musculares como un vehículo a través del cual se manifiestan diferentes sistemas, elementos o expresiones corporales, sean éstas psicocomportamentales, antiálgicas o viscerales. Además, trataremos la relación del sistema postural y los captores sensoriales, — siendo el pie, uno de los principales captores — cuya información eferente es integrada por el sistema nervioso central (SNC) para poder mantener la postura y el equilibrio. El sistema postural efectúa una acción y coordinación conjunta de las cadenas musculares y del sistema miofascial, las alteraciones de las mismas pueden ser tanto la causa como la consecuencia de disfunciones o patologías de otros sistemas corporales. Podemos describir el sistema postural como el resultado de múltiples factores relacionados (genéticos, psicosomáticos, viscerales, traumáticos) en la que, en cualquier caso, el sistema podopostural es fundamental.

Treballs Finals de Grau de Farmàcia, Facultat de Farmàcia i Ciències de l'Alimentació, Universitat de Barcelona, 2018. Tutora: Magdalena Rafecas., [eng] The relationship between the gut microbiota and the central nervous system (the microbiota-gut-brain axis) is an area of increasing interest and research. Studies based on germ-free models have provided a big amount of evidence of the connection between the gut and the brain. This research has done a great step forward in recent years, due to the application of metagenomics and bioinformatics. We now know that the human gut microbiome can be classified into three enterotypes, characterized by the variation in three genera: Bacteroides, Bacteroidetes, and Prevotella. Type of birth, formula feeding, and antibiotic intake are among the main factors that impact on infant microbiome assembly. Moreover, the composition of the gut microbiota is strongly associated with diet. A review of the bibliographical evidence connecting the alterations in the microbiome and some central nervous system disorders (as Alzheimer disease, Parkinson disease or autism spectrum disorder) shows us that the levels of Prevotella and the Firmicutes/Bacteroidetes ratio are altered in these pathologies. Accumulating data reveals that the microbiota-gut-brain axis can be modulated by the administration of probiotics and prebiotics. Moreover, some traditional fermented food has been seen to have probiotic properties and high-fiber containing diets have been associated with a lower Firmicutes/Bacteroidetes ratio and higher levels of Prevotella., [cat] La relació entre la microbiota intestinal i el sistema nerviós central (l'eix microbiota-intestí-cervell) és una àrea d'interès i investigació creixents. Els estudis basats en models lliures de gèrmens han proporcionat gran quantitat d’evidències de la connexió entre l'intestí i el cervell. Aquesta investigació ha fet un gran avenç en els últims anys gràcies a la metagenòmica i la bioinformàtica. Ara sabem que el microbioma intestinal humà es pot classificar en tres enterotips, caracteritzats per la variació en tres gèneres: Bacteroides, Bacteroidetes i Prevotella. Els tipus de naixement, el tipus d’alletament i la ingesta d'antibiòtics són els principals factors que afecten el desenvolupament del microbioma infantil. A més, la composició de la microbiota intestinal està fortament relacionada amb la dieta. Una revisió de les evidències bibliogràfiques que connecten les alteracions en el microbioma i alguns trastorns del sistema nerviós central (com la malaltia d'Alzheimer, la malaltia de Parkinson o el trastorn de l'espectre autista) ens mostra que els nivells de Prevotella i la relació Firmicutes/Bacteroidetes estan alterats en aquestes patologies. Cada cop hi ha més dades que revelen que l'eix microbiota-intestí-cervell pot ser modulat per l'administració de probiòtics i prebiòtics. D'altra banda, s'ha observat que alguns aliments fermentats tradicionals tenen propietats probiòtiques i que les dietes amb alt contingut de fibra s’associen a nivells inferiors de la raó Firmicutes /Bacteroidetes i superiors de Prevotella.

Overweight and stress are both related to brain structural abnormalities. The allostatic load model states that frequent disruption of homeostasis is inherently linked to oxidative stress and inflammatory responses that in turn can damage the brain. However, the effects of the allostatic load on the central nervous system remain largely unknown. The current study aimed to assess the relationship between the allostatic load and the composition of whole-brain white matter tracts in overweight subjects. Additionally, we have also tested for grey matter changes regarding allostatic load increase. Thirty-one overweight-to-obese adults and 21 lean controls participated in the study. Our results showed that overweight participants presented higher allostatic load indexes. Such increases correlated with lower fractional anisotropy in the inferior fronto-occipital fasciculi and the right anterior corona radiata, as well as with grey matter reductions in the left precentral gyrus, the left lateral occipital gyrus, and the right pars opercularis. These results suggest that an otherwise healthy overweight status is linked to long-term biological changes potentially harmful to the brain.

Under pathological conditions, microglia, the resident CNS immune cells, become reactive and release pro-inflammatory cytokines and neurotoxic factors. We investigated whether this phenotypic switch includes changes in the expression of the L-type voltage-gated calcium channel (VGCC) in a rat model of N-methyl-d-aspartate-induced hippocampal neurodegeneration. Double immunohistochemistry and confocal microscopy evidenced that activated microglia express the L-type VGCC. We then analyzed whether BV2 microglia express functional L-type VGCC, and investigated the latter's role in microglial cytokine release and phagocytic capacity. Activated BV2 microglia express the CaV1.2 and CaV1.3 subunits of the L-type VGCC determined by reverse transcription-polymerase chain reaction, Western blot and immunocytochemistry. Depolarization with KCl induced a Ca2+ entry facilitated by Bay k8644 and partially blocked with nifedipine, which also reduced TNF-α and NO release by 40%. However, no nifedipine effect on BV2 microglia viability or phagocytic capacity was observed. Our results suggest that in CNS inflammatory processes, the L-type VGCC plays a specific role in the control of microglial secretory activity.

[cat] En els últims anys s’ha produït un increment de l’esperança de vida que ha despertat un gran interès en promoure un envelliment saludable. Habitualment, la senescència va acompanyada de diferents trastorns i malalties en la base de les quals hi ha una desregulació del sistema immunitari. Aquesta desregulació implica un increment de l’activació basal del sistema immunitari que comporta un augment de l’estat inflamatori de baixa intensitat, conegut com a inflammaging. Alguns suplements dietètics redueixen l’activació del sistema immunitari, prevenint el deteriorament cognitiu associat a l’envelliment a través d’una connexió entre l’intestí i el cervell. En animals acabats de deslletar, la suplementació dietètica amb proteïnes plasmàtiques (SDP) millora la funció de barrera de la mucosa intestinal i modula el grau d’activació del teixit limfoide associat a l’intestí durant un procés inflamatori. En aquest estudi hem demostrat que, als 6 mesos d’edat, els ratolins amb envelliment accelerat (SAMP8) presenten una activació inespecífica del sistema immunitari intestinal així com una menor capacitat de resposta front a l’enterotoxina B d’S. aureus. La suplementació dietètica amb SDP durant 4 mesos disminueix l’activació inespecífica del sistema immunitari intestinal dels ratolins senescents, ja que hi ha una menor expressió de citocines proinflamatòries; a més, la resposta immunitària intestinal d’aquests animals front a l’enterotoxina B d’S. aureus és similar a la dels ratolins joves. Els ratolins SAMP8 de 6 mesos presenten un deteriorament de la memòria a curt i a llarg termini que s’associa a un increment en l’expressió de citocines proinflàmatòries, a la presència d’agents oxidants a l’encèfal i a una major permeabilitat de la barrera hematoencefàlica. Els ratolins senescents presenten una major fosforilació de la proteïna Tau que els ratolins joves, fet que està relacionat amb trastorns cognitius com els que s’observen en la malaltia d’Alzheimer. La suplementació dietètica amb proteïnes plasmàtiques atenua la majoria d’aquests efectes de l’envelliment sobre la funcionalitat cerebral, gràcies a la connexió que existeix entre el sistema immunitari intestinal i altres mucoses i òrgans sistèmics., [eng] The increase in life expectancy that has occurred in recent years has generated a great interest in promoting a healthy aging. Usually, senescence is accompanied by different disorders and diseases, at the base of which there is a deregulation of the immune system. This deregulation implies an increase in the basal activation of the immune system which involves an increment of the low intensity inflammatory state known as inflammaging. Some dietary supplements may reduce the activation of the immune system, preventing the cognitive deterioration associated with aging through the connection between the intestine and the brain. In weaned animals, plasma protein dietary supplementation (SDP) improves the barrier function of the intestinal mucosa and modulates the degree of activation of the intestinal lymphoid tissue during an intestinal inflammation. In this study we have demonstrated that, at 6-month-old, mice with accelerated aging (SAMP8) present a non-specific activation of the intestinal immune system as well as a lower response capacity to S. aureus enterotoxin. Dietary supplementation with SDP for 4 months decreases nonspecific activation of the intestinal immune system of senescent mice, as there is a lower expression of proinflammatory cytokines. In addition, the intestinal immune response of these animals to S. aureus enterotoxin is similar to that of young mice. The 6-month-old SAMP8 mice present short- term and long-term memory impairment associated with increased proinflammatory cytokines, the presence of oxidizing agents in the brain and increased permeability of the blood-brain barrier. Senescent mice show higher phosphorylation of Tau protein than young mice, which is related to cognitive disorders such as those observed in Alzheimer's disease. Dietary supplementation with plasma proteins attenuates most of these effects of aging on brain functionality, through the connection between the intestinal immune system and other mucosal and systemic organs.

Sistema Nerviós Autònom; Risc de malalties cardiovasculars; Procediments mèdics quirúrgics i invasius Sistema nervioso autónomo; Riesgo de enfermedad cardiovascular; Procedimientos médicos quirúrgicos e invasivos Autonomic Nervous System; Cardiovascular disease risk; Surgical and invasive medical procedures Background Neurological complications after lung transplantation are common. The full spectrum of neurological complications and their impact on clinical outcomes has not been extensively studied. Methods We investigated the neurological incidence of complications, categorized according to whether they affected the central, peripheral or autonomic nervous systems, in a series of 109 patients undergoing lung transplantation at our center between January 1 2013 and December 31 2014. Results Fifty-one patients (46.8%) presented at least one neurological complication. Critical illness polyneuropathy-myopathy (31 cases) and phrenic nerve injury (26 cases) were the two most prevalent complications. These two neuromuscular complications lengthened hospital stays by a median period of 35.5 and 32.5 days respectively. However, neurological complications did not affect patients’ survival. Conclusions The real incidence of neurological complications among lung transplant recipients is probably underestimated. They usually appear in the first two months after surgery. Despite not affecting mortality, they do affect the mean length of hospital stay, and especially the time spent in the Intensive Care Unit. We found no risk factor for neurological complications except for long operating times, ischemic time and need for transfusion. It is necessary to develop programs for the prevention and early recognition of these complications, and the prevention of their precipitant and risk factors. JG is the recipient of a grant from the Spanish Fondo de Investigaciones Sanitarias (FIS PI13-01272-FEDER).

The blood-brain barrier is a well-coordinated and highly selective barrier whose main function is to regulate brain homeostasis and the transport of endogenous and exogenous compounds between the blood and the brain. It permits the selective brain uptake of nutrients and impedes the entrance of potentially harmful substances and pathogenic organisms into the brain. Due to this restrictive nature of the blood-brain barrier, the transport of neurotherapeutics from the blood to the brain results extremely difficult, and has become a major pharmaceutical challenge in recent decades. Only lipophilic molecules with a molecular weight under 400-600 Da are able to cross the blood-brain barrier. For this reason, 98% of all small molecule drugs and almost 100% of large molecule drugs are unable to cross the blood-brain barrier, and hence, most neurological and neurodegenerative diseases currently have few or no treatment options. During the last decades, numerous strategies have been proposed to overcome the blood-brain barrier and efficiently deliver therapeutic agents to the brain. One of these strategies consists in linking the pharmacologically active substance to a molecular vector that acts as a molecular Trojan Horse and is capable of crossing the blood-brain barrier using a receptor-mediated transcellular transport system of the brain capillary endothelial cells. These molecular vectors can be natural ligands, peptides or monoclonal antibodies (mAbs) that bind to a particular receptor and trigger endocytosis or transcytosis processes. Several mAbs directed against the transferrin receptor (TfR), which is abundant in brain capillaries, have been extensively studied. However, there is still no consensus regarding their transcytotic capacity and their ability to transport substances across the blood-brain barrier. Moreover, the intracellular mechanisms that these antibodies or constructs undergo inside the endothelial cells remain unclear. To gain insight on this strategy, this thesis aimed to study the potential of the 8D3 monoclonal antibody, directed against the murine TfR, to transport substances across the blood-brain barrier in mice. On this basis, a series of experiments were performed where the 8D3 antibody was conjugated to different cargoes, the resulting constructs were administered in vivo to mice, and the distribution and intracellular mechanisms that these constructs undergo at the blood-brain barrier were studied. Overall results suggest, firstly, that the 8D3 antibody is able to bind to the TfR and trigger endocytosis of the 8D3-TfR complex, but does not complete transcytosis except in rare occasions. Secondly, the 8D3 antibody is capable of internalizing the conjugated cargo inside the brain capillary endothelial cells through a clathrin-dependent endocytosis process, and hence, of overcoming the first obstacle in the transport across these cells. However, once inside the endothelial cells, the constructs tend to progressively accumulate in mature endosomal structures of large size and great complexity. These results can be mainly explained by the high affinity of the peptidomimetic mAbs for their receptors and their consequent difficulty to dissociate from them. Only a small percentage of the endocytic vesicles fuse with the abluminal membrane and open up to the basal lamina. Thus, in these cases, the constructs complete transcytosis. Nevertheless, they remain attached to the abluminal membrane and never reach the brain parenchyma, probably, in this case as well, because of the high affinity of the 8D3 for the TfR. Based on this premise, different aspects can be approached regarding the design of the constructs. Nanocarrier functionalization, development of acid-cleavable linkages between mAb and cargo, and reducing mAb’s affinity for the target receptor are some of the ideas that are currently under development for the optimization of this receptor-mediated transport based strategy to overcome the blood-brain barrier. These aspects are today main focus of research and starting point towards new perspectives.

A relatively large number of studies have investigated the power of structural magnetic resonance imaging (sMRI) data to discriminate patients with schizophrenia from healthy controls. However, very few of them have also included patients with bipolar disorder, allowing the clinically relevant discrimination between both psychotic diagnostics. To assess the efficacy of sMRI data for diagnostic prediction in psychosis we objectively evaluated the discriminative power of a wide range of commonly used machine learning algorithms (ridge, lasso, elastic net and L0 norm regularized logistic regressions, a support vector classifier, regularized discriminant analysis, random forests and a Gaussian process classifier) on main sMRI features including grey and white matter voxel-based morphometry (VBM), vertex-based cortical thickness and volume, region of interest volumetric measures and wavelet-based morphometry (WBM) maps. All possible combinations of algorithms and data features were considered in pairwise classifications of matched samples of healthy controls (N = 127), patients with schizophrenia (N = 128) and patients with bipolar disorder (N = 128). Results show that the selection of feature type is important, with grey matter VBM (without data reduction) delivering the best diagnostic prediction rates (averaging over classifiers: schizophrenia vs. healthy 75%, bipolar disorder vs. healthy 63% and schizophrenia vs. bipolar disorder 62%) whereas algorithms usually yielded very similar results. Indeed, those grey matter VBM accuracy rates were not even improved by combining all feature types in a single prediction model. Further multi-class classifications considering the three groups simultaneously made evident a lack of predictive power for the bipolar group, probably due to its intermediate anatomical features, located between those observed in healthy controls and those found in patients with schizophrenia. Finally, we provide MRIPredict (http://www.mripredict.com/) a free tool for SPM, FSL and R, to easily carry out voxelwise predictions based on VBM images. The following directories are included in the compresed files: /rois: It contains a single text file (rois1.txt) with volumes of cortical and subcortical structures for each one of the subjects. /vbm_gm: It contains the grey matter probability maps for the three groups of subjects in format NIFTI compressed (nii.gz extension). /vbm_wm: It contains the white matter probability maps for the three groups of subjects in format NIFTI compressed (nii.gz extension). /thickness: It contains two binary files (thickness_lh.bin and thickness_rh.bin) which contain cortical thickness data for the left hemsiphere (lh) and right hemisphere (rh) for all subjects included in the study. Specifically, cortical thickness data is stored in a 4 byte real format (149955 points per subject (lh), and 149926 points per subject (rh), with the 383 subjects stored sequentially in the same order as in the other data formats. /volume: Two files containing cortical volume information with the same format as for thickness (volume_lh.bin and volume_rh.bin).

[eng] INTRODUCTION Multimodal neuromonitoring increases the knowledge of the physiopathology underlying the pathological slow vasogenic waves known as ‘plateau waves’ of intracranial pressure (ICP). The transcranial Doppler (TCD) pulsatility index (PI) describes changes in the morphology of the blood flow velocity (FV) waveform and is classically consider a descriptor of the distal cerebrovascular resistance (CVR). Critical closing pressure (CCP) or zero-flow pressure denotes a threshold of arterial blood pressure (ABP) at which small cerebral vessels collapse and cerebral blood flow (CBF) ceases increasing the ischemic risk. The difference between CCP and ICP is explained by the tone of the small cerebral vessels, so-called wall tension (WT). Although it has inspired theoretical interest, its clinical applicability is limited for methodological reasons. HYPOTHESIS 1) PI is a complex function determined by the interaction of multiple haemodynamic variables, and is not solely determined by distal CVR; 2) CCP and WT estimated with a cerebrovascular impedance model, could accurately define the pathophysiological changes during plateau waves. AIMS 1) to clarify the relationship between PI and CVR; to define which factors truly influence PI; 2) to calculate CCP and arterial WT with a novel multiparametric mathematical model in order to examine the proposed vasodilatory pathophysiology of plateau waves; to evaluate its possible clinical appliance. SUBJECTS AND METHODS Recordings from patients with severe head-injury undergoing monitoring of ABP, ICP, cerebral perfusion pressure (CPP), and TCD assessed CBF velocities (FV) were analysed. The Gosling PI was compared between baseline and ICP plateau waves (n = 20 patients) or short-term (30–60 min) hypocapnia (n = 31). In addition, a modeling study was conducted with the ‘‘spectral’’ PI (calculated using fundamental harmonic of FV) resulting in a theoretical formula expressing the dependence of PI on balance of cerebrovascular impedances. Multimodality neuromonitoring integrated with bio-informatics analysis (ICM+™ Software, www.neurosurg.cam.ac.uk/icmplus). Both studies are based in a multiparametric method new model of cerebrovascular impedance; first a retrospective study of 2 opposing physiological conditions comparing basal PI to: a) plateau waves (n= 20 patients, 38 plateau waves); and b) moderate hyperventilation (n=31); next CCP was calculated in the plateau waves group (n= 20). According to Burton’s model, wall tension was estimated as: WT = CCP-ICP. RESULTS 1) PI increased significantly (p< 0.001) while CVR decreased (p< 0.001) during plateau waves. During hypocapnia both PI and CVR increased (p< 0.001). The modeling formula explained more than 65 % of the variability of Gosling PI and 90% of the variability of the ‘spectral’ PI (R=0.81 and R=0.95, respectively); 2) During the vasodilatory loop of the plateau waves, there is a rise in CCP and reduction in WT (both significant, p < 0.001). Change in CCP was correlated to ICP changes (R=0.80, p, [spa] ANTECEDENTES: la neuromonitorización multimodal aumenta el conocimiento de la fisiopatología subyacente a las ondas plateau de hipertensión intracraneal. El índice de pulsatilidad (IP) del Doppler transcraneal (DTC) se considera un descriptor de las resistencias cerebrovasculares (RCV) distales. La presión crítica de cierre (critical closing pressure, CCP) es un umbral de presión arterial por debajo del cual los vasos cerebrales pequeños se colapsan interrumpiendo el flujo sanguíneo cerebral (FSC) y aumentando la lesión secundaria. Teóricamente útil, su aplicabilidad clínica está limitada por razones metodológicas. HIPÓTESIS: 1) el IP está determinado por la interacción de múltiples variables hemodinámicas y no solo por las RCV; 2) la CCP (estimada mediante un modelo de impedancia cerebrovascular) aumenta durante las ondas plateau. OBJETIVOS: 1) definir qué factores determinan el IP de la velocidad del FSC; 2) medir la CCP y verificar un nuevo método para su cálculo durante las ondas plateau. METODOLOGÍA: pacientes con traumatismo craneoencefálico (TCE) monitorizados con presión arterial continua, presión intracraneal (PIC) y DTC. Análisis con un software específico (ICM+®) validado. Ambos trabajos se basan en un método multiparamétrico basado en un modelo de impedancia cerebrovascular: 1) estudio retrospectivo de 2 situaciones fisiológicas contrapuestas comparando el IP basal con: a) ondas plateau (n= 20 pacientes, 38 ondas) y b) hiperventilación moderada (n=31); 2) cálculo de la CCP en el grupo de ondas plateau (n= 20). Según el modelo de Burton la tensión de la pared arterial (wall tension, WT) se estimó como: WT = CCP-PIC. RESULTADOS: 1) durante las ondas plateau el IP aumenta de forma significativa pero las RCV disminuyen. Durante la hipocapnia aumentan el IP y las RCV; 2) La CCP aumenta significativamente en el plateau de la onda y la WT disminuye un 34.3%. El nuevo método matemático es más fisiológico. CONCLUSIONES: 1) El IP no es solo un descriptor de la RCV distales, existiendo una compleja relación matemática con múltiples variables hemodinámicas; 2) La CCP aumenta durante las ondas plateau, pero la WT disminuye. Se aplica un nuevo modelo matemático para calcular la CCP que permite una interpretación más fisiológica de sus valores.

The work of this thesis is based on research on peptides able to cross the blood-brain barrier and their use as tools to enable the delivery of drugs into the brain. The blood-brain barrier (BBB) is a permeable but selective barrier that tightly regulates the transport into the central nervous system (CNS). In this regard, therapeutic treatments at the CNS are hampered by the presence of this barrier (BBB). Thus, diverse strategies have been developed to overcome it. Blood-brain barrier shuttles are peptides able to cross this barrier and deliver drugs into the brain. Peptides are privileged structures from the therapeutic point of view, they share properties from small organic molecules and large biologics: the synthesis through solid-phase peptide synthesis (SPPS) enables a straightforward method to obtain them with high purity and at the same time they can be purified and characterized like small organic compound. In addition, their structure is present in nature and thus the risk of toxicity is lower or more predictable compared with organic compounds, and their larger structure enables to obtain more selective and stronger interactions with targets. In addition, peptides have been shown to cross the BBB by diverse transport mechanisms and thus enabling to select the best one for each therapy and drug. In this thesis a family of BBB shuttles crossing by passive diffusion (based on phenylproline) have been improved from a parent peptide shuttle (based on N-methyl-phenylalanine). The solubility was three orders of magnitude superior and the transport capacity was maintained upon cargo attachment. In addition, the role of stereochemistry in passive diffusion in biological membranes was demonstrated. A method which combined the use of MALDI-TOF MS and in vitro cell-based models of the BBB enabled the increase in sensitivity for transport quantification of three orders of magnitude compared to RP-HPLC-PDA. Additionally, a BBB shuttle library was evaluated and quantified by this novel methodology. Two new analogs showed better performance when evaluated in these in vitro cell-based models. Immunogenicity of BBB shuttle peptides made by L- or D-amino acids was evaluated and compared. Both peptide shuttles showed low immunogenic response in mice, however, the response to those made with D- amino acids was lower. Finally, the applicability of these peptide shuttles for a therapeutic use was considered for Friedreich’s Ataxia, a monogenic recessive disease. Both a protein replacement therapy and a gene therapy for the central nervous system were attempted by coupling covalently BBB shuttles to the affected protein or viruses, respectively. The protein replacement therapy was impeded by the high rate of proteolysis of the protein used. On the other hand, novel methods of conjugation of BBB shuttles into enveloped viruses (Herpes simplex Virus type 1; HSV- 1) were developed. These modified viral particles were subsequently characterized through a range of methods comprising molecular biology tools (SDS-PAGE, western blots), proteomics (mass spectrometry) and biophysical tools (dynamic light scattering and z-potential)., La barrera hematoencefàlica (BHE) actua com a protecció del sistema nerviós central (SNC) regulant el transport de molècules d’una manera selectiva. Això dificulta el tractament de malalties que afecten al SNC, ja que la BHE també evita que fàrmacs que serien efectius no siguin transportats al cervell. Per això, s’estan desenvolupant mètodes que permetin enviar selectivament fàrmacs a través de la BHE. És el cas dels pèptids llançadora. Aquests es poden dissenyar per creuar per algun dels mecanismes de transport existents en la BHE. En aquesta tesi es desenvolupen uns pèptids que creuen per difusió passiva (basats en fenilprolines), que respecte al disseny anterior (basats en N‐ metilfenilalanines) milloren la solubilitat en aigua en tres ordres de magnitud i al transport un cop s’hi enganxa el fàrmac. Per una altra banda, es desenvolupa una metodologia per a la quantificació del transport basada en la combinació d’espectrometria de masses MALDI‐TOF amb models de BHE in vitro (cel∙lulars), millorant la sensibilitat respecte a la detecció per RP‐HPLC‐PDA en tres ordres de magnitud. L’avaluació d’una peptidoteca derivada d’un pèptid llançadora mitjançant aquesta metodologia permet el descobriment de dos anàlegs del pèptid original que milloren el transport. Addicionalment, s’estudia la immunogenicitat de pèptids llançadora formats per aminoàcids L o D. S’observa que encara que ambdós mostren una baixa immunogenicitat, la resposta dels pèptids amb aminoàcids D és encara menor. Finalment, s’estudia de forma preliminar la possibilitat de desenvolupar una teràpia de reemplaçament proteic i una teràpia gènica per atàxia de Friedreich al SNC mitjançant l’ús de pèptids llançadora.

Diffusion-weighted imaging (DWI) quantifies water molecule diffusion within tissues and is becoming an increasingly used technique. However, it is very challenging as correct quantification depends on many different factors, ranging from acquisition parameters to a long pipeline of image processing. In this work, we investigated the influence of voxel geometry on diffusion analysis, comparing different acquisition orientations as well as isometric and anisometric voxels. Diffusion-weighted images of one rat brain were acquired with four different voxel geometries (one isometric and three anisometric in different directions) and three different encoding orientations (coronal, axial and sagittal). Diffusion tensor scalar measurements, tractography and the brain structural connectome were analyzed for each of the 12 acquisitions. The acquisition direction with respect to the main magnetic field orientation affected the diffusion results. When the acquisition slice-encoding direction was not aligned with the main magnetic field, there were more artifacts and a lower signal-to-noise ratio that led to less anisotropic tensors (lower fractional anisotropic values), producing poorer quality results. The use of anisometric voxels generated statistically significant differences in the values of diffusion metrics in specific regions. It also elicited differences in tract reconstruction and in different graph metric values describing the brain networks. Our results highlight the importance of taking into account the geometric aspects of acquisitions, especially when comparing diffusion data acquired using different geometries.

Los ácidos grasos omega 3 y 6 se consideran esenciales, ya que nuestro organismo no tiene las enzimas necesarias para producirlos. De tal forma, deben obtenerse de la dieta o sintetizarse a partir de sus precursores. La gran importancia de los ácidos grasos omega 3 y 6 durante la gestación, la lactancia y los primeros años de vida en la formación del sistema nervioso central, radica en que son los más abundantes en el cerebro y su adecuado aporte se ha relacionado con un adecuado crecimiento y desarrollo durante las diferentes etapas en los primeros años de vida. Els àcids grassos omega 3 i 6 es consideren essencials, ja que el nostre organisme no té els enzims necessaris per produir-los. De tal forma, s'han d'obtenir de la dieta o sintetitzar a partir dels seus precursors. La gran importància dels àcids grassos omega 3 i 6 durant la gestació, la lactància i els primers anys de vida a la formació del sistema nerviós central, radica que són els més abundants en el cervell i la seva adequada aportació s'ha relacionat amb un adequat creixement i desenvolupament durant les diferents etapes durant els primers anys de vida. S'han realitzat múltiples estudis sobre la suplementació d'aquest tipus d'àcids grassos en les mares gestants, durant la lactància i a les fórmules infantils i la seva relació amb el neurodesenvolupament. No obstant això, no s'ha aconseguit evidenciar que la suplementació durant aquestes etapes del desenvolupament, tingui algun benefici. Omega 3 and 6 fatty acids are considered essential because our body does not have the necessary enzymes to produce them. Therefore, they must be obtained from the diet or synthesized from their precursors. The great importance of omega 3 and 6 during pregnancy, lactation and the early years of life in the formation of the central nervous system, is that they are the most abundant in the brain and adequate intake has been linked with proper growth and development during different stages during the first years of life.

El procés d’envelliment causa nombrosos canvis a l’organisme, reduint les capacitats funcionals màximes i l’homeòstasi, i augmentant la probabilitat de desenvolupar malalties degeneratives. Tots aquests canvis probablement s’originen en un nombre petit de causes, que operen contínuament al llarg de tota la vida i sembla que l’estrès oxidatiu és un factor clau. Les pèrdues cognitives i psicomotores són manifestacions comuns del procés d’envelliment del sistema nerviós, no obstant això, l’inici i severitat d’aquestes pèrdues semblen ser relativament independents una de l’altra, suggerint que diferents regions del sistema nerviós tenen una vulnerabilitat neuronal regió-dependent. En aquest sentit, l’objectiu ha estat definir les bases moleculars de la vulnerabilitat neuronal selectiva a l’estrès oxidatiu en regions del sistema nerviós central (SNC) humà; determinar l’efecte de l’envelliment del SNC humà; i avaluar el mecanisme d’acció de la restricció de metionina en el SNC de rata, com a intervenció nutricional que redueix l’estrès oxidatiu i el grau d’envelliment. Els resultats mostren l’existència d’una vulnerabilitat neuronal selectiva en les diferents regions del SNC humà, sent l’escorça frontal la regió que presenta un perfil metabolòmic i molecular més diferencial a la resta de regions que conformen el SNC humà., El proceso de envejecimiento causa numerosos cambios en el organismo, reduciendo las capacidades funcionales máximas y la homeostasis, y aumentando la probabilidad de desarrollar enfermedades degenerativas. Todos estos cambios probablemente se originan en un número pequeño de causas, que operan continuamente a lo largo de toda la vida y parece que el estrés oxidativo es un factor clave. Pérdidas cognitivas y psicomotoras son manifestaciones comunes del proceso de envejecimiento del sistema nervioso, sin embargo, el inicio y la severidad de tales pérdidas parecen ser relativamente independientes una de la otra, sugiriendo que diferentes regiones del sistema nervioso tienen una vulnerabilidad neuronal región-dependiente. En este sentido, el objetivo se ha centrado en definir las bases moleculares de la vulnerabilidad neuronal selectiva al estrés oxidativo en regiones específicas del sistema nervioso central (SNC) humano; determinar el efecto del envejecimiento del SNC humano; y evaluar el mecanismo de acción de la restricción de metionina en el SNC de rata, como intervención nutricional que reduce el estrés oxidativo y la tasa de envejecimiento. Los resultados muestran la existencia de una vulnerabilidad neuronal selectiva en las diferentes regiones del SNC humano, siendo la corteza frontal la región que presenta un perfil metabolómico y molecular más diferencial al resto de regiones que conforman el SNC humano., The aging process causes numerous changes in the organism, reducing the maximum functional capabilities and homeostasis, and increasing the probability of developing degenerative diseases. All these changes are likely to originate from a small number of causes, operating continuously throughout life span and it seems that oxidative stress plays an important role. Losses in cognition and psychomotor functions are common manifestations in mammals, however, the onset and severity of such casualities seem to be relatively independent of each other, suggesting that different regions of the nervous system have a region-dependent selective neuronal vulnerability. In this sense, this thesis has been designed with the aim of: provide insights into the molecular basis of the selective neuronal vulnerability to oxidative stress in specific regions of the human central nervous system (CNS); to determine the effect of aging in the human CNS; and to evaluate the mechanism of action of methionine restriction in rat CNS, as nutritional intervention that decreases oxidative stress and aging rate. The results show the existence of a selective neuronal vulnerability in different regions of the human CNS, and the frontal cortex is the region that has specific metabolomic and molecular hallmark comparing to all the other regions that comprise the human CNS.

Blastic plasmacytoid dendritic cell neoplasm (BPDCN) is a rare aggressive myeloid neoplasm which shows a high rate of central nervous system (CNS) recurrence and overall survival (OS) of < 1 year. Despite this, screening for CNS involvement is not routinely performed at diagnosis and intrathecal (IT) prophylaxis is not regularly administered in BPDCN. Here, we prospectively evaluated 13 consecutive BPDCN patients for the presence of CNS involvement by flow cytometry. Despite none of the patients presented with neurological symptoms, occult CNS involvement was detected in 6/10 cases evaluated at diagnosis and 3/3 studied at relapse/progression. BPDCN patients evaluated at diagnosis received IT treatment -either CNS prophylaxis (n = 4) or active therapy (n = 6)- and all but one remain alive (median follow-up of 20 months). In contrast, all three patients assessed at relapse/progression died. The potential benefit of IT treatment administered early at diagnosis on OS and CNS recurrence-free survival of BPDCN was further confirmed in a retrospective cohort of another 23 BPDCN patients. Our results show that BPDCN patients studied at diagnosis frequently display occult CNS involvement; moreover, they also indicate that treatment of occult CNS disease might lead to a dramatically improved outcome of BPDCN., This work was supported by grants RD06/0020/0035 and RD12/0036/0048 from RETICS (Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Madrid, Spain and Fondos FEDER) and grant EDU/878/2004 from Junta de Castilla y León and Fondo Social Europeo.

Background: It has recently become evident that activating/inhibitory cell surface immune receptors play a critical role in regulating immune and inflammatory processes in the central nervous system (CNS). The immunoreceptor CD300f expressed on monocytes, neutrophils, and mast cells modulates inflammation, phagocytosis, and outcome in models of autoimmune demyelination, allergy, and systemic lupus erythematosus. On the other hand, a finely regulated inflammatory response is essential to induce regeneration after injury to peripheral nerves since hematogenous macrophages, together with resident macrophages and de-differentiated Schwann cells, phagocyte distal axonal and myelin debris in a well-orchestrated inflammatory response. The possible roles and expression of CD300f and its ligands have not been reported under these conditions. Methods: By using quantitative PCR (QPCR) and CD300f-IgG2a fusion protein, we show the expression of CD300f and its ligands in the normal and crush injured sciatic nerve. The putative role of CD300f in peripheral nerve regeneration was analyzed by blocking receptor-ligand interaction with the same CD300f-IgG2a soluble receptor fusion protein in sciatic nerves of Thy1-YFP-H mice injected at the time of injury. Macrophage M1/M2 polarization phenotype was also analyzed by CD206 and iNOS expression. Results: We found an upregulation of CD300f mRNA and protein expression after injury. Moreover, the ligands are present in restricted membrane patches of Schwann cells, which remain stable after the lesion. The lesioned sciatic nerves of Thy1-YFP-H mice injected with a single dose of CD300f-IgG2a show long lasting effects on nerve regeneration characterized by a lower number of YFP-positive fibres growing into the tibial nerve after 10 days post lesion (dpl) and a delayed functional recovery when compared to PBS- or IgG2a-administered control groups. Animals treated with CD300f-IgG2a show at 10 dpl higher numbers of macrophages and CD206-positive cells and lower levels of iNOS expression than both control groups. At later time points (28 dpl), increased numbers of macrophages and iNOS expression occur. Conclusions: Taken together, these results show that the pair CD300f ligand is implicated in Wallerian degeneration and nerve regeneration by modulating both the influx and phenotype of macrophages.

Xenacoelomorpha is, most probably, a monophyletic group that includes three clades: Acoela, Nemertodermatida and Xenoturbellida. The group still has contentious phylogenetic affinities; though most authors place it as the sister group of the remaining bilaterians, some would include it as a fourth phylum within the Deuterostomia. Over the past few years, our group, along with others, has undertaken a systematic study of the microscopic anatomy of these worms; our main aim is to understand the structure and development of the nervous system. This research plan has been aided by the use of molecular/developmental tools, the most important of which has been the sequencing of the complete genomes and transcriptomes of different members of the three clades. The data obtained has been used to analyse the evolutionary history of gene families and to study their expression patterns during development, in both space and time. A major focus of our research is the origin of ‘cephalized’ (centralized) nervous systems. How complex brains are assembled from simpler neuronal arrays has been a matter of intense debate for at least 100 years. We are now tackling this issue using Xenacoelomorpha models. These represent an ideal system for this work because the members of the three clades have nervous systems with different degrees of cephalization; from the relatively simple sub-epithelial net of Xenoturbella to the compact brain of acoels. How this process of ‘progressive’ cephalization is reflected in the genomes or transcriptomes of these three groups of animals is the subject of this paper.

The application of induced pluripotent stem cell (iPSC) technologies in cell based strategies, for the repair of the central nervous system (with particular focus on the spinal cord), is moving towards the potential use of clinical grade donor cells. The ability of iPSCs to generate donor neuronal, glial and astrocytic phenotypes for transplantation is highlighted here, and we review recent research using iPSCs in attempts to treat spinal cord injury in various animal models. Also discussed are issues relating to the production of clinical grade iPSCs, recent advances in transdifferentiation protocols for iPSC-derived donor cell populations, concerns about tumourogenicity, and whether iPSC technologies offer any advantages over previous donor cell candidates or tissues already in use as therapeutic tools in experimental spinal cord injury studies.

Pleiotrophin (PTN) is a secreted growth factor, and also a cytokine, associated with the extracellular matrix, which has recently starting to attract attention as a significant neuromodulator with multiple neuronal functions during development. PTN is expressed in several tissues, where its signals are generally related with cell proliferation, growth, and differentiation by acting through different receptors. In Central Nervous System (CNS), PTN exerts post-developmental neurotrophic and -protective effects, and additionally has been involved in neurodegenerative diseases and neural disorders. Studies in Drosophila shed light on some aspects of the different levels of regulatory control of PTN invertebrate homologs. Specifically in hippocampus, recent evidence from PTN Knock-out (KO) mice involves PTN functioning in learning and memory. In this paper, we summarize, discuss, and contrast the most recent advances and results that lead to proposing a PTN as a neuromodulatory molecule in the CNS, particularly in hippocampus.

Background: Psychopathy is characterized by a distinctive interpersonal style that combines callous-unemotional traits with inflexible and antisocial behavior. Traditional emotion-based perspectives link emotional impairment mostly to alterations in amygdala-ventromedial frontal circuits. However, these models alone cannot explain why individuals with psychopathy can regularly benefit from emotional information when placed on their focus of attention and why they are more resistant to interference from nonaffective contextual cues. The present study aimed to identify abnormal or distinctive functional links between and within emotional and cognitive brain systems in the psychopathic brain to characterize further the neural bases of psychopathy. Methods: High-resolution anatomic magnetic resonance imaging with a functional sequence acquired in the resting state was used to assess 22 subjects with psychopathy and 22 control subjects. Anatomic and functional connectivity alterations were investigated first using a whole-brain analysis. Brain regions showing overlapping anatomic and functional changes were examined further using seed-based functional connectivity mapping. Results: Subjects with psychopathy showed gray matter reduction involving prefrontal cortex, paralimbic, and limbic structures. Anatomic changes overlapped with areas showing increased degree of functional connectivity at the medial-dorsal frontal cortex. Subsequent functional seed-based connectivity mapping revealed a pattern of reduced functional connectivity of prefrontal areas with limbic-paralimbic structures and enhanced connectivity within the dorsal frontal lobe in subjects with psychopathy. Conclusions: Our results suggest that a weakened link between emotional and cognitive domains in the psychopathic brain may combine with enhanced functional connections within frontal executive areas. The identified functional alterations are discussed in the context of potential contributors to the inflexible behavior displayed by individuals with psychopathy.

INTRODUCCIÓN Los mecanismos celulares y moleculares a través de los cuales produce sus efectos son, por ahora, mal conocidos. En los últimos años se está valorando con gran interés la interacción existente entre el etanol y el glutamato. Se han realizado múltiples experimentos con animales de laboratorio que han demostrado que el etanol es un potente inhibidor de la acción del glutamato sobre los receptores NDMA, surgiendo la hipótesis de que la relación etanol-NDMA condiciona parte de los efectos lesivos del tóxico sobre el sistema nervioso central (SNC). OBJETIVOS 1.- Estudiar, mediante la técnica de la autorradiografía de receptores, la distribución y la densidad de los receptores NMDA del glutamato en diferentes áreas del SNC de un grupo de personas consumidoras regulares de diferentes dosis de alcohol, y comparar los resultados con los obtenidos en controles, pacientes no consumidores de alcohol 2.- Valorar los cambios en la densidad de receptores NMDA entre los consumidores crónicos de alcohol no abstinentes (consumidores de alcohol hasta horas antes de su defunción) y abstinentes 3.- Analizar las posibles diferencias en la densidad de receptores NMDA entre los consumidores crónicos de alcohol según la intensidad de la ingesta habitual, diferenciando entre consumidores moderados (40-80g/dia) y consumidores de dosis altas de alcohol (>80g/dia) 4.- Estudiar las posibles diferencias en la densidad de receptores entre consumidores de alcohol atribuible a las circunstancias de la defunción (fallecidos por muerte súbita o no) PACIENTES Y MÉTODOS Fueron seleccionados para el estudio un total de 16 pacientes alcohólicos y 16 controles. Las áreas cerebrales incluidas en los diferentes experimentos fueron las siguientes: Córtex frontal, núcleo estriado, hipocampo, y vermis y hemiferio cerebeloso Los experimentos de autorradiografía de receptores se realizaron en el Departament de Neuroquímica del Consell Superior d'Investigacions Científiques (CSIC). RESULTADOS Y CONCLUSIONES 1.- El consumo prolongado de alcohol no modifica significativamente la densidad de receptores NMDA en las áreas estudiadas. A la luz de los recientes estudios sobre las subunidades del receptor NMDA, el etanol probablemente actúa modificando la composición y proporción de las mismas, y a través de estos cambios provoca las alteraciones funcionales en el sistema glutamatérgico, sin que se observen cambios en la densidad global de estos receptores. 3.- No se han detectado diferencias significativas entre la densidad de receptores NMDA en pacientes consumidores abstinentes y no abstinentes. Debido a los fenómenos asociados de sobre-regulación adaptativa e inhibición funcional de los receptores NDMA bajo los efectos del etanol, la densidad global de los mismos puede mantenerse estable mientras el individuo siga manteniendo la ingesta de forma regular. Durante la fase de abstinencia aguda, la liberación brusca de receptores inhibidos hasta ese momento, y el incremento real del número de los mismos (sobre-regulación) provocarían la hiperestimulación glutamatérgica y la excitotoxicidad. En ese momento probablemente sí se detectarían alteraciones en la densidad de receptores, durante un período de varios días. Al cabo de unas semanas en ausencia del tóxico en el medio, los receptores NDMA retornarían a sus niveles previos. 4.- No se han detectado diferencias en la densidad de receptores NMDA entre las muestras de tejido provenientes de alcohólicos fallecidos por muerte súbita y no súbita., Specific binding of [3H]MK801 to N-methyl-D-aspartate (NMDA) receptors in the frontal cortex, cerebellum, striatum and hippocampus (CA1 and gyrus dentatus) was measured by receptor autoradiography in 16 Caucasian chronic alcohol consumers free of clinical manifestations of alcoholism, and compared to 16 Caucasian control subjects. Binding densities were not significantly different between heavy and moderate drinkers, neither between alcohol consumers that were abstinent or non-abstinent before death, nor between ethanol drinkers and controls. Continued alcohol consumption, in the absence of hepatic, neurologic or psychiatric disorders related to alcoholism, does not alter the binding properties of NMDA receptors in the brain areas studied.

The mode of action of trans-resveratrol, a promising lead compound for the development of neuroprotective drugs, is unknown. Data from a functional genomics study were retrieved with the aim to find differentially expressed genes that may be involved in the benefits provided by trans-resveratrol. Genes that showed a significantly different expression (p

En el sistema nerviós central (SNC) la dopamina es comporta com la catecolamina prototip controlant el moviment i l’estat de motivació de l’individu, així com l’alliberació de prolactina. L’objectiu principal d’aquesta tesi doctoral ha estat estudiar la utilitat de tres biomarcadors relacionats amb el metabolisme de la dopamina (àcid homovaníl•lic, les pterines i el piridoxal fosfat) com a eines pel diagnòstic i la investigació de malalties neurològiques en la infància. La tesi ha estat estructurada en tres objectius, i per tant en tres treballs. En el primer treball, es va avaluar la utilitat de l’àcid homovaníl•lic, el metabòlit principal i més estable de la dopamina, per estudiar l’estat dopaminèrgic. El seu ús com a biomarcador en el líquid cefaloraquidi (LCR) ens ha permès diagnosticar varis errors congènits del metabolisme en els últims anys. No obstant això, la necessitat d’aquest estudi va originar-se pel fet que hi ha molts pacients amb simptomatologia de deficiència dopaminèrgica que romanen sense un diagnòstic definitiu confirmat. La descripció d’aquests pacients des d’un punt de vista bioquímic, buscant associacions amb les característiques clíniques, radiològiques i electrofisiològiques ens ha permès millorar el coneixement científic respecte aquest marcador bioquímic en l’àmbit neuropediàtric. Seguint en l’avaluació del metabolisme de la dopamina, cal remarcar el paper essencial que tenen els cofactors tetrahidrobiopterina i vitamina B6 en el metabolisme dopaminèrgic. Les alteracions quantitatives d’aquests cofactors també poden provocar deficiències en la via dopaminèrgica i, per tant, també tenen un important valor a la hora d’estudiar aquests pacients. D’altra banda, s’ha demostrat el paper que té la neopterina (metabòlit de la tetrahidrobiopterina) com a marcador bioquímic en LCR en el diagnòstic de processos immuno-inflamatoris del SNC. D’aquí neix el plantejament del segon treball, la finalitat del qual és estudiar les pterines (neopterina i biopterina) com a eina d’ajut en pacients neurològics sense un diagnòstic confirmat però que presenten alteracions en el valor de pterines en LCR. Alteracions que poden estar associades a malalties genètiques d’aquest cofactor o bé causades per processos immuno-inflamatoris. Finalment l’últim treball es basa en la utilitat de quantificar els valors del cofactor que participa en l´últim pas de la síntesi dopaminèrgica, el piridoxal fosfat (la forma activa de la vitamina B6). Les seves deficiències poden provocar trastorns marcats de la síntesis de dopamina, però també d’altres metabòlits, com aminoàcids o l’àcid gamma-aminobutíric. Clínicament, aquests pacients presenten greus alteracions neurològiques sent l’epilèpsia refractària als anticonvulsivants habituals, la simptomatologia més freqüent. La necessitat d’aquest treball esdevé en què hi ha varis pacients que presenten deficiències de piridoxal fosfat els quals han respòs favorablement a suplements de vitamina B6. Els diferents estudis que s’han presentat en aquesta tesi s’han realitzat en pacients procedents del Hospital Sant Joan de Déu, però també d’altres hospitals nacionals i internacionals, entre els quals n’hi ha de Portugal, Argentina, Grècia, Turquia i la Índia. La col•laboració amb altres serveis del nostre hospital ha donat un component traslacional als tres treballs presentats en aquesta tesi, que inclouen dades clíniques, bioquímiques i moleculars, aportant un aspecte més holístic i tenint una aplicació pràctica i valuosa per l’estudi de pacients amb malalties neuropediàtriques greus., Our objective was to study the utility of three biomarkers related to dopamine metabolism (homovanillic acid (HVA), pterins and pyridoxal phosphate (PLP)) as a diagnostic and investigation tool of neurologic disorders in infancy. In the first study our objective was to determine and assess the HVA concentrations in cerebrospinal fluid (CSF) in a large neuropediatric cohort as a biochemical tool to identify dopaminergic abnormalities. To achieve this aim, we studied biogenic amines in 1388 CSF samples from children with neurological disorders analysed by high pressure liquid chromatography with electrochemical detector. This study allowed us to determine the prevalence of dopaminergic abnormalities, as well as we defined several neurologic diseases with CSF HVA abnormalities, besides from 21 primary dopaminergic defects. In the second study, our objective was to analyse the CSF pterins (neopterin and biopterin) concentrations in children with neurologic disorders to identify primary or secondary diseases about these coenzymes. We also evaluated the CSF neopterin utility as a biomarker in inflammatory-immune mediated processes establishing a new cut-off for it. After that, the next step was to assess data from 606 neuropediatric patients presenting CSF neopterin values above or below the new established cut-off value. Finally, our last study was to analyse the CSF PLP concentrations in neuropediatric patients without an aetiological diagnosis in order to identify primary or secondary diseases related to PLP metabolism. After the analyses of PLP in CSF of 147 patients, we detected our first PNPO gene defect. Moreover, we recruited a series of 10 patients who disclosed low-normal CSF PLP values, that the main clinical feature was epilepsy. Additionally we also assessed the response to PLP treatment in all of them. These different studies have been performed in patients from Hospital Sant Joan de Déu, as well as from other national and international hospitals, such as Portugal, Argentina, Greece, Turkey and India. Moreover, the collaboration with other services of our hospital has allowed this thesis to have a holistic component including clinical, radiological, biochemical and molecular data.

La Mucopolisacaridosi tipus IIIB (MPSIIIB) és una malaltia minoritària d’acumulació lisosòmica, d’herència autosòmica recessiva, causada per la deficiència de l’enzim α-N-acetilglucosaminidasa (NAGLU), implicat en la via de degradació del glicosaminoglicà (GAG) Heparan Sulfat (HS). Aquesta pèrdua d’activitat comporta una acumulació progressiva de HS intralisosòmic que finalment dóna lloc a la disfunció i mort cel·lular. La MPSIIIB és principalment una malaltia neurodegenerativa amb una lleu afectació somàtica, en comparació amb d’altres malalties d’acumulació lisosòmica. Després d’un primer període asimptomàtic durant els primers mesos de vida, la malaltia es caracteritza per una fase inicial entre el primer i quart any de vida amb alentiment del desenvolupament mental. Aquesta fase és seguida per l’aparició de problemes de comportament severs conjuntament amb un deteriorament intel·lectual que s’inicia al voltant del tercer o quart any de vida. Finalment, amb l’aparició de demència severa, els problemes de comportament comencen a desaparèixer i les funcions motores inicien un alentiment progressiu, que finalment resulta en una pèrdua total de locomoció, disfàgia i lesions en el sistema piramidal. La mort se sol produir entre la segona i tercera dècada de vida, tot i que formes més lleus de la malaltia presenten una evolució més lenta amb esperances de vida més llargues. Actualment no hi ha cap tractament específic ni efectiu per la MPSIIIB. Totes les teràpies i tractaments actuals estan basats en el control simptomàtic i en la millora de la qualitat de vida dels pacients i de l’entorn familiar. Per aquesta raó, i degut a l’absència de teràpies eficaces, es fa necessari el desenvolupament de noves estratègies terapèutiques per al tractament de la malaltia. La teràpia gènica in vivo basada en l’administració de vectors virals derivats dels virus adenoassociats (AAV) ofereix una alternativa atractiva per al tractament de malalties minoritàries d’origen genètic com la MPSIIIB. D’una banda, les estratègies de teràpia gènica basades en vectors virals AAV han mostrat resultats prometedors com a eina per a la transferència gènica in vivo. D’altra banda, aquestes estratègies també han mostrat expressió a llarg termini en models animals de malalties així com també en pacients afectats de malalties genètiques després d’una única administració del vector. Per tant, i degut a la seva baixa immunogenicitat, a la seva expressió a llarg termini i al fet que poden transduir cèl·lules quiescents, els vectors virals AAV es presenten com a bons candidats per al tractament de malalties genètiques amb afectació del Sistema Nerviós Central (SNC). Entre tots els vectors AAV identificats i caracteritzats, aquells derivats del serotip 9 (AAV9) es caracteritzen per presentar un ampli tropisme. L’administració d’aquest serotip directament al Líquid Cefaloraquidi (LCR) ha demostrat ser eficaç en transduir diferents tipus cel·lulars del SNC juntament amb una àmplia distribució. A més, part del vector administrat al LCR pot passar a circulació sanguínia i transduir el fetge, entre d’altres teixits, donant lloc a l’expressió del transgèn no només al SNC, sinó que també a nivell somàtic. Per tant, en la present tesi doctoral s’ha desenvolupat una estratègia de teràpia gènica centrada en l’administració al LCR d’un AAV9 codificant per la proteïna NAGLU murina (AAV9-mNAGLU), amb la finalitat de tractar la patologia neurològica i somàtica característiques de la MPSIIIB. Una única administració del vector viral AAV9-mNAGLU a la cisterna magna de ratolins models de la MPSIIIB, va donar lloc a una àmplia transducció del SNC i del fetge, que alhora es va traduir en un augment en els nivells d’activitat NAGLU en aquest òrgans i en la correcció de la patologia neuronal i somàtica. A nivell del SNC, l’augment d’activitat enzimàtica NAGLU en els ratolins MPSIIIB tractats va donar lloc a la normalització del contingut de GAGs del cervell. També es van corregir els signes de neuroinflamació característics d’aquest model animal, amb la desaparició de l’astrocitosi i la microgliosi al cervell dels ratolins MPSIIIB tractats. També es va dur a terme un estudi d’expressió gènica del cervell, el qual va permetre observar una desregulació en l’expressió gènica de gens implicats en la immunitat innata, l’estrès oxidatiu i la funció lisosòmica, entre d’altres, al cervell dels ratolins MPSIIIB no tractats. El tractament amb AAV9-mNAGLU va donar lloc a la normalització de l’expressió de la majoria dels gens diferencialment expressats, fent similars els perfils d’expressió gènica entre ratolins MPSIIIB tractats i ratolins control sans (ratolins WT). Aquests resultats van proporcionar una prova més de l’eficàcia terapèutica d’aquesta aproximació de teràpia gènica. A nivell somàtic, l’enzim NAGLU produït al fetge de ratolins MPSIIIB tractats va donar lloc a un augment de l’activitat NAGLU circulant, fent accessible l’enzim a la resta d’òrgans i teixits somàtics. Alhora, aquest augment d’activitat NAGLU circulant va permetre la normalització del contingut en GAGs de la majoria d’òrgans i teixits analitzats. A banda d’aquest efecte, el tractament amb el vector viral AAV9-mNAGLU també va comportar una normalització de les alteracions de comportament característiques de la malaltia així com també un augment significatiu de la supervivència dels ratolins MPSIIIB tractats d’ambdós sexes. Demostrada l’eficàcia del tractament en ratolins MPSIIIB, es va avaluar la translacionabilitat de l’aproximació terapèutica en models animals més grans. Gossos Beagle adults van ser utilitzats per a aquesta finalitat, ja que la mida del cervell d’aquests és molt similar a la mida dels pacients humans en edat pediàtrica a qui aniria destinada una possible teràpia. Es va administrar el vector viral AAV9 codificant per la proteïna NAGLU canina (AAV9-cNAGLU) al LCR de 4 gossos sans, de tal manera que es va poder observar una producció i secreció elevada i estable de l’enzim NAGLU actiu al LCR, sense signes de toxicitat associada. Un percentatge elevat de la població humana és seropositiva per anticossos neutralitzants (NAbs) contra la càpsida proteica dels vectors virals AAV9. La presència de NAbs circulants podria suposar l’eliminació del vector viral de la circulació sanguínia abans de transduir cap tipus cel·lular, limitant així l’eficàcia terapèutica del tractament proposat. Degut a aquest fet, també es va utilitzar el model caní per tal d’avaluar l’efecte de la immunitat preexistent sobre els nivells d’activitat NAGLU mesurats al LCR. Els resultats van mostrar com després de l’administració intracisterna del vector AAV9-cNAGLU, els nivells d’activitat en LCR es mantenien elevats i constants a llarg termini independentment de la presència d’elevats títols circulants de NAbs. Conjuntament, els resultats van mostrar l’eficàcia terapèutica de l’administració al LCR de vectors AAV9 codificants per la proteïna NAGLU i la translacionabilitat d’aquesta aproximació cap a models animals de major mida, així com també la persistència d’activitat al LCR independentment de la presència de NAbs circulants contra la càpsida del vector viral. Per tant, l’aproximació de teràpia gènica descrita en aquest treball pot esdevenir un possible tractament, no només per a la MPSIIIB, sinó que també per a altres malalties d’acumulació lisosòmica amb afectació del SNC., Mucopolysaccharidosis type IIIB (MPSIIIB) is a rare Lysosomal Storage Disease (LSD) caused by the lack of the α-N-acetylglucosaminidase (NAGLU) activity, an enzyme involved in the stepwise degradation of the Glycosaminoglycan (GAG) Heparan Sulfate (HS). This leads to a pathological accumulation of HS in the lysosomes of cells that ultimately results in cell dysfunction and/or death. MPSIIIB is mainly a neurodegenerative disease, and has a mild somatic involvement compared with other LSDs. After an initial asymptomatic period during the first few months of life, the disease manifests between the first and fourth year with slowing of mental development. This is followed by severe behavioral problems along with intellectual impairment during the second phase, which starts around the third or fourth year of life. Finally, with the onset of severe dementia, behavioral alterations begin to disappear and a progressive impairment of motor functions starts, which ultimately results in a total loss of locomotion, dysphagia and pyramidal system lesions. Death usually occurs between the second and third decades of life, although milder forms of the disease with slower progression and longer life expectancies have been described. There is currently no effective treatment for MPSIIIB. All available therapies and treatments are symptomatic and aimed at improving the quality of life of patients and their families. For this reason, it is necessary to develop new therapeutic strategies for this disease. In this regard, in vivo gene therapy based on the administration of adeno-associated viral vectors (AAV) offers an attractive alternative for the treatment of rare genetic diseases such as MPSIIIB. On the one hand, strategies based on AAV gene therapy have shown promising results as a tool for gene transfer in vivo. On the other hand, these strategies have showed long term expression in animal models as well as in patients affected of genetic diseases after a single vector administration. In addition, and due to their low immunogenicity, their long-term expression and their ability to transduce different cell types in the brain, AAV vectors are good candidates for the treatment of genetic diseases with CNS involvement. Among all the AAV serotypes described and characterized so far, those derived from serotype 9 (AAV9) show a broad tropism. Administration of AAV9 directly to the cerebrospinal fluid (CSF) results in transduction of different cell types in the CNS with a widespread distribution. Moreover, after its passage through the CNS, part of the vector reaches the bloodstream, resulting in transduction of the liver and other somatic tissues, allowing transgene expression not only in the CNS but also in the periphery. Therefore, the aim of the present Doctoral Thesis was to develop a therapeutic strategy based on the intra-CSF delivery of an AAV9 vector coding for murine NAGLU (mNAGLU) to treat both CNS and somatic MPSIIIB pathology. A single intra-CSF administration of AAV9-mNAGLU viral vector to MPSIIIB male and female mice resulted in a broad transduction of CNS and liver which led to an increase of NAGLU activity above normal levels in those organs and correction of neurologic and somatic pathology. At a CNS level, the restoration of NAGLU activity in MPSIIIB-treated mice lead to normalization of the content of GAGs in the brain. AAV9-mNAGLU administration also completely corrected all signs of neuroinflamation characteristic of this animal model, with disappearance of astrocytosis and microgliosis in MPSIIIB-treated mice. Moreover, the AAV9 treatment also normalized brain lisosomal homeostasis as reflected by the normalization of the activities of other lisosomal enzymes. Gene expression analysis were performed on whole brain and revealed a dysregulation of gene expression of genes related to innate immunity, lisosomal functions and oxidative stress in untreated MPSIIIB. Interestingly, a representative group of up-regulated genes (~50%) were related to microglia, indicating the critical role of this cellular type in the development of the disease. Treatment with AAV9-mNAGLU normalized gene expression of the majority of these deregulated genes, so that the expression profile of AAV9-treated MPSIIIB mice resembled that of healthy WT littermates, providing further evidence of the therapeutic efficacy of the approach. At a somatic level, the NAGLU enzyme produced by the liver led to increased NAGLU circulating activity, making the enzyme available for all the affected organs and tissues. This led to the normalization of GAG content in most of the somatic organs and tissues analyzed. Intra-CSF AAV9-mNAGLU administration also normalized typical behavioral deficits observed in MPSIIIB untreated mice and increased survival both in males and females. Once the efficacy of the AAV9 treatment was demonstrated in the mouse model, we evaluated the feasibility of the approach in a larger animal model. Healthy adult Beagle dogs were used for this purpose, since the brain of dogs is closer in size to that of the human pediatric patients who are the target of this therapy. A high and stable production and secretion of active NAGLU was documented in the CSF of dogs that received an intracisternal administration of AAV9 vectors coding for the canine NAGLU, in the absence of any signs of toxicity. In addition, due to the fact that a high percentage of the human population is seropositive for neutralizing antibodies (NAbs) against the AAV9 capsid proteins, which could potentially limit the efficacy of the treatment by clearing the vector from the circulation before it enters any cell, we also evaluated the effect of pre-existing immunity on the levels of NAGLU activity measured in the CSF. The results showed that the levels of enzymatic activity achieved in the CSF were the same regardless of the presence of high NAbs titers in serum prior to the treatment. Together, these results demonstrate the efficacy of intra-CSF AAV9-mNAGLU delivery in correcting MPSIIIB disease and the scalability of the approach to larger animal models, as well as the lack of effect of pre-exiting immunity on CNS efficacy when the vector is delivered directly to the CSF. Thus, the therapeutic approach described herein may become a possible treatment not only for MPSIIIB, but also for other LSDs with CNS involvement.

Methylone is a cathinone derivative that has recently emerged as a designer drug of abuse in Europe and the USA. Studies on the acute and long-term neurotoxicity of cathinones are starting to be conducted. We investigated the neurochemical/enzymatic changes indicative of neurotoxicity after methylone administration (4 × 20 mg/kg, subcutaneously, per day with 3 h intervals) to adolescent rats, to model human recreational use. In addition, we studied the effect of methylone on spatial learning ad memory using the Morris water maze paradigm. Our experiments were carried out at a high ambient temperature to simulate the hot conditions found in dance clubs where the drug is consumed. We observed a hyperthermic response to methylone that reached a peak 30 min after each dose. We determined a serotonergic impairment in methylone-treated rats, especially in the frontal cortex, where it was accompanied by astrogliosis. Some serotonergic alterations were also present in the hippocampus and striatum. No significant neurotoxic effect on the dopaminergic system was identified. Methylone-treated animals only displayed impairments in the probe trial of the Morris water maze, which concerns reference memory, while the spatial learning process seemed to be preserved.

Mephedrone is a drug of abuse marketed as 'bath salts'. There are discrepancies concerning its long-term effects. We have investigated the neurotoxicity of mephedrone in mice following different exposition schedules. Schedule 1: four doses of 50 mg/kg. Schedule 2: four doses of 25 mg/kg. Schedule 3: three daily doses of 25 mg/kg, for two consecutive days. All schedules induced, in some animals, an aggressive behavior and hyperthermia as well as a decrease in weight gain. Mephedrone (schedule 1) induced dopaminergic and serotoninergic neurotoxicity that persisted 7 days after exposition. At a lower dose (schedule 2) only a transient dopaminergic injury was found. In the weekend consumption pattern (schedule 3), mephedrone induced dopamine and serotonin transporter loss that was accompanied by a decrease in tyrosine hydroxylase and tryptophan hydroxylase 2 expression one week after exposition. Also, mephedrone induced a depressive-like behavior, as well as a reduction in striatal D2 density, suggesting higher susceptibility to addictive drugs. In cultured cortical neurons, mephedrone induced a concentration-dependent cytotoxic effect. Using repeated doses for 2 days in an elevated ambient temperature we evidenced a loss of frontal cortex dopaminergic and hippocampal serotoninergic neuronal markers that suggest injuries at nerve endings.

Ack1 es una proteína tirosina quinasa citoplasmática que pertenece a la familia de proteínas Ack que está formada por 9 miembros. Ack1 está compuesta por varios dominios de interacción proteína-proteína y un dominio catalítico tirosina quinasa cuya autofosforilación regula parcialmente la función de la proteína (Lougheed et al., 2004). Ack1 se encuentra altamente expresada en cerebro (Urena et al., 2005; La Torre et al., 2006), principalmente en las zonas del hipocampo, corteza cerebral, bulbo olfativo y cerebelo (Urena et al., 2005). Además, se ha demostrado que Ack1 se localiza tanto en dendritas como en regiones presinápticas y su expresión se regula a la alza por un incremento de actividad neuronal (Urena et al., 2005). Las funciones fisiológicas de Ack1 son bastante desconocidas. En conjunto, los datos publicados hasta la actualidad destacan la capacidad de la proteína Ack1 para interaccionar con una gran variedad de proteínas, que indica que Ack1 puede participar en diferentes rutas de transducción de señales, y por tanto, participar en diferentes procesos fisiológicos de las células. Por ejemplo, se ha descrito que puede participar en la regulación del citoesqueleto de actina (Burbelo et al., 1995), en los procesos de endocitosis (Teo et al., 2001), en las cascadas de señalización que resultan de la adhesión celular (Bourdoulous et al., 1998), en la migración celular (Modzelewska et al., 2006), en la proliferación (Nur et al., 2005) y en la supervivencia (Mahajan et al., 2005). La mayoría de funciones de Ack1 conocidas se deducen de las interacciones moleculares de ésta y hasta la actualidad se dispone de muy pocos datos respecto a las competencias biológicas de esta proteína, especialmente a nivel de SNC. Por eso, en esta tesis hemos intentado determinar el papel de la tirosina quinasa Ack1 en las funciones del SNC mediante 5 capítulos de resultados. En el capítulo I explicamos la producción de un anticuerpo monoclonal contra Ack1 que produjimos con el objetivo de poder estudiar la proteína Ack1 a nivel funcional. Este anticuerpo lo produjimos contra la región rica en prolinas de Ack1 con el fin que nos permitiera aumentar la especificidad de nuestros experimentos. En el capítulo II caracterizamos la proteína Ack1 a nivel más funcional. Demostramos que Ack1 es un componente de la vía de señalización de las neurotrofinas, siendo fosforilada en respuesta a neurotrofinas e interaccionando con sus receptores Trk. Además, también describimos que cambios de expresión de Ack1 alteran la neuritogénesis en células PC12 y los patrones de ramificación axonal y dendrítico en neuronas de hipocampo y células granulares de cerebelo. En el capítulo III examinamos la interacción de Ack1 con las proteínas de la densidad postsináptica CaMKII y PSD-95, su fosforilación en respuesta a los estímulos excitadores NMDA y glutamato, y la afectación de los botones axonales de las ramas colaterales de Schaffer por una falta de expresión de Ack1. Los resultados obtenidos en este capítulo apuntan a una posible implicación de Ack1 a nivel de terminales sinápticos. En el capítulo IV nos centramos en estudiar la interacción de Ack1 con la proteína FAK y su participación en procesos de quimioatracción mediados por Netrina-1. Los datos obtenidos sugieren una interacción entre ambas proteínas, que Ack1 se fosforila en respuesta a Netrina-1 y una implicación de la proteína Ack1 en los procesos de quimioatracción mediados por Netrina-1 en células de hipocampo. Finalmente, siendo las proteínas Ack1 y FAK unas proteínas que dependen de su estado de fosforilación para regular su actividad, en el capítulo V de resultados de esta tesis estudiamos las dianas de fosforilación y posibles proteínas de interacción de ambas proteínas, mediante la técnica de espectrometría de masas en muestras de cerebro de ratón en desarrollo, de cerebro de ratón adulto y de cerebro de ratón adulto hiperestimulado. - Bibliografía Bourdoulous S, Orend G, MacKenna DA, Pasqualini R, Ruoslahti E (1998) Fibronectin matrix regulates activation of RHO and CDC42 GTPases and cell cycle progression. The Journal of cell biology 143:267-276. Burbelo PD, Drechsel D, Hall A (1995) A conserved binding motif defines numerous candidate target proteins for both Cdc42 and Rac GTPases. The Journal of biological chemistry 270:29071-29074. La Torre A, del Rio JA, Soriano E, Urena JM (2006) Expression pattern of ACK1 tyrosine kinase during brain development in the mouse. Gene Expr Patterns 6:886-892. Lougheed JC, Chen RH, Mak P, Stout TJ (2004) Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1. The Journal of biological chemistry 279:44039-44045. Mahajan NP, Whang YE, Mohler JL, Earp HS (2005) Activated tyrosine kinase Ack1 promotes prostate tumorigenesis: role of Ack1 in polyubiquitination of tumor suppressor Wwox. Cancer research 65:10514-10523. Modzelewska K, Newman LP, Desai R, Keely PJ (2006) Ack1 mediates Cdc42-dependent cell migration and signaling to p130Cas. The Journal of biological chemistry 281:37527-37535. Nur EKA, Zhang A, Keenan SM, Wang XI, Seraj J, Satoh T, Meiners S, Welsh WJ (2005) Requirement of activated Cdc42-associated kinase for survival of v-Ras-transformed mammalian cells. Mol Cancer Res 3:297-305. Teo M, Tan L, Lim L, Manser E (2001) The tyrosine kinase ACK1 associates with clathrin-coated vesicles through a binding motif shared by arrestin and other adaptors. The Journal of biological chemistry 276:18392-18398. Urena JM, La Torre A, Martinez A, Lowenstein E, Franco N, Winsky-Sommerer R, Fontana X, Casaroli-Marano R, Ibanez-Sabio MA, Pascual M, Del Rio JA, de Lecea L, Soriano E (2005) Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain. The Journal of comparative neurology 490:119-132., Ack1 is a cytoplasmic tyrosine kinase highly expressed in Central Nervous System (Urena et al., 2005; La Torre et al., 2006) that has several protein-protein interaction domains and a catalytic domain that is autophosphorylated, and this process regulates, at least in part, the action of this protein (Lougheed et al., 2004). Moreover, it has been demonstrated that Ack1 is localized in dendrites and presynaptic regions and that its expression is up-regulated by an increase of neuronal activity (Urena et al., 2005). Most of the known functions of Ack1 have been elucidated from interactions of Ack1 with several proteins. But, most of the physiologically functions of Ack1 remain to be described, especially in CNS. For this reason, the aim of this thesis has been to unravel some of these functions on CNS that are described through 5 chapters. In the 1st chapter we have explained the production of a monoclonal antibody against Ack1 that allowed us to improve the specificity of our experiments and to study Ack1 at a functional level. In the 2nd chapter we have demonstrated that Ack1 is a component of the neurotrophin pathway, because it is phosphorylated as a response to neurotrophins and interacts with Trk receptors. Moreover, we also have described how changes in Ack1 expression alter neuritogenesis and axonal and dendritic ramification. In the 3th chapter we have analyzed the interaction of Ack1 with the postsynaptical proteins CaMKII and PSD-95, its phosphorylation in response to excitatory stimulus such as NMDA or glutamate and we have described that a lack of Ack1 expression decrease the size of axonal buttons. These data suggest an implication of Ack1 at a synaptic level. In the 4th chapter we focused on the determination of the interaction of Ack1 and FAK, the Ack1 phosphorylation in response to Netrin-1 and the effect of Ack1 in chemoattraction regulated by Netrin-1. Finally, in the 5th chapter we studied the phosphorylation sites and the possible interacting proteins of FAK and Ack1 by mass spectrometry in samples of mice brain in development, of adult mice brain and of adult mice overstimulated brain. - Bibliography La Torre A, del Rio JA, Soriano E, Urena JM (2006) Expression pattern of ACK1 tyrosine kinase during brain development in the mouse. Gene Expr Patterns 6:886-892. Lougheed JC, Chen RH, Mak P, Stout TJ (2004) Crystal structures of the phosphorylated and unphosphorylated kinase domains of the Cdc42-associated tyrosine kinase ACK1. The Journal of biological chemistry 279:44039-44045. Urena JM, La Torre A, Martinez A, Lowenstein E, Franco N, Winsky-Sommerer R, Fontana X, Casaroli-Marano R, Ibanez-Sabio MA, Pascual M, Del Rio JA, de Lecea L, Soriano E (2005) Expression, synaptic localization, and developmental regulation of Ack1/Pyk1, a cytoplasmic tyrosine kinase highly expressed in the developing and adult brain. The Journal of comparative neurology 490:119-132.

The aims of this thesis are: Aim 1. The involvement of A(1)R-A(2A)R heteromer in glial cells and its study at a molecular level. Aim 2. To find evidence for allosteric interactions between partner receptors in the A(2A)RD(2)R receptor heteromer which confer specific pharmacological characteristics to the heteromer. Aim 3. Search for selective antagonists of A(2A)R for presynaptic A(1)R-A(2A)R heteromers versus postsynaptic A(2A)R-D(2)R heteromers that can be useful for treatment of neurological disorder’s treatment, particularly Huntington’s disease. Aim 4. Investigate the pharmacological and functional properties of A(2A)R in the A(2A)CB1 heteromer. Aim 5. Compound screening of different A(2A)R antagonists in stable CHO cell lines expressing A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R or A(2A)R-CB(1)R heteromers. The conclusions of the objectives are: Conclusions derived from the first aim: The involvement of A(1)R-A(2A)R heteromer in glial cells and its study at a molecular level. - Upon GABA uptake, adenosine has a biphasic effect, which is mediated by A(1)RA(2A)R heteromers coupled to both Gi/0 and Gs proteins. Extracellular adenosine acting on these A(1)R-A(2A)R functional units operates in a concerted way to balance a PKA-dependent action on GABA uptake. The neural output would thus be inhibitory at low firing rates and facilitatory at high firing rates. - Adenosine by acting on adenosine receptors in astrocytes may significantly contribute to neurotransmission in a dual manner, which depends on the concentration of the nucleoside that is in turn dependent on neuronal firing activity. - BRET and single molecule tracking with TIRF microscope show that the minimal GPCR heteromer unit may consist of four protomers and two G proteins. The strong similarity between GPCRs suggests that the molecular model proposed could apply to other receptors. - These heteromers can be formed in the plasma membrane and are stable on the order of minutes. Such stability suggests that designing ways to target these heteromers may indeed be a viable strategy. - The orientation of the alpha-subunits of the G proteins is on the distal receptors, suggesting that G proteins cross-talk could occur via receptors across the heteromer complex. - The heteromeric unit described, with its dynamic and structural limitations, provides the molecular framework to understand why heteromers are functionally distinct units and not merely the aggregation of two entities with independent functions. Conclusions derived from the second aim: To find an evidence for allosteric interactions between partner receptors in the A(2A)R-D(2)R receptor heteromer which confer specific pharmacological characteristics to the heteromer. In cell culture, the agonist and antagonist binding to the adenosine A(2A)R diminish the affinity of dopamine D(2)R agonists and antagonists. - Those negative interactions between ligands are consequence of allosteric interactions between both receptors conforming the A(2A)R-D(2)R heteromer and constitute a unique biochemical property of this heteromer. - In ex vivo tissue, using these allosteric interactions as a heteromer fingerprint, it has been demonstrated the expression of A(2A)R-D(2)R heteromer in human striatum. - The fact that the A(2A)R antagonists are able to modulate dopamine D(2)R pharmacology has to be taken into account to understand pathologies such as Parkinson’s disease or for human PET neuroimaging. Conclusions derived from the third aim: Search for selective antagonists of A(2A)R for presynaptic A(1)R-A(2A)R heteromers versus postsynaptic A(2A)R-D(2)R heteromers that can be useful for neurological disorder’s treatment, particularly Huntington’s disease. - The physical presence of dopamine D(2)R in the A(2A)R-D(2)R heteromer induced a strong negative cooperativity in the A(2A)R that was detected by SCH-442416. This cooperativity indicates that A(2A)R-A(2A)R homodimers are present in the A(2A)R-D(2)R heteromer. - Based on in vitro and in vivo approaches, the compound SCH-442416 was classified as a preferential presynaptic A(2A)R antagonist, and the compound KW- 6002 was classified as a preferential postsynaptic A(2A)R antagonist. Considering this, SCH-442416 can be used as a lead compound in the development of antidyskinetic drugs in Huntington’s disease; meanwhile KW-6002 can be beneficial in Parkinson’s disease. Conclusions derived from the fourth aim: Investigate the pharmacological and functional properties of A(2A)R in the A(2A)R-CB1R heteromer. - Adenosine A(2A)R changes its G-protein coupling from stimulatory Gs to inhibitory Gi when it forms heteromer with CB1R and a synergistic cross-talk in G-protein activation is observed when both receptors are coactivated. - CB1R mainly controls the ERK1/2 signaling under the A(2A)R-CB1R heteromer. - The A(2A)R-CB1R heteromer does not show allosteric effects at the ligand binding level. Conclusions derived from the fifth aim: Compound screening of different A(2A)R antagonists in stable CHO cell lines expressing A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R or A(2A)R-CB1R heteromers. - Compound number 9 could be a good candidate to treat Parkinson’s disease due to its preferential binding to A(2A)R forming A(2A)R-D(2)R heteromer., Els objectius de la tesi doctoral són: - Objectiu 1. Implicacions de l’heteròmer A(1)R-A(2A)R a les cèl•lules glials i el seu estudi a nivell molecular. - Objectiu 2. Trobar evidències de modulacions al•lostèriques entre receptors a l’heteròmer A(2A)DD(2)R que confereixen característiques farmacològiques específiques a l’heteròmer. - Objectiu 3. Recerca d’antagonistes selectius de A(2A)R per a heteròmers presinàptics A(1)R-A(2A)R versus heteròmers postsinàptics A(2A)R-D(2)R que poden ser útils en el tractament de malalties neurològiques, particularment en la malaltia de Huntington. - Objectiu 4. Investigar les propietats funcionals i farmacològiques de A(2A)R en l’heteròmer A(2A)RCB(1)R. - Objectiu 5. Anàlisi de diferents compostos antagonistes de A(2A)R en línies cel•lulars estables CHO expressant A(2A)R, A(1)R-A(2A)R, A(2A)R-D(2)R o A(2A)R-CB(1)R. Les conclusions de la tesi són les següents. Primer objectiu: - En la recaptació de GABA, l’adenosina te un efecte bifàsic, el qual està mediat pels heteròmers A(1)R-A(2A)R que es troben acoblats a proteïnes Gi/o i Gs. L’adenosina extracel•lular actuant sobre aquests heteròmers opera en el balanç de recaptació de GABA depenent de l’activitat PKA. La senyalització neural serà inhibitòria a baixa activació neuronal i facilitadora a alta activitat neuronal. - BRET i experiments de “single molecule tracking” amb microscopi TIRF demostren que l’expressió mínima d’aquest heteròmer consta de quatre protomers i dues proteïnes G. La gran similitud entre GPCR suggereix que aquest model molecular podria ser aplicable a altres receptors. - Aquests heteròmers poden formar-se a la membrana plasmàtica i són estables durant minuts. Aquesta estabilitat suggereix que el disseny de fàrmacs dirigits contra aquests heteròmers és una estratègia viable. Segon objectiu: - En cultius cel•lulars, la unió d’agonistes i antagonistes a A(2A)R fa disminuir l’afinitat d’agonistes i antagonistes per al D(2)R. - Aquestes interaccions negatives entre lligands són conseqüència d’interaccions al•lostèriques entre ambdós receptors que conformen l’heteròmer A(2A)R-D(2)R i constitueixen una unitat bioquímica. - El fet de que els antagonistes de A(2A)R són capaços de modular la farmacologia de D(2)R ha de ser tingut en compte per poder entendre patologies com la malaltia de Parkinson i per a la neuroimatge per PET. Tercer objectiu: - La presència física de D(2)R en l’heteròmer A(2A)R-D(2)R indueix una forta cooperativitat negativa a A(2A)R la qual va ser detectada per SCH 442416. Aquesta cooperativitat indica que el homodimers A(2A)R-A(2A)R es troben presents a l’heteròmer A(2A)R-D(2)R. - Basant-nos en experiments in vitro i in vivo, el compost SCH 442416 va ser classificat com a preferentment antagonista de receptor A(2A) presinàptic, i el compost KW 6002 va ser classificat com a antagonista preferentment postsinàptic. Considerant això, SCH 442416 pot ser utilitzat pel desenvolupament de fàrmacs antidiscinètics pel tractament de la malaltia de Huntington, mentre que KW 6002 pot ser beneficiós per a tractar la malaltia de Parkinson. Quart objectiu: - A(2A)R canvia el seu acoblament a proteïna Gs per Gi quan passa a formar heteròmers amb CB(1)R i un “cross-talk” sinergístic en activació de proteïna G s’observa quan tots dos receptors es troben co-activats. Cinquè objectiu: - El compost número nou podria ser un bon candidat per tractar la malaltia de Parkinson degut a la seva unió preferencial al receptor A(2A) present a l’heteròmer A(2A)R-D(2)R.