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Background and purpose Oxaliplatin-induced neuropathy (OIN) implies axonal damage of both small and large sensory nerve fibers. We aimed at comparing the neurophysiological changes occurred after treatment and the capability to recovery based on histological marker of re-innervation GAP-43. Methods 48 patients with cancer were assessed before and after chemotherapy (at 3 months and 12 months if available). We recorded ulnar and sural sensory nerve action potentials (SNAP), determined quantitative sensory thresholds for warm and cold (WDT, CDT), pain thresholds and collected a distal biopsy of skin to assess the intra-epidermal nerve fiber density (IENFD) with PGP9.5 and GAP-43 markers (in a subgroup of 19 patients). Results Increased WDT and CDT as well as diminished IENFD at distal leg were already found in 30% of oncologic patients before treatment. After oxaliplatin, there was a significant increase in thermal thresholds in 52% of patients, and a decrease of SNAP amplitude in the sural nerve in 67% patients. IENFD was reduced in 47% and remained unchanged in 37% after oxiplatin. The density of GAP-43 + fibers and GAP-43/PGP 9.5 ratio was similar before and after treatment showing that cutaneous re-innervation is preserved despite no clinical recovery was observed after one year. Conclusion Non-selective axonal loss affects sensory fibers in OIN. However, the presence of intra-epidermal regenerative sprouts detected by GAP-43 may reduce the impact of neurotoxicity in the small fibers with long-term sequelae mostly on myelinated nerve endings. Pre-oxaliplatin GAP-43 failed to identify patients with higher risk of damage or worse recovery after treatment.

Introduction: Three-dimensional (3D) bioprinting is a promising technique for the development of neuronal in vitro models because it controls the deposition of materials and cells. Finding a biomaterial that supports neural differentiation in vitro while ensuring compatibility with the technique of 3D bioprinting of a self-standing construct is a challenge.Methods: In this study, gelatin methacryloyl (GelMA), methacrylated alginate (AlgMA), and hyaluronic acid (HA) were examined by exploiting their biocompatibility and tunable mechanical properties to resemble the extracellular matrix (ECM) and to create a suitable material for printing neural progenitor cells (NPCs), supporting their long-term differentiation. NPCs were printed and differentiated for up to 15 days, and cell viability and neuronal differentiation markers were assessed throughout the culture.Results and Discussion: This composite biomaterial presented the desired physical properties to mimic the ECM of the brain with high water intake, low stiffness, and slow degradation while allowing the printing of defined structures. The viability rates were maintained at approximately 80% at all time points. However, the levels of β-III tubulin marker increased over time, demonstrating the compatibility of this biomaterial with neuronal cell culture and differentiation. Furthermore, these cells showed increased maturation with corresponding functional properties, which was also demonstrated by the formation of a neuronal network that was observed by recording spontaneous activity via Ca2+ imaging.

Background: Neurodegenerative diseases, such as Parkinson’s disease, are heterogeneous disorders with multifactorial nature involving impaired bioenergetics; that are on the rise with the increasing global population and average lifespan. Without definite therapeutic options, both stem regenerative medicine and bioenergetics have been proposed as promising therapeutic targets in the neurologic field. The rationale of the present study was to assess human derived adipose stem cells (hASC) potential to transdifferentiate into neuronal-like cells (NhASC and neurospheres), as well as to explore hASC bioenergetic profile. Methods: To this purpose, hASC derived from the forearm of both healthy controls and clinical diagnosed Parkinson’s disease patients (PD) were included in this study and transdifferentiated through neuronal induction. Results: Morphological, growth features and neuronal protein expression markers of differentiated neuron-like NhASC and neurospheres were achieved. Increased MAP-2 neuronal marker protein expression upon neuronal induction (pConclusions: We have confirmed the capability of transdifferentiation to neuronal-like profile of hASC derived from the forearem of human subjects and characterized the bioenergetic oxidative profile of hASC. Despite the latter did not lead to significant differential respiration profiles, trends to suboptimal maximal respiratory capacity in PD were found. The neuronal induction leading to positive neuronal protein expression markers is a relevant issue that encourages the suitability of the NhASC models in neurodegeneration.

Forkhead box (Fox) genes belong to the “winged helix” transcription factor superfamily. The function of some Fox genes is well known, such as the role of foxO in controlling metabolism and longevity and foxA in controlling differentiation of endodermal tissues. However, the role of some Fox factors is not yet well characterized. Such is the case of FoxK genes, which are mainly studied in mammals and have been implicated in diverse processes including cell proliferation, tissue differentiation and carcinogenesis. Planarians are free-living flatworms, whose importance in biomedical research lies in their regeneration capacity. Planarians possess a wide population of pluripotent adult stem cells, called neoblasts, which allow them to regenerate any body part after injury. In a recent study, we identified three foxK paralogs in the genome of Schmidtea mediterranea. In this study, we demonstrate that foxK1 inhibition prevents regeneration of the ectodermal tissues, including the nervous system and the epidermis. These results correlate with foxK1 expression in neoblasts and in neural progenitors. Although the triggering of wound genes expression, polarity reestablishment and proliferation was not affected after foxK1 silencing, the apoptotic response was decreased. Altogether, these results suggest that foxK1 would be required for differentiation and maintenance of ectodermal tissues.

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

[eng] Spinal cord injuries (SCI) are a major cause of paralysis in young adults. In this type of injuries recovery is impaired as adult central nervous system (CNS) axons fail to regenerate. This results from both a loss of intrinsic growing capacities in developmental axons when they mature, together with the presence of extrinsic factors hampering this regeneration, including a glial scar together with the production of growth-inhibitory molecules, as well as a lack of injury resolution leading to a chronic inflammation. Unfortunately, despite research efforts, current therapies for this type of injuries only lead to mild improvements and among them, activity-based therapies seem to raise above the others. Activity- based therapies try to induce recovery by increasing neuronal activity, however, a proper physiological and molecular characterization of the rationale behind their success is still missing. Neuronal activity has been described to regulate transcriptional and epigenetic mechanisms; moreover, it also alters neuronal secretion with an impact on cellular dialogues. These characteristics indicate neuronal activity may be modulating both of the CNS barriers for regeneration. During this doctoral thesis we aimed to explore the influence of neuronal activity on SCIs, hypothesizing specific neuronal activations were the principal responsible for success in activity-based therapies. Particularly, we studied the role of precise manipulations of neuronal activity, using optogenetic and chemogenetic tools, in axonal growth of stimulated neurons as well as the impact these activations could have on neuronal extrinsic signalling. Our results show that optogenetic and chemogenetic stimulations of neuronal activity enhanced growth in both regenerating and refractory to regenerate neurons. However, this growth was hampered by the inhibitory molecules present in the injured CNS and did not result in functional recovery in rodent models of SCI. Our data indicated that the growth induction in specifically stimulated neurons resulted from local adjustments rather than inducing a pro-regenerative transcriptional state, as seen by our gene expression analysis of regeneration-associated genes (RAGs). Altogether, our results suggest recovery in activity-based therapies derives from the summation of various forms of plasticity, induced by their simultaneous recruitment of several circuits. In parallel, we observed that these precise modulations of neuronal activity, while unable to alter the predominant environment after SCI, could initiate previously undescribed intricate cellular dialogues. Specifically, we found an increase in the chemokine CCL21 upon nociceptor activation which triggered the response of several cell types in the injury. In proprioceptors, this CCL21 was responsible for a growth induction after CCR7 activation, which required the MEK-ERK pathway as well as the modulation of the actin cytoskeleton. Meanwhile, the CCL21 interaction with CXCR3 in other cells effectively aborted this regeneration. All in all, our work reveals the existence of a complex plethora of synergic mechanisms, far from understood, contributing to the outcome of activity-based therapies and reinforces the need for further mechanistic studies which would allow the optimization of their success.

Activating and inhibitory immune receptors play a critical role in regulating systemic and central nervous system (CNS) immune and inflammatory processes. The CD200R1 immunoreceptor induces a restraining signal modulating inflammation and phagocytosis in the CNS under different inflammatory conditions. However, it remains unknown whether CD200R1 has a role in modulating the inflammatory response after a peripheral nerve injury, an essential component of the successful regeneration. Expression of CD200R1 and its ligand CD200 was analyzed during homeostasis and after a sciatic nerve crush injury in C57Bl/6 mice. The role of CD200R1 in Wallerian Degeneration (WD) and nerve regeneration was studied using a specific antibody against CD200R1 injected into the nerve at the time of injury. We found an upregulation of CD200R1 mRNA after injury whereas CD200 was downregulated acutely after nerve injury. Blockade of CD200R1 significantly reduced the acute entrance of both neutrophils and monocytes from blood after nerve injury. When long term regeneration and functional recovery were evaluated, we found that blockade of CD200R1 had a significant effect impairing the spontaneous functional recovery. Taken together, these results show that CD200R1 has a role in mounting a successful acute inflammatory reaction after injury, and contributes to an effective functional recovery.

The frequency-following response (FFR) is an auditory evoked potential (AEP) that follows the periodic characteristics of a sound. Despite being a widely studied biosignal in auditory neuroscience, the neural underpinnings of the FFR are still unclear. Traditionally, FFR was associated with subcortical activity, but recent evidence suggested cortical contributions which may be dependent on the stimulus frequency. We combined electroencephalography (EEG) with an inhibitory transcranial magnetic stimulation protocol, the continuous theta burst stimulation (cTBS), to disentangle the cortical contribution to the FFR elicited to stimuli of high and low frequency. We recorded FFR to the syllable /ba/ at two fundamental frequencies (Low: 113 Hz; High: 317 Hz) in healthy participants. FFR, cortical potentials, and auditory brainstem response (ABR) were recorded before and after real and sham cTBS in the right primary auditory cortex. Results showed that cTBS did not produce a significant change in the FFR recorded, in any of the frequencies. No effect was observed in the ABR and cortical potentials, despite the latter known contributions from the auditory cortex. Possible reasons behind the negative results include compensatory mechanisms from the non-targeted areas, intraindividual variability of the cTBS effectiveness, and the particular location of our target area, the primary auditory cortex.

Understanding the basic mechanisms of neural regeneration after injury is a pre-requisite for developing appropriate treatments. Traditional approaches to model axonal lesions, such as high intensity power laser ablation or sharp metal scratching, are complex to implement, have low throughputs, and generate cuts that are difficult to modulate. We present here a novel reproducible microfluidic approach to model in vitro mechanical lesion of tens to hundreds of axons simultaneously in a controlled manner. The dimensions of the induced axonal injury and its distance from the neuronal cell body are precisely controlled while preserving both the proximal and distal portions of axons. We have observed that distal axons undergo Wallerianlike anterograde degeneration after axotomy; in contrast, proximal portions of the axons remain un-degenerated, possessing the potential to re-grow. More importantly, surpassing the previous axotomy methods performed in petridish in which local microenvironments cannot be tailored, our platform holds the capability to implement fine-tuned treatments to lesioned axon stumps in a local, controlled manner. Specifically, molecules such as chondroitin sulphate proteoglycans and its degrading enzyme Chondroitinase ABC, hydrogels, and supporting cells have been shown to be deliverable to the lesioned site of injured axons. In addition, this system also permits double interventions at the level of the lesioned axons and the perikaryon. This proves the potentiality of our model by demonstrating how axonal regrowth can be evaluated under circumstances that are better mimicking biological problems. We believe that this novel mechanical microfluidic axotomy approach is easy to perform, yields high throughput axon lesions, is physiologically relevant, and offers a simplified platform for screening of potential new neurological drugs.

Diazoxide, a well-known mitochondrial KATP channel opener with neuroprotective effects, has been proposed for the effective and safe treatment of neuroinflammation. To test whether diazoxide affects the neurogenesis associated with excitotoxicity in brain injury, we induced lesions by injecting excitotoxic N-methyl-d-aspartate (NMDA) into the rat hippocampus and analyzed the effects of a daily oral administration of diazoxide on the induced lesion. Specific glial and neuronal staining showed that NMDA elicited a strong glial reaction associated with progressive neuronal loss in the whole hippocampal formation. Doublecortin immunohistochemistry and bromo-deoxyuridine (BrdU)-NeuN double immunohistochemistry revealed that NMDA also induced cell proliferation and neurogenesis in the lesioned non-neurogenic hippocampus. Furthermore, glial fibrillary acidic protein (GFAP)-positive cells in the injured hippocampus expressed transcription factor Sp8 indicating that the excitotoxic lesion elicited the migration of progenitors from the subventricular zone and/or the reprograming of reactive astrocytes. Diazoxide treatment attenuated the NMDA-induced hippocampal injury in rats, as demonstrated by decreases in the size of the lesion, neuronal loss and microglial reaction. Diazoxide also increased the number of BrdU/NeuN double-stained cells and elevated the number of Sp8-positive cells in the lesioned hippocampus. These results indicate a role for KATP channel activation in regulating excitotoxicity-induced neurogenesis in brain injury.

Glibenclamide is neuroprotective against cerebral ischemia in rats. We studied whether glibenclamide enhances long-term brain repair and improves behavioral recovery after stroke. Adult male Wistar rats were subjected to transient middle cerebral artery occlusion (MCAO) for 90 minutes. A low dose of glibenclamide (total 0.6 μg) was administered intravenously 6, 12, and 24 hours after reperfusion. We assessed behavioral outcome during a 30-day follow-up and animals were perfused for histological evaluation. In vitro specific binding of glibenclamide to microglia increased after pro-inflammatory stimuli. In vivo glibenclamide was associated with increased migration of doublecortin-positive cells in the striatum toward the ischemic lesion 72 hours after MCAO, and reactive microglia expressed sulfonylurea receptor 1 (SUR1) and Kir6.2 in the medial striatum. One month after MCAO, glibenclamide was also associated with increased number of NeuN-positive and 5-bromo-2-deoxyuridine-positive neurons in the cortex and hippocampus, and enhanced angiogenesis in the hippocampus. Consequently, glibenclamide-treated MCAO rats showed improved performance in the limb-placing test on postoperative days 22 to 29, and in the cylinder and water-maze test on postoperative day 29. Therefore, acute blockade of SUR1 by glibenclamide enhanced long-term brain repair in MCAO rats, which was associated with improved behavioral outcome.

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.

[spa] Las células de la glía envolvente olfatoria surgen hace algunos años como una estrategia prometedora para reparar la médula espinal lesionada. Sin embargo, diversas moléculas inhibitorias están presentes durante largo tiempo en la médula espinal lesionada siendo capaces de inhibir tanto la migración de las OECs como la regeneración axonal. Dos de estas familias de moléculas, los condroitín sulfato proteoglicanos (CSPGs) y las moléculas inhibitorias de la mielina (MAIs) son capaces de desencadenar respuestas inhibitorias redundantes en los axones. Aquí nosotros analizamos las propiedades de las MAIs y el CSPG en la migración de las OECs. Además, con tal de evitar el efecto inhibitorio de las MAIs se ha generado una línea estable de OECs capaces de sobreexpresar el ectodominio de Nogo Receptor (NgR). Los resultados indican que las células manipuladas genéticamente son capaces de migrar distancias más largas que las células no modificadas sobre las MAIs. Además, las células que expresan el ectodominio NgR también migran distancias más largas en la médula espinal lesionada. Complementariamente, hemos utilizado un sistema de microfluídica abierto con pocillos de cultivo de células para controlar con precisión microambientes neuronales, con tal de ser capaces de imitar el transporte axonal y la formación sináptica. Hemos demostrado que el uso de sistemas lab-on-a-chip para co-cultivos de motoneuronas con células C2C12, línea celular de miotubos, permite biomimetizar la unión neuromuscular. Además, mediante la integración de técnicas de microscopía en tiempo real de Ca+2 hemos demostrado funcionalidad de los NMJ en los chips a través de transientes de calcio inducidos por KCl en miotubos conectados. Esta plataforma puede potencialmente convertirse en una herramienta útil para análisis experimental in vitro para investigación básica de la NMJ., [eng] Olfactory ensheathing cell transplantation emerged some years ago as a promising therapeutical strategy to repair injured spinal cord. However, inhibitory molecules are present for long times in lesioned spinal cord inhibiting both OEC migration and axonal regrowth. Two of these families of molecules, chondroitin sulphate proteoglycans (CSPG) and myelinderived molecules (MAIs), are able to trigger redundant inhibitory responses in lesioned axons. Here we analysed the inhibitory properties of MAIs o CSPGs in OEC migration. In addition, to avoid MAIs associated inhibition we generated a stable OEC cell line overexpressing the Nogo Receptor (NgR) ectodomain. Results indicate that engineered cells migrate longer distances than unmodified OECs over MAIs-coated substrates. In addition, cells expressing the NgR ectodomain also migrate longer distances in lesioned spinal cord. Complementarily, we have utilized a microfluidic system with large open cell culture reservoirs to precisely control neuronal microenvironments, capable of mimicking axon transport and synapse formation and to facilitate their analysis. We demonstrate using this labon-a-chip system for long-term motoneuron co culture with C2C12-derived myotubes to mimic neuro-muscular junction (NMJ) formation. Furthermore, by integration with a calcium (Ca+2) imaging technique, we have proved the NMJ functionality in-chip through KCl-induced Ca+2 transient in connected myotubes. This platform can potentially become a useful tool as a straightforward, reproducible, and high-throughput in vitro model for basic NMJ research, and for high-throughput drug screening

As olfactory receptor axons grow from the peripheral to the central nervous system (CNS) aided by olfactory ensheathing cells (OECs), the transplantation of OECs has been suggested as a plausible therapy for spinal cord lesions. The problem with this hypothesis is that OECs do not represent a single homogeneous entity, but, instead, a functionally heterogeneous population that exhibits a variety of responses, including adhesion and repulsion during cell-matrix interactions. Some studies report that the migratory properties of OECs are compromised by inhibitory molecules and potentiated by chemical gradients. In this paper, we report a system based on modified OECs carrying magnetic nanoparticles as a proof of concept experiment enabling specific studies aimed at exploring the potential of OECs in the treatment of spinal cord injuries. Our studies have confirmed that magnetized OECs (i) survive well without exhibiting stress-associated cellular responses; (ii) in vitro, their migration can be modulated by magnetic fields; and (iii) their transplantation in organotypic slices of spinal cord and peripheral nerve showed positive integration in the model. Altogether, these findings indicate the therapeutic potential of magnetized OECs for CNS injuries.

Lesioned axons do not regenerate in the adult mammalian central nervous system, owing to the overexpression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3 (GSK3beta) and ERK1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3beta and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: i) cerebellar granule cells and ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3beta inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Lastly these regenerative effects were corroborated in the lesioned EHP in NgR1 -/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections. On the other hand, and focused in the OMgp, by using recording electrophysiological nano-devices we found that, OMgp has a role in synaptic transmission, since it can induce excitatory postsynaptic potentials (EPSPs) in cultured hippocampal neurons.

Planarian flatworms are an exception among bilaterians in that they possess a large pool of adult stem cells that enables them to promptly regenerate any part of their body, including the brain. Although known for two centuries for their remarkable regenerative capabilities, planarians have only recently emerged as an attractive model for studying regeneration and stem cell biology. This revival is due in part to the availability of a sequenced genome and the development of new technologies, such as RNA interference and next-generation sequencing, which facilitate studies of planarian regeneration at the molecular level. Here, we highlight why planarians are an exciting tool in the study of regeneration and its underlying stem cell biology in vivo, and discuss the potential promises and current limitations of this model organism for stem cell research and regenerative medicine.

Adult mammalian central nervous system (CNS) axons have a limited regrowth capacity following injury. Myelin-associated inhibitors (MAIs) limit axonal outgrowth and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo-A, MAG and OMgp, share two common neuronal receptors: NgR1, together with its co-receptors (p75(NTR), TROY and Lingo-1), and the recently described paired immunoglobulin-like receptor B (PirB). These proteins impair neuronal regeneration by limiting axonal sprouting. Some of the elements involved in the myelin inhibitory pathways may still be unknown, but the discovery that blocking both PirB and NgR1 activities leads to near-complete release from myelin inhibition, sheds light on one of the most competitive and intense fields of neuroregeneration study during in recent decades. In parallel with the identification and characterization of the roles and functions of these inhibitory molecules in axonal regeneration, data gathered in the field strongly suggest that most of these proteins have roles other than axonal growth inhibition. The discovery of a new group of interacting partners for myelin-associated receptors and ligands, as well as functional studies within or outside the CNS environment, highlights the potential new physiological roles for these proteins in processes such as development, neuronal homeostasis, plasticity and neurodegeneration.

Lesioned axons do not regenerate in the adult mammalian central nervous system, owing to the overexpression of inhibitory molecules such as myelin-derived proteins or chondroitin sulphate proteoglycans. In order to overcome axon inhibition, strategies based on extrinsic and intrinsic treatments have been developed. For myelin-associated inhibition, blockage with NEP1-40, receptor bodies or IN-1 antibodies has been used. In addition, endogenous blockage of cell signalling mechanisms induced by myelin-associated proteins is a potential tool for overcoming axon inhibitory signals. We examined the participation of glycogen synthase kinase 3 (GSK3) and ERK1/2 in axon regeneration failure in lesioned cortical neurons. We also investigated whether pharmacological blockage of GSK3 and ERK1/2 activities facilitates regeneration after myelin-directed inhibition in two models: i) cerebellar granule cells and ii) lesioned entorhino-hippocampal pathway in slice cultures, and whether the regenerative effects are mediated by Nogo Receptor 1 (NgR1). We demonstrate that, in contrast to ERK1/2 inhibition, the pharmacological treatment of GSK3 inhibition strongly facilitated regrowth of cerebellar granule neurons over myelin independently of NgR1. Lastly these regenerative effects were corroborated in the lesioned EHP in NgR1 -/- mutant mice. These results provide new findings for the development of new assays and strategies to enhance axon regeneration in injured cortical connections.

[spa] El potencial regenerativo del sistema nervioso central (SNC), a diferencia del sistema nervioso periférico (SNP), desaparece gradualmente conforme avanza el desarrollo, coincidiendo con el inicio de la mielinización. Hasta el momento, se ha descrito la existencia de tres proteínas asociadas a la mielina (MAIPs) que estarían implicadas en este proceso. Estas proteínas, Nogo-A, MAG y OMgp, se unen, de forma independiente, a un complejo receptor neuronal común formado por NgR1, p75 o TAJ-1/TROY y Lingo-1, y la activación de este complejo receptor desencadena una señalización intracelular que causa la pérdida de plasticidad en el SNC adulto y la inhibición de la regeneración axonal tras lesión. Sin embargo, los estudios publicados en el momento de iniciarse el presente trabajo sugerían que, tanto los ligandos como los componentes del complejo receptor, podrían ejercer otras funciones no relacionadas con la inhibición de la regeneración axonal en el adulto. Además, aunque se acababa de describir que Lingo-1 era miembro esencial del complejo receptor para las MAIPs, no se había demostrado si los tres componentes propuestos para formar el complejo receptor compartían el mismo patrón de expresión, ni tampoco si podían coexistir en el mismo tipo celular y compartimento. Por ello, nos propusimos analizar en detalle el patrón de expresión de Lingo-1 y de su distribución celular en relación al resto de componentes del complejo. Nuestros resultados muestran la coexistencia de Lingo-1, NgR1, p75 y TROY en el cerebro de ratón durante una ventana temporal postnatal muy definida. También hemos determinado la distribución de Lingo-1 en determinadas subpoblaciones neuronales. Sorprendentemente, Lingo-1 se expresa en estadios tempranos del desarrollo en los cuales no se expresa NgR1. Finalmente, mostramos que el dominio intracelular de Lingo-1 contribuye a la señalización y además interacciona con la proteína postmitótica neuronal Myt1l, sugiriendo que Lingo-1 puede regular la actividad de este factor de transcripción al afectar a su localización subcelular. Por otra parte, existían ciertas discrepancias en cuanto al tipo celular en el que se expresaba OMgp. Mientras algunos estudios sostenían una localización exclusiva en oligodendrocitos, otros proponían una expresión neuronal. Además, todos los estudios de expresión descritos hasta la fecha se centraban en estadios postnatales y adultos, pero no en estadios embrionarios. Así, uno de nuestros objetivos fue profundizar en la distribución celular y regional de las proteínas OMgp a lo largo del desarrollo del telencéfalo de ratón y determinar posibles funciones adicionales. Observamos que, a nivel celular, OMgp se localiza en membranas neuronales de dendritas y axones y también en fracciones sinaptosomales de cerebro y en estructuras similares a botones sinápticos. Además, la aparición de la inmunoreactividad en la corteza durante el desarrollo coincide con el establecimiento de la conexión tálamo-cortical y, por tanto, de la formación de los barriles (barrel-field) de la corteza. El análisis del barrel-field en ratones deficientes en OMgp revela que, aunque la conexión tálamo-cortical está formada, el destino final de numerosos axones talámicos está alterado ya que invaden ectópicamente las capas II-III de corteza en lugar de capa IV. Por último, algunos estudios habían descrito la implicación de algunas proteínas de la mielina, como Nogo-A, en diversas enfermedades neurodegenerativas como epilepsia del lóbulo temporal, esclerosis múltiple, esclerosis lateral amiotrófica y esquizofrenia. Estas evidencias llevaron a plantearnos la posibilidad de que la expresión de Nogo-A pudiera estar alterada en la enfermedad de Alzheimer (AD), situación patológica en la cual ocurre una gran pérdida celular y una significante reorganización sináptica en áreas relacionadas con el aprendizaje y la memoria. Por ello, nos propusimos analizar la expresión de Nogo-A en la formación hipocámpica humana en una situación de envejecimiento normal y en una situación de AD. Nuestros resultados indican que en AD, Nogo-A es sobreexpresado por neuronas hipocámpicas y aparece asociado a depósitos amiloideos en placas seniles. Estos resultados refuerzan la idea de que la expresión temprana de OMgp y Lingo-1 juega un importante papel durante el desarrollo y apuntan una participación de OMgp en el establecimiento de la conexión tálamo-cortical. Además, sugieren una posible participación de las proteínas asociadas a mielina, así como de los componentes del complejo receptor, en las respuestas asociadas al envejecimiento y en particular a enfermedades neurodegenerativas como AD.

Tesis del Departament de Química Orgànica de la Universitat de Barcelona (UB).-- Fecha de defensa 17-07-2007., [CAT] La recerca en el camp del desenvolupament de fàrmacs peptídics i peptidomimètics ha experimentat una evolució important durant els darrers anys. Els fàrmacs peptídics sovint presenten baixa disponibilitat oral, una vida mitjana curta, accions múltiples i àmplies fluctuacions en el seu comportament farmacocinètic. D’aquí la necessitat de desenvolupar pèptids modificats o peptidomimètics amb propietats que millorin aquests inconvenients. Una de les idees més interessants en aquest camp va ser el trasllat de la cadena lateral de l’àtom de carboni de l’aminoàcid a l’àtom de nitrogen de l’enllaç amida, tot conduint a oligòmers de glicines N substituïdes, compostos que van ser anomenats peptoides. Aquestes estructures presenten clars avantatges davant dels pèptids, com ara una estabilitat metabòlica més elevada que els fa resistents a la degradació per proteases. A més, es poden sintetitzar amb una gran variabilitat de grups funcionals, són més flexibles degut a l’absència de ponts d’hidrogen augmentant l’espai conformacional capaç d’interaccionar amb diferents dianes terapèutiques, i presenten un esquelet estructural molt apropiat per aplicar-hi estratègies combinatòries., L’absència de regeneració neuronal en el sistema nerviós central adult és un greu problema sanitari i social. Estudis recents indiquen que el bloqueig d’inhibidors endògens del recreixement axonal pot constituir una nova via per estimular la regeneració neuronal. En aquest sentit, el descobriment de la família de les semaforines (i els seus receptors: les neuropilines), els inhibidors més potents del creixement axonal, ofereix un ampli ventall de noves possibilitats experimentals i terapèutiques en regeneració neuronal. Les semaforines (SEMA) són una gran família de proteïnes secretades o associades a la membrana que actuen com a quimiorepel•lents o inhibidors dels axons, tot afectant la seva direcció de creixement, la seva fascicul•lació i ramificació així com la formació de la sinapsi., Fruit de la col•laboració amb altres grups d’investigació es va identificar, mitjançant el cribratge d’una quimioteca de peptoides, el peptoide SICHI com a inhibidor de la quimiorepulsió induïda per semaforines. La síntesi posterior d’aquest compost ens va conduir a la detecció de la formació de subproductes cíclics que impedien l’obtenció del peptoide desitjat amb rendiments de reacció acceptables. Per aquest motiu, es va plantejar la síntesi de SICHI mitjançant la combinació de monòmers d’N-alquilglicines. L’aplicació d’aquesta estratègia va obligar a desenvolupar una metodologia de síntesi dels mencionats monòmers. Es van desenvolupar un total de tres rutes sintètiques, utilitzant en cadascuna d’elles un grup protector diferent. Una vegada optimitzades, es va fer la síntesi de SICHI mitjançant l’acoblament d’aquests monòmers. El rendiment global de la síntesi va ser satisfactori, la qual cosa va permetre completar els estudis de funcionalitat d’aquest peptoide., En la segona part de la tesi, es va decidir sintetitzar una nova quimioteca optimitzada de 625 pentàmers d’N-alquilglicines i 20 mescles controlades, en fase sòlida i en format de rastreig posicional. La síntesi dels peptoides es va fer seguint l’estratègia del submonòmer, mitjançant cicles successius constituïts per una etapa d’acilació amb àcid bromoacètic i una etapa de desplaçament del bromoderivat corresponent per una amina primària. La innovació d’aquesta quimioteca radica en la utilització de la radiació de microones per accelerar les etapes de reacció. Per a les posicions de diversitat química es van escollir un total de 10 amines primàries, tenint en compte diferents criteris com hidrofobicitat, presència de grups aromàtics, possibilitat de formar ponts d’hidrogen, etc. D’acord amb el format del rastreig posicional, es va realitzar un estudi de reactivitat de les amines primàries utilitzades en les etapes d’introducció de diversitat per tal d’assegurar reactivitats similars entre elles. La quimioteca ha estat assajada fins al moment contra diverses dianes terapèutiques d’interés, amb resultats satisfactoris en l’àrea de la diabetis i el càncer., [EN] Peptoids are oligomers of N-substituted glycines residues that have been developed as peptidomimetics but with improved stability and better pharmacological properties. For these reasons, peptoids have also been subjected of application of combinatorial approaches to identify bioactive molecules., In this context, the screening of a peptoid library identified a peptoid called SICHI as a potent inhibitor of semaphorin induced chemorepulsion activity. Subseqüent synthesis of this peptoid drove to detection of cyclic byproducts avoiding peptoid obtention in acceptable yield. For that reason, we proposed SICHI’s synthesis through the combination of N-alkylglycine monomers. The application of this strategy forced us to develope a methodology of monomers synthesis. We developed three synthetic routes, using a different protecting group on each one. Once all of them were optimized, we synthesized SICHI by coupling those monomers. The yield was satisfactory, allowing us to complete functionality studies of this peptoid., In the second part of the thesis, we decided to synthesize a new optimized library of pentamers containing 625 compounds in 20 controlled mixtures, constructed on solid-phase by using the positional scanning format following the submonomer strategy. An important innovation was introduced by the application of microwave irradiation to accelerate the reaction steps. We selected 10 primary amines for diversity positions considering hydrofobic properties, presence of aromatic goups, possibility of hydrogen bridge formation, etc. As positional scanning requires, we developed a reactivity study of the primary amines used to insure similar reactivity between them. The library has been tested against different therapeutic targets, obtaining satisfactory results in the fields of diabetes and cancer.

The adult central nervous system (CNS) has a very little capability to regrow its connections after lesion. The lack of axonal regeneration is a multifactorial problem, which is mainly due to the presence of inhibitory molecules in the CNS. Two main families of inhibitors exist, that of the so-called myelin-associated inhibitors, and that of the chondoritin-sulfate proteoglycans (CSPG). The question this thesis aims to address is the role of myelin-associated inhibitors in the regeneration of cortical connections. To do so, the model we have used is the entorhino-hippocampal connection and the main questions addressed were i) characterization of the temporal expression of MAG, Nogo-A and NgR, ii) analysis of their regulation after lesion, and iii) study of the effect of their blockade on axonal regeneration.Our results demonstrate that developmental expression of the myelin-associated inhibitors MAG and Nogo-A temporally coincides with the loss of regeneration capability of the entorhino-hippocampal connection also known as perforant pathway (PP). Moreover, expression of NgR (the common receptor for MAG and Nogo-A) also matches regeneration loss. After lesioning of the PP, MAG is overexpressed by mature oligodendrocytes which experiment a process of glial reactivity. Similarly, Nogo-A is re-expressed by reactive astrocytes in the deafferented hippocampus. The regulation of both inhibitors following injury supports a direct implication in the preventing axonal regeneration. To confirm this implication, we performed the blockade of MAG and the inhibitory domains of Nogo-A in an in vitro model (entorhino-hippocampal organotypic cultures). Both the blockade of MAG with Neuraminidase and the blockade of Nogo-A with NEP1-40 induced regeneration of the PP. Moreover, Blockade of CSPG with ChABC proved to be efficient in the same model. However, combination of NEP1-40 and ChABC, in order to block simultaneously Nogo-A-induced and CSPG-induced inhibition, did not display additive effects, suggesting a convergence in the inhibition mechanisms employed by both inhibitors. Last, we addressed the possibility of delaying treatment delivery as a therapeutic approach. While we confirmed that PP keeps its capability to regenerate during at least five days after lesion, we also determined that ChABC-based treatments are greatly compromised and lose efficiency when treatment is delayed.In conclusion, we have characterized the developmental expression pattern of the inhibitors MAG and Nogo-A and their common receptor NgR. The expression of the three proteins is strongly regulated following lesion and the blockade of MAG and Nogo-A is proved to be successful to promote axonal regeneration. We also determined the convenience of combined treatments and the most successful delivery schedule. Our data provide an insight for the development of new strategies addressed to induce axonal regeneration of injured cortical connections.

Peripheral nerve injury is typically associated with long-term disturbances in sensory localization, despite nerve repair and regeneration. Here, we investigate the extent of correct reinnervation by back-labeling neuronal soma with fluorescent tracers applied in the target area before and after sciatic nerve injury and repair in the rat. The subpopulations of sensory or motor neurons that had regenerated their axons to either the tibial branch or the skin of the third hindlimb digit were calculated from the number of cell bodies labeled by the first and/or second tracer. Compared to the normal control side, 81% of the sensory and 66% of the motor tibial nerve cells regenerated their axons back to this nerve, while 22% of the afferent cells from the third digit reinnervated this digit. Corresponding percentages based on quantification of the surviving population on the experimental side showed 91%, 87%, and 56%, respectively. The results show that nerve injury followed by nerve repair by epineurial suture results in a high but variable amount of topographically correct regeneration, and that proportionally more neurons regenerate into the correct proximal nerve branch than into the correct innervation territory in the skin.

Objetivo Evaluar el efecto a largo plazo del trasplante de células de la glía envolvente (GE) del bulbo olfatorio tras lesión de la médula espinal. Material y método Se practicó una laminectomía dorsal T8, en 16 ratas adultas Sprague-Dawley, dejando al descubierto la médula espinal subyacente, la cual se bañó con rosa de Bengala durante 10 minutos, antes de lesionarla por iluminación con una fibra óptica acoplada a una lámpara halógena, durante 2,5 minutos. A la mitad de los animales se les inyectó 180.000 células de GE, en 10 μl de medio (grupo GE), y a la otra mitad sólo 10 μl de DMEM (Dulbecco's modified Eagle's medium) (grupo DM). Los animales se sacrificaron a los 90 días de efectuar la lesión y se evaluó el área de médula espinal preservada, la recuperación locomotora y la sensibilidad nociceptiva. Resultados Los animales del grupo GE mostraron un nivel de locomoción superior y retiraron antes la pata al estímulo nociceptivo que los del grupo DM. También hubo una mayor preservación de parénquima medular y más células p75 positivas en el grupo GE que en el DM. Conclusiones El trasplante de GE favorece la preservación de parénquima medular y evita la pérdida de funciones motoras y sensoriales en la rata.

Selective reinnervation of peripheral targets after nerve injury might be assessed by injecting a first tracer in a target before nerve injury to label the original neuronal population, and applying a second tracer after the regeneration period to label the regenerated population. However, altered uptake of tracer, fading, and cell death may interfere with the results. Furthermore, if the first tracer injected remains in the target tissue, available for 're-uptake' by misdirected regenerating axons, which originally innervated another region, then the identification of the original population would be confused. With the aim of studying this problem, the sciatic nerve of adult rats was sectioned and sutured. After 3 days, to allow the distal axon to degenerate avoiding immediate retrograde transport, one of the dyes: Fast Blue (FB), Fluoro-Gold (FG) or Diamidino Yellow (DY), was injected into the tibial branch of the sciatic nerve, or in the skin of one of the denervated digits. Rats survived 2-3 months. The results showed labelled dorsal root ganglion (DRG) cells and motoneurones, indicating that late re-uptake of a first tracer occurs. This phenomenon must be considered when the model of sequential labelling is used for studying the accuracy of peripheral reinnervation.

We present a method for using long-term organotypic slice co-cultures of the entorhino-hippocampal formation to analyze the axon-regenerative properties of a determined compound. The culture method is based on the membrane interphase method, which is easy to perform and is generally reproducible. The degree of axonal regeneration after treatment in lesioned cultures can be seen directly using green fluorescent protein (GFP) transgenic mice or by axon tracing and histological methods. Possible changes in cell morphology after pharmacological treatment can be determined easily by focal in vitro electroporation. The well-preserved cytoarchitectonics in the co-culture facilitate the analysis of identified cells or regenerating axons. The protocol takes up to a month.

Adult mammalian central nervous system (CNS) axons have very limited capacity of regrowth after injury. In recent years, advances in the field of axonal regeneration have proved that neurons do not regenerate, mainly because of the presence of inhibitory molecules. Myelin-associated proteins limit axonal outgrowth and their blockage improves the regeneration of damaged fiber tracts. Three of these proteins, Nogo, MAG and OMgp, share a common neuronal receptor (NgR), and together represent one of the main hindrances to neuronal regeneration. The recent molecular cloning of Nogo and its receptors opened a new door to the study of axon regeneration. However, many of the elements involved in the myelin inhibitory pathway are still unknown, and the preliminary experiments with knockout mice are rather contradictory. Because of this complexity, Nogo and NgR need to be characterized before precise strategies to promote axon regeneration in the CNS can be designed.

In the current global climate change scenario, stressors overlap in space and time, and knowledge on the effects of their interaction is highly needed to understand and predict the response and resilience of organisms. Corals, among many other benthic organisms, are affected by an increasing number of global change-related stressors including warming and invasive species. In this study, the cumulative effects between warming and invasive algae were experimentally assessed on the temperate reef-builder coral Cladocora caespitosa. We first investigated the potential local adaptation to thermal stress in two distant populations subjected to contrasting thermal and necrosis histories. No significant differences were found between populations. Colonies from both populations suffered no necrosis after long-term exposure to temperatures up to 29 °C. Second, we tested the effects of the interaction of both warming and the presence of invasive algae. The combined exposure triggered critical synergistic effects on photosynthetic efficiency and tissue necrosis. At the end of the experiment, over 90% of the colonies subjected to warming and invasive algae showed signs of necrosis. The results are of particular concern when considering the predicted increase of extreme climatic events and the spread of invasive species in the Mediterranean and other seas in the future.