Regenerating cortical connections in a dish: the entorhino-hippocampal organotypic slice co-culture as tool for pharmacological screening of molecules promoting axon regeneration

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

Nogo, myelin and axonal regeneration

El sistema nerviós dels mamífers té una baixa capacitat de reparació axonal després d'una lesió. En els últims anys, diversos estudis han demostrat que els axons lesionats no poden recréixer a causa de la presència d'un gran nombre de molècules inhibitòries. Les molècules associades a la mielina limiten el creixement axonal i el seu bloqueig afavoreix la regeneració de diverses connexions. Tres d'aquestes proteïnes, Nogo, MAG i OMgp, comparteixen un mateix receptor: NgR. El clonatge recent de Nogo ha obert noves vies per estudiar la regeneració axonal. No obstant això, molts dels elements involucrats en la via inhibitòria de la mielina són desconeguts, i els primers estudis amb animals knockout són, a més, contradictoris. Per aquesta raó, Nogo i el seu receptor han de caracteritzar-se abans de desenvolupar noves tècniques per promoure regeneració axonal.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

TrkB signaling is required for postnatal survival of CNS neurons and protects hippocampal and motor neurons from axotomy-induced cell death

Newborn mice carrying targeted mutations in genes encoding neurotrophins or their signaling Trk receptors display severe neuronal deficits in the peripheral nervous system but not in the CNS. In this study, we show that trkB (¿/¿) mice have a significant increase in apoptotic cell death in different regions of the brain during early postnatal life. The most affected region in the brain is the dentate gyrus of the hippocampus, although elevated levels of pyknotic nuclei were also detected in cortical layers II and III and V and VI, the striatum, and the thalamus. Furthermore, axotomized hippocampal and motor neurons of trkB (¿/¿) mice have significantly lower survival rates than those of wild-type littermates. These results suggest that neurotrophin signaling through TrkB receptors plays a role in the survival of CNS neurons during postnatal development. Moreover, they indicate that TrkB receptor signaling protects subpopulations of CNS neurons from injury- and axotomy-induced cell death

Distinct X-chromosome SNVs from some sporadic AD samples

Sporadic Alzheimer disease (SAD) is the most prevalent neurodegenerative disorder. With the development of new generation DNA sequencing technologies, additional genetic risk factors have been described. Here we used various methods to process DNA sequencing data in order to gain further insight into this important disease. We have sequenced the exomes of brain samples from SAD patients and non-demented controls. Using either method, we found a higher number of single nucleotide variants (SNVs), from SAD patients, in genes present at the X chromosome. Using the most stringent method, we validated these variants by Sanger sequencing. Two of these gene variants, were found in loci related to the ubiquitin pathway (UBE2NL and ATXN3L), previously do not described as genetic risk factors for SAD

Regulation of patterned dynamics of local exocytosis in growth cones by netrin-1

Axonal guidance and synaptic specification depends on specific signaling mechanisms that occur in growth cones. While several signaling pathways implicated in cone navigation have been identified, membrane dynamics in growth cones remains largely unknown. We took advantage of SynaptopHluorin and high-speed optical recordings to monitor the patterns of membrane dynamics in rat hippocampal growth cones. We show that exocytosis occurs both at the peripheral and central domains, including filopodia, and that SynaptopHluorin signals occur as spontaneous patterned peaks. Such transients average approximately two per minute and last ∼30 s. We also demonstrate that the chemoattractant Netrin-1 elicits increases in the frequency and slopes of these transients, with peaks averaging up to six per minute in the peripheral domain. Netrin-1-dependent regulation of exocytotic events requires the activation of the Erk1/2 and SFK pathways. Furthermore, we show that domains with high SynaptopHluorin signals correlate with high local calcium concentrations and that local, spontaneous calcium increases are associated with higher SynaptopHluorin signals. These findings demonstrate highly stereotyped, spontaneous transients of local exocytosis in growth cones and that these transients are positively regulated by chemoattractant molecules such as Netrin-1

Nystatin Regulates Axonal Extension and Regeneration by Modifying the Levels of Nitric Oxide

Nystatin is a pharmacological agent commonly used for the treatment of oral, mucosal and cutaneous fungal infections. Nystatin has also been extensively applied to study the cellular function of cholesterol-enriched structures because of its ability to bind and extract cholesterol from mammalian membranes. In neurons, cholesterol level is tightly regulated, being essential for synapse and dendrite formation, and for axonal guidance. However, the action of Nystatin on axon regeneration has been poorly evaluated. Here, we examine the effect of Nystatin on primary cultures of hippocampal neurons, showing how acute dose (minutes) of Nystatin increases the area of growth cones, and chronic treatment (days) enhances axon length, axon branching and axon regeneration post-axotomy. We describe two alternative signaling pathways responsible for the observed effects, and activated at different concentrations of Nystatin. At elevated concentrations, Nystatin promotes growth cone expansion through phosphorylation of Akt; whereas at low concentrations, Nystatin enhances axon length and regrowth by increasing nitric oxide levels. Together, our findings indicate new signaling pathways of Nystatin and propose this compound as a novel regulator of axon regeneration

Validation of suspected somatic Single Nucleotide Variations in the brain of Alzheimer disease patients

Next-generation sequencing techniques and genome-wide association study analyses have provided a huge amount of data, thereby enabling the identification of DNA variations and mutations related to disease pathogenesis. New techniques and software tools have been developed to improve the accuracy and reliability of this identification. Most of these tools have been designed to discover and validate single nucleotide variants (SNVs). However, in addition to germ-line mutations, human tissues bear genomic mosaicism, which implies that somatic events are present only in low percentages of cells within a given tissue, thereby hindering the validation of these variations using standard genetic tools. Here we propose a new method to validate some of these somatic mutations. We combine a recently developed software with a method that cuts DNA by using restriction enzymes at the sites of the variation. The non-cleaved molecules, which bear the SNV, can then be amplified and sequenced using Sanger's technique. This procedure, which allows the detection of alternative alleles present in as few as 10% of cells, could be of value for the identification and validation of low frequency somatic events in a variety of tissues and diseases

The Eutherian Armcx genes regulate mitochondrial trafficking in neurons and interact with Miro and Trak2

This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivative Works 3.0 Unported License.-- et al.Brain function requires neuronal activity-dependent energy consumption. Neuronal energy supply is controlled by molecular mechanisms that regulate mitochondrial dynamics, including Kinesin motors and Mitofusins, Miro1-2 and Trak2 proteins. Here we show a new protein family that localizes to the mitochondria and controls mitochondrial dynamics. This family of proteins is encoded by an array of armadillo (Arm) repeat-containing genes located on the X chromosome. The Armcx cluster is unique to Eutherian mammals and evolved from a single ancestor gene (Armc10). We show that these genes are highly expressed in the developing and adult nervous system. Furthermore, we demonstrate that Armcx3 expression levels regulate mitochondrial dynamics and trafficking in neurons, and that Alex3 interacts with the Kinesin/Miro/Trak2 complex in a Ca 2+ -dependent manner. Our data provide evidence of a new Eutherian-specific family of mitochondrial proteins that controls mitochondrial dynamics and indicate that this key process is differentially regulated in the brain of higher vertebrates. © 2012 Macmillan Publishers Limited. All rights reserved.This project was supported by grant BFU2008-3980 (Ministerio de Ciencia e Innovación (MICINN), Spain) and grants from the 'Marató de TV3' and 'Caixa Catalunya-Obra Social' Foundations to E. S., by grant BFU2011-23921 (MICINN) and the ICREA Academia Prize (Generalitat de Catalunya) to J.G.-F., by grants PI07/0715 (Fondo de Investigación Sanitaria) to F. B., by grant SAF2008-03514 to R. T. and by grants from the EU-ERA-Net program and MICINN (SAF2008-03175-E, BFU2010-17379 and Red de Terapia Celular, RD06/0010/0000) to J.G.-S. I. S. holds an IEF Postdoctoral fellowship funded by the Marie Curie FP7 'People' Programme.Peer Reviewe

Similarities and differences between exome sequences found in a variety of tissues from the same individual

DNA is the most stable nucleic acid and most important store of genetic information. DNA sequences are conserved in virtually all the cells of a multicellular organism. To analyze the sequences of various individuals with distinct pathological disorders, DNA is routinely isolated from blood, independently of the tissue that is the target of the disease. This approach has proven useful for the identification of familial diseases where mutations are present in parental germinal cells. With the capacity to compare DNA sequences from distinct tissues or cells, present technology can be used to study whether DNA sequences in tissues are invariant. Here we explored the presence of specific SNVs (Single Nucleotide Variations) in various tissues of the same individual. We tested for the presence of tissue-specific exonic SNVs, taking blood exome as a control. We analyzed the chromosomal location of these SNVs. The number of SNVs per chromosome was found not to depend on chromosome length, but mainly on the number of protein-coding genes per chromosome. Although similar but not identical patterns of chromosomal distribution of tissue-specific SNVs were found, clear differences were detected. This observation supports the notion that each tissue has a specific SNV exome signature

Tareas de la asignatura de climatología utilizando el programa Walking-Weather (WW)

[EN] New technologies allow the use of applicable tools in different areas of knowledge adapted to the educational needs of the subject (Domingo, et al., 2011). In this sense can be applied to tasks and work done by the students, such as measures to demonstrate the existence of heat islands in the cities, or differences in temperature or other climatic variables depending on exposure. The measurements of climatic variables on tours made with different instrumentation and accompanied by their location are transferred to the platform where you can visualize the path of the student and the places where there have been measures. The tool is a technological resource to support active learning and to be included in a web environment favors the existence of an active space of easy consultation for any discipline and specifically for the implementation of case studies in climatology. Our goal is to facilitate the learning process based on the case method applied to measures of climate variables and using the WW platform.[ES] Las nuevas tecnologías permiten el uso de herramientas aplicables en diferentes áreas de conocimiento adaptándolas a las necesidades docentes de la asignatura (Domingo, et al., 2011). En este sentido se pueden aplicar a tareas y trabajos realizados por los alumnos, como son las medidas para la demostración de la existencia de las islas de calor en las ciudades, o las diferencias de temperatura o de otras variables climáticas en función de la exposición. Las medidas de variables climáticas en los recorridos realizadas con diferente instrumentación y acompañada de su localización son transferidas a la plataforma donde se puede visualizar el recorrido del alumno y los lugares donde se han realizado las medidas.La herramienta es un recurso tecnológico de apoyo al aprendizaje activo y al incluirse en un entorno web, favorece la existencia de un espacio activo de fácil consulta para cualquier disciplina y concretamente para la aplicación de case studies en climatología.Nuestro objetivo es facilitar el proceso de aprendizaje basado en el método del caso aplicado a medidas de las variables climáticas y utilizando la plataforma WW.Soriano Soto, MD.; García España, L.; Blasco, E. (2015). Tareas de la asignatura de climatología utilizando el programa Walking-Weather (WW). En In-Red 2015 - CONGRESO NACIONAL DE INNOVACIÓN EDUCATIVA Y DE DOCENCIA EN RED. Editorial Universitat Politècnica de València. https://doi.org/10.4995/INRED2015.2015.1573OC