IKAP/Elp1 Is Required In Vivo for Neurogenesis and Neuronal Survival, but Not for Neural Crest Migration

Familial Dysautonomia (FD; Hereditary Sensory Autonomic Neuropathy; HSAN III) manifests from a failure in development of the peripheral sensory and autonomic nervous systems. The disease results from a point mutation in the IKBKAP gene, which encodes the IKAP protein, whose function is still unresolved in the developing nervous system. Since the neurons most severely depleted in the disease derive from the neural crest, and in light of data identifying a role for IKAP in cell motility and migration, it has been suggested that FD results from a disruption in neural crest migration. To determine the function of IKAP during development of the nervous system, we (1) first determined the spatial-temporal pattern of IKAP expression in the developing peripheral nervous system, from the onset of neural crest migration through the period of programmed cell death in the dorsal root ganglia, and (2) using RNAi, reduced expression of IKBKAP mRNA in the neural crest lineage throughout the process of dorsal root ganglia (DRG) development in chick embryos in ovo. Here we demonstrate that IKAP is not expressed by neural crest cells and instead is expressed as neurons differentiate both in the CNS and PNS, thus the devastation of the PNS in FD could not be due to disruptions in neural crest motility or migration. In addition, we show that alterations in the levels of IKAP, through both gain and loss of function studies, perturbs neuronal polarity, neuronal differentiation and survival. Thus IKAP plays pleiotropic roles in both the peripheral and central nervous systems

Connections : safe spaces for women and youth in Latin America and The Caribbean

RESUMEN: Este libro se puede leer en muchos niveles. Uno de ellos puede no ser muy obvio para aquellos que están acostumbrados a leer sobre violencia e inseguridad en América Latina. Es el nivel que le da a este libro un estatus de originalidad y una contribución que va más allá de la región: el ser una forma de conocimiento destinada no solo a interpretar el mundo, sino a cambiarlo […], visibiliza la importancia de un proceso de investigación ajustado al tipo de conocimiento que produce. Aquí se conectan el proceso y el resultado, lo que debería propiciar un debate más amplio con respecto a cómo y qué sabemos de la naturaleza de la violencia y la agencia social para reducirla […]. Esta visión es particularmente relevante en contextos donde el Estado reproduce la violencia, con terribles impactos, en especial en periferias excluidas. […] El proceso de investigación abordado en este libro transgredió muchas fronteras. Hubo fronteras entre países, barreras lingüísticas, fronteras en torno a la educación, el conocimiento y la experiencia, y entre etnias, géneros y generaciones. […] este proceso reunió a académicos, activistas y líderes comunitarios de cinco países de América Latina y uno del Caribe, incluyendo comunidades indígenas en México y Guatemala […]. La violencia está en el tiempo y en el espacio y se reproduce entre las generaciones en diversos espacios de socialización. Este proceso de investigación que trasciende las fronteras, plantea una discusión que atraviesa los diferentes casos sobre cómo los déficits y las desigualdades materiales, las violencias estatales en nombre de la ‘seguridad’, las especificidades culturales, de género y generacionales de la experiencia y la comprensión de la violencia, así como las diversas formas de criminalidad, se cruzan y se reproducen a través del tiempo y el espacio. Jenny Pearce, investigadora y profesora en el Latin American and Caribbean Centre (LACC) de la London School of Economics and Political ScienceABSTRACT: This book can be read on many levels. One level may not be so obvious to those who are used to reading about violence and insecurity in Latin America. It is the level which gives this book a claim to true originality and a contribution beyond the region. This contribution is to form of scholarship aimed not only to interpret the world but to change it […], this text visibilizes the significance of the research process to the kind of knowledge that is produced. It connects process and outcome, and this should start a wider debate about how as well as what we know about the nature of violence and the social agency to reduce it […]. This is particularly relevant in contexts where the State reproduces violence, with terrible impacts on the margins. The research process discussed in this book transgressed many boundaries. There were intercountry borders, linguistic barriers, boundaries around education, knowledge and experience and between ethnicities, genders and generations. […] the research process brought together scholars and community activists and actors from five Latin American and one Caribbean country. And within Latin America there were indigenous communities in Mexico and Guatemala who participated […]. Violence is located in time and space. It is reproduced inter-generationally through varied socialisation spaces. The boundary crossing research process, raises cross case discussion about how material deficits and inequalities, state violences in the name of ‘security’, cultural, gender and generational specificities of experience and understanding of violence, and varied forms of criminality, intersect and reproduce through time and space. Professor Jenny Pearce. Latin American and Caribbean Centre (LACC), London School of Economics and Political Scienc

Nociceptive sensory neurons derive from contralaterally migrating, fate-restricted neural crest cells

Neural crest cells (NCCs) are a transient population of multipotent progenitors that give rise to numerous cell types in the embryo. An unresolved issue is the degree to which the fate of NCCs is specified prior to their emigration from the neural tube. In chick embryos, we identified a subpopulation of NCCs that, upon delamination, crossed the dorsal midline to colonize spatially discrete regions of the contralateral dorsal root ganglia (DRG), where they later gave rise to nearly half of the nociceptor sensory neuron population. Our data indicate that before emigration, this NCC subset is phenotypically distinct, with an intrinsic lineage potential that differs from its temporally synchronized, but ipsilaterally migrating, cohort. These findings not only identify a major source of progenitor cells for the pain- and temperature-sensing afferents, but also reveal a previously unknown migratory pathway for sensory-fated NCCs that requires the capacity to cross the embryonic midline

IKAP is expressed in PNS and CNS neurons, but not in neural crest cells.

<p>Chick embryo sections were immunolabeled with commercial anti-IKAP antibody (Santa Cruz H302; red, A–I and with either HNK-1 (A; to label neural crest cells, green) or Tuj-1 (to label neurons; B). IKAP protein (red, A–I) is not expressed in HNK+ neural crest cells (green, A), but is expressed in nascent neurons in the DRG anlagen (A–C, E, arrow) and neural tube (B–C, E; arrowhead), and its expression is maintained through the period of programmed cell death (G–I). IKAP is also expressed in progenitor cells in the ventricular zone (D,J open arrowheads) and in motor neurons (arrowhead) and axons (arrow) (F–H,J,K). IKAP is also strongly expressed on the pre-ganglionic Column of Terni cells (arrow, H). In situ hybridization for IKBKAP confirms the protein expression pattern (J,K).</p

IKBKAP shRNAs reduce levels of IKAP protein and IKBKAP mRNA <i>in ovo</i>.

<p>(A) Schematic combines the known partial chicken IKBKAP sequence with the human IKBKAP sequence and indicates the regions of chicken IKBKAP to which shRNAs were designed and the regions used as the immunogens for generation of both antibodies (P1D8 and Santa Cruz H301). The cytoplasmic terminus of the chicken IKAP was used for the CT-IKAP His construct overexpression experiments. QPCR indicated that approximately 50% of the IKBKAP mRNA was reduced following a 24 hr transfection <i>in ovo</i> with the IKBKAP shRNAs, when compared to control levels of IKBKAP (B). In situ hybridization following IKBKAP shRNAs transfection <i>in ovo</i> also showed reduced levels of IKBKAP mRNA (C) in the transfected half of the spinal cord (arrow); sense probe (D). Transfection <i>in ovo</i> with IKBKAP shRNAs reduced levels of IKAP protein (E–H) on the transfected side only (compare the control half to the EGFP+ half of the spinal cord).</p

Reductions in IKAP decreases proliferation of progenitors and increases cell death in the developing neural tube.

<p>Embryos were transfected/electroporated with IKBKAP shRNAs (A,B) or control shRNAs (C,D) at St. 13 and fixed at St. 23 (ca 48 hrs). Reductions in IKAP increase the death of CNS progenitors as indicated by cleaved-caspase 3+ (A–D; vertical line indicates the midline). (E–H) The number of pH3+ cells was determined in the ventricular zone on the transfected and control sides of each embryo and a ratio for each embryo obtained. The graph depicts those ratios from embryos transfected with either control shRNA or IKBKAP shRNA or IKBKAP shRNA and a mouse IKBKAP cDNA. There are significantly fewer pH3+ cells in IKBKAP shRNA transfected embryos (n = 4 embryos, 3300 cells counted) than in control shRNA transfected embryos (n = 3 embryos, 4,331 cells counted; p = 0.004), an effect that is rescued (red bar) with co-transfection with a plasmid encoding full length mouse IKBKAP (E; n = 3 embryos, 2153 cells counted; p = 0.005 by ANOVA). There is no significant difference between the number of pH3+ cells in the rescued embryos vs. the control shRNA transfected embryos (p = 0.103).</p

Confirmation of IKAP expression with an anti-chick IKAP antibody.

<p>(A) Generation of a monoclonal antibody to chicken IKAP, P1D8, confirms the expression pattern obtained with anti-human IKAP commercial antibody (H302). Both antibodies demonstrate that the majority of IKAP is in the cytoplasm, primarily perinuclear, and in axons (A, C–E) both <i>in vivo</i> (A, C) and in DRG <i>in vitro</i> (D,E). Incubation with the peptide immunogen used to generate P1D8 completely blocks labeling (compare B to C). Both IKAP antibodies label a band of approximately 140 kDa in lysates of chick embryo spinal cord (F).</p

Medición de la actividad física por medio del IPAQ corto versión Colombia de los estudiantes del primer semestre de la Universidad San Buenaventura Medellín, 2019-1

He investigation consisted of a cross-sectional analysis in which it was intended to examine the levels of physical activity of the population who entered the first semester of the different programs offered by the University of San Buenaventura sectional Medellin by means of the questionnaire short IPAQ validated version for Colombia. The tests were carried out in the spaces arranged by the university in the week of induction of the year 2019-1. The participants, all of them of age, were informed of the objective of the investigation, had a brief description of how the questionnaire was filled by the investigators and signed an informed consent authorizing the management of the Information. The project was endorsed by the Bioethical Committee of the Universidad San Buenaventura MedellínLa investigación consto de un análisis de corte transversal en el cual se pretendió examinar los niveles de actividad física de la población que ingresó a cursar primer semestre de los diferentes programas ofrecidos por la Universidad de San Buenaventura seccional Medellín por medio del cuestionario corto IPAQ versión validada para Colombia. Las pruebas se llevaron a cabo en los espacios dispuestos por parte de la universidad en la semana de inducciones del año 2019-1. Los participantes, todos mayores de edad, fueron informados del objetivo de la investigación, tuvieron una breve descripción de cómo se llenaba el cuestionario por parte de los investigadores y firmaron un consentimiento informado autorizando el manejo de la información. El proyecto fue avalado por el comité bioético de la Universidad San Buenaventura Medellí

Reductions in IKAP alter neural tube development and induce ectopic branching of motor axons.

<p>All transfected cells (control shRNA transfected embryos in A, H, I; IKBKAP shRNA in B–G) are EGFP+; right side of spinal cord is transfected in all embryos (asterisk). (A) control shRNA transfected spinal cord develops normally in contrast to (B–D) IKBKAP shRNA transfected spinal cords in which cells at the ventricular zone (VZ) often evaginate into the neural tube lumen rather than migrate laterally away from the VZ as in (A). IKBKAP shRNA-transfected evaginated cells typically express neural markers such as Ben (C: box in B contains the Ben+ evaginated neurons in the VZ that are EGFP+ and magnified in C) and Islet1/2 (D). Spinal interneuron populations are reduced in IKBKAP shRNA-transfected spinal cords including Engrailed+ cells (E) and the dorsal population of Islet1+ cells – compare the left, non-transfected side of the spinal cords to the right, transfected sides (*asterisk) of the spinal cord. (F, I) The ratio of islet 1+ dorsal interneurons on the transfected vs. non-transfected sides of the spinal cords was determined in multiple sections from 2 control shRNA transfected embryos (n = 263 Islet+ cells for the control shRNA transfected side vs. n = 252 Islet+ cells for the non-transfected side) and in 4 IKBKAP shRNA transfected embryos (n = 356 Islet+ cells for the transfected side of the IKBKAP shRNA spinal cord vs. n = 658 Islet+ cells for the non-transfected sides of the IKBKAP shRNA transfected embryos); p = 0.006. (G, H) IKBKAP shRNA transfected motor axons branch abnormally (arrow in G) as they exit the neural tube in the ventral root (compare H to I).; vertical line indicates spinal cord midline.</p