Disfunción de las células precursoras dérmicas asociada al envejecimiento cutáneo: SOX2 como regulador del destino del linaje celular

204 p.Durante el envejecimiento se produce una disminución funcional progresiva de los tejidos, a la cual subyace la pérdida del potencial regenerativo por alteraciones en el funcionamiento de las células madre/precursoras residentes del tejido. En la piel, una población de células precursoras dérmicas es trazada por la expresión de Sox2, conocido por su función en el mantenimiento de las células madre. En este tejido, los niveles de Sox2 modulan la transición entre diferentes estadios celulares gliales y perivasculares con capacidad multipotente. En este trabajo hipotetizamos que la disminución funcional dérmica que ocurre en el envejecimiento es consecuencia de alteraciones en las poblaciones de células precursoras Sox2+. Utilizando los modelos animales C57BL/6J y Sox2EGFP envejecidos cronológicamente, hemos encontrado alteraciones en los nichos dérmicos nervioso y vascular, en conjunto con una disminución in vitro en la capacidad proliferativa, en el potencial de diferenciación, y un aumento de la senescencia celular. Más aún, la cantidad total de células precursoras Sox2+ disminuye con la edad, pero esta pérdida es acusada en la fracción celular con altos niveles de expresión de Sox2, mientras que aquella con bajos niveles de expresión parece estar menos afectada por el envejecimiento. Estos cambios, producto de alteraciones proliferativas selectivas, afectan al potencial de diferenciación hacia los linajes neural y mesodérmico. De hecho, en el animal heterocigoto Sox2EGFP hay un mayor compromiso adiposo, incluso desde edades jóvenes, que es concomitante con una disminución de la celularidad, resultando en alteraciones tempranas de la estructura de la matriz extracelular dérmica. Los resultados de este trabajo proponen la importancia de Sox2 en el mantenimiento de la homeostasis dérmica durante el envejecimiento, a través de la regulación de diversas poblaciones celulares en la piel

Efecto del déficit de GDAP1 en motoneuronas aisladas de un modelo murino asociado a la neuropatía Charcot-Marie-Tooth

[EN] Charcot-Marie-Tooth (CMT) is the most frequently inherited peripheral neuropathy with highly genetic and clinic heterogeneity. It is classified in two main groups: axonal and demyelinating, both been related with at least 40 genes. Our interest is focused in the forms of CMT disease caused by mutations in GDAP1 (Ganglioside-induced associated protein 1): autosomal recessive demyelinating (CMT4A), autosomal recessive axonal (AR-CMT2K) and dominant axonal (CMT2K). The protein GDAP1 is located in the mitochondrial outer membrane and has being related to mitochondrial dynamics. This study is focused on the effect caused by deficiency of GDAP1 in primary cell cultures of embryonic motorneurons (MN) from a murine model of CMT (Gdap1-/-). Results suggest that the absence of GDAP1 has no effect on MN cell morphology, but important changes in mitochondrial structure and other mitochondrial parameters were observed.[ES] Charcot-Marie-Tooth (CMT) es la neuropatía periférica de tipo sensitivo-motora hereditaria más frecuente, con gran heterogeneidad clínica y genética. Se presenta en formas desmielinizantes o axonales, estando relacionadas a más de 40 genes. Son de nuestro interés las formas desmielinizantes autosómicas recesivas (CMT4A), axonales autosómicas recesivas (AR-CMT2K) y axonales dominantes (CMT2K), ya que son las causadas por mutaciones en GDAP1 (Ganglioside-induced associated protein 1). La proteína GDAP1 se encuentra en la membrana mitocondrial externa y está relacionada con la dinámica de este organelo. El presente estudio se centra en el efecto de la deficiencia de GDAP1 en cultivos primarios de motoneuronas (MN) embrionarias, provenientes de un modelo murino de CMT (Gdap1-/-). Nuestros resultados sugieren que la ausencia de GDAP1 no tiene efectos sobre la morfología celular de las MN, pero produce cambios importantes en la estructura de la mitocondria y en otros parámetros mitocondriales.Yndriago Acha, L. (2014). Efecto del déficit de GDAP1 en motoneuronas aisladas de un modelo murino asociado a la neuropatía Charcot-Marie-Tooth. http://hdl.handle.net/10251/51484Archivo delegad

Generation of <i>Gdap1</i>^<i>-/-</i> mice.

<p><b>(A)</b> Schematic representation of <i>Gdap1</i>^<i>-/-</i> targeting strategy. Diagram is not to scale. Hatched rectangles represent <i>Gdap1</i> exons 1 to 6, solid line represents mouse chromosome 1. FRT sites are represented by double triangles and <i>lox</i>P sites are right-faced triangles. <b>(B)</b> GDAP1 protein expression was assessed by immunoblotting of selected tissue homogenates prepared from 2 months-old wild-type (WT), <i>Gdap1</i>^<i>+/-</i> (+/-) and <i>Gdap1</i>^-/- (-/-) mice.</p

Mitochondrial quantitation and network distribution in cultured MNs and sciatic nerves from WT and <i>Gdap1</i>^<i>-/-</i> mice.

<p>Number of mitochondria <b>(A)</b> and network interconnectivity <b>(B)</b> in cultured MNs are represented. The study was performed in the proximal segments (p) and distal segments (d) of WT (black bars) and <i>Gdap1</i>^<i>-/-</i> (gray bars) axons after 24 hour and 48 hour of cell culture. Error bars represent S.E.M. Student’s <i>t</i> test *p<0.05, **p<0.01 and ***p<0.001 <b>(C)</b> Left panel shows semi-thin cross sections of the sciatic nerve from five months old WT and <i>Gdap1</i>^-/- mice. Mitochondria are clearly visible on higher magnification images of transversal section (right panel). Mitochondrial axonal content was quantified by electron microscopy on proximal and distal cross sections of the sciatic nerve. (n = 4; Error bars represent S.E.M.; asterisks indicate significant differences between WT and <i>Gdap1</i>^<i>-/-</i> mice, Mann-Whitney test, **p<0.01,***p<0.001). <b>(D)</b> Measurement of mitochondrial DNA (mtDNA) copy number in sciatic nerves.</p

Postranscriptional modification of the tubulin cytoskeleton in primary sensory and motor neuron cultures.

<p><b>(A)</b> DRG sensory neurons and <b>(B)</b> embryonic MNs were double-stained for acetylated α-tubulin (acetylated α-tub, green) and β-III tubulin (β-III tub, red). As indicated by respective histograms there is a significant reduction of acetylated α-tubulin in both MN and sensory neurites in <i>Gdap1</i>^<i>-/-</i> mice. Graph represents means and S.E.M of 3 independent culture preparation per genotype. Student’s <i>t</i> test ***p<0.001.</p

Behavioural testing and electrophysiological measurements on <i>Gdap1</i>^<i>-/-</i> mice.

<p><b>(A)</b> Upper panel shows photographs of 3 months-old mice suspended by its tail. WT mice show a characteristic response trying to escape by splaying its hind limbs away from the trunk of its body. In contrast, hind limbs of <i>Gdap1</i>^-/- mice are held tonically against its trunk in an abnormal dystonic posture. Lower panels display a low body position and a dragging tail present in <i>Gdap1</i>^<i>-/-</i> mice as compared to age-matched WT mice. <b>(B)</b> Motor coordination was assessed by rotarod test, (n = 10 for each genotype and at each age group). <b>(C)</b> Representative hind limb walking patterns of 5 months-old WT and <i>Gdap1</i>^-/- mice where the stride length (SL) and stride angle (SA) have been depicted. Footprints revealed that <i>Gdap1</i>^<i>-/-</i> mice walk with an abnormal gait. The scheme of a hindpaw footprint indicating measured parameters (PL: plantar length; TS: toe spreading) has been included. <b>(D)</b> Quantification of various parameters obtained from the gait analysis of WT (black columns) and <i>Gdap1</i>^<i>-/-</i> (grey columns) animals at 5 and 12 months of age. Upper graphs show stride length (left) and stride angle (right). Lower graphs show the quantitative analysis of the hindpaw footprint parameters toe spreading (left) and plantar length (right). Analysis was conducted on 10 clearly visible footprints at 5 animals per genotype. Determination of sciatic nerve compound muscle action potential (CMAP) amplitudes at both distal and proximal <b>(E)</b> as well as motor nerve conduction velocities (MNCV) <b>(F)</b> measured in WT and <i>Gdap1</i>^-/- mice at 2 and 5 months of age (n = 4). Error bars indicate standard error of the mean (S.E.M.). <i>p</i> values were calculated using Student's <i>t</i> test,*p<0.05, **p<0.001, ***p<0.0001.</p

Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.

<p>Mitochondrial shape descriptors were measured in 20 WT and 30 <i>Gdap1</i>^-/- motorneurons. Student’s t test was performed for normal distributed parameters (number of mitochondria, circularity, roundness and aspect ratio) and Mann-Whitney U test for those that were non-normal distributed (surface area, Feret´s diameter and perimeter). See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005115#pgen.1005115.g006" target="_blank">Fig 6</a> for a visual representation.</p><p>*p<0.05,</p><p>**p<0.01.</p><p>Detailed morphological parameters for WT and Gdap1^-/- mitochondria in mouse motorneuron primary culture.</p

Lack of GDAP1 leads to loss of motor neurons and abnormal neuromuscular junctions.

<p><b>(A)</b> Anterior horns from lumbar spinal cord of 5 and 12-months-old mice were stained by Nissl staining. Reduced number of MNs and evidence of chromatolysis are visible in <i>Gdap1</i>^<i>-/-</i> mice. <b>(B)</b> Progressive graphics representing the number of healthy motor neurons in anterior horns per section in WT (black line) and <i>Gdap1</i>^<i>-/-</i> (gray line) mice at several ages (n = 3). <b>(C)</b> Representative confocal stack images of NMJs from the gastrocnemius muscle. Axons were immunostained with anti-β-III tubulin (β-III tub, green) and the postsynaptic acetyl-choline receptor was stained with AF488-coupled α-bungarotoxin (BTX, red). <b>(D)</b> Histograms show the percentage of NMJ occupancy by terminal axons in WT (black bars) and <i>Gdap1</i>^<i>-/-</i> (gray bars) mice. <b>(E)</b> Magnification of tangle-like abnormal structures (white arrows) at the terminal axons closed to the NMJ observed in <i>Gdap1</i>^<i>-/-</i> mice muscles. * represents significant differences between WT and <i>Gdap1</i>^<i>-/-</i> mice; <b>&</b> indicates differences between ages of WT mice; and <b>#</b> indicates differences between ages of <i>Gdap1</i>^<i>-/-</i> animals (Student’s <i>t</i> test, data are presented as means ±S.E.M.).</p

Ultrastructural analysis of embryonic motor neurons.

<p>WT <b>(A)</b> and <i>Gdap1</i>^<i>-/-</i><b>(B-D)</b> cultured embryonic MNs are shown. Medium and right panels show higher magnifications of frames of whole cell in left panels. N: nucleus; M: mitochondria; PS: perinuclear space; LD: lipid droplet; AV: autophagic vacuole; *: autophagolysosome; #: dilated endoplasmatic reticulum cisternae; arrowheads: endoplasmatic reticulum.</p