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

SOCE alteration in <i>Gdap1</i>^<i>-/-</i> embryonic motor neurons <b>(A)</b> Fura-2 [Ca²⁺] signals of embryonic MNs from WT (black) and <i>Gdap1</i>^<i>-/-</i> (grey) mice.

<p>After Ca²⁺ release from cell stores with 5 μM thapsigargin (TG) treatment during 7 min in Ca²⁺ free medium, SOCE was activated by adding 2 mM of CaCl₂. Traces were used to obtain <b>(B)</b> maximum Ca²⁺ peak during SOCE and <b>(C)</b> SOCE Ca²⁺ influx (slope). <b>(D)</b> Fura-2 recordings of 5 μM ionomycin elicited [Ca²⁺]_cyt peak in Ca²⁺-free medium. <b>(E)</b> Maximum [Ca²⁺]_cyt peak obtained in Ca²⁺-free medium represents total amount of cytoplasmic Ca²⁺ after cell stores Ca²⁺ release. Traces were obtained averaging at least 100 cells from each genotype. Error bars represent S.E.M. (***p<0.001, Student’s <i>t</i> test).</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

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

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

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