Diagnostic performance of metabolic biomarkers.

<p>ROC curves were plotted to describe performance characteristics of the indicated metabolites in the 57 subject cohort. The Area under the curve (AUC), 95% range of the interval of confidence (IC) and P values are indicated.</p

Identification of metabolites by co-elution with commercial standards.

<p>Extracted ion chromatograms of selected metabolites in plasma samples (purple lines) and commercial standards (blue lines) are shown. The metabolites are defined by their name, ID number, m/z (M+H), retention time, Human Metabolome Data Base ID number and molecular formula.</p

Scatter plots showing significant correlations between plasma metabolite levels and severity of the disease in CMT1A patients.

<p>Determination of the plasma levels of the metabolites was carried out by UHPLC-MS and its levels correlated with severity of the disease as assessed by CMTNSv2. Healthy controls (blue circles, n = 15), Mild (red squares, n = 15), Moderate (green diamonds, n = 18) and Severe (black triangles, n = 9) groups of CMT1A patients are represented. Metabolites related to (i) protein catabolism (upper row); (ii) mobilization of membrane lipids (middle row) and (iii) muscle biogenesis and the anti-apoptotic function (lower row) are represented. P-value is calculated by analysis of variance; r is the Spearman coefficient.</p

Summary of the major changes in the expression of skin and plasma metabolites during progression of CMT1A.

<p>The loss of muscle and nerves is shown during severity of the disease as revealed by the CMT neuropathy score v2. The changes in biomarkers are illustrated by row thicknesses.</p

Protein expression in skin biopsies of CMT1A patients.

<p>Protein expression in skin biopsies of CMT1A patients.</p

Analysis of plasma metabolome in CMT1A patients.

<p>(A), Representative UHPLC-MS total ion chromatogram of plasma samples. (B), Plot in a two-dimensional Cartesian coordinate system, with the axes (principal components, PC) representing the greatest variations in the data of Control (blue), Mild (red), Moderate (green) and Severe (black) states related to CMT1A. Three quality control (QC) injections per group are also represented in the plot for the four groups of individuals. 95% confidence ellipses are also included. Triplicate outliers of one of the samples in the Moderate group fall out of the ellipse. (C), Plot of distribution of the plasma samples defined by the two canonical variables (CV1 and CV2) obtained by Canonical Variate Analysis considering the 12 selected metabolites after forward stepwise Linear Discriminant Analysis. The 95% canonical ellipses are also included. Control subjects and mild, moderate and severe CMT1A patients are represented by blue circles, red squares, green diamonds and black triangles, respectively. (D), Histogram showing the content of the 12 metabolites in plasma samples of healthy (blue, n = 15) and CMT1A patients (yellow, n = 42). The results shown are the mean values ± S.E.M. *, P<<i>0</i>.<i>05</i> by <i>Student’s t test</i>.</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

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