Additional file 1: Figure S1. of Gene-specific mitochondria dysfunctions in human TARDBP and C9ORF72 fibroblasts

Mitochondria morphology in mutant TARDBP and C9ORF72 fibroblasts after 48 h in galactose medium. Representative confocal images of mitochondrial network in primary fibroblasts from three healthy controls (C1, C2, C3), three ALS patients carrying mutation in TARDBP gene (p.A382T; T1, T2, T3) and three ALS//FTD patients with pathological expansion of the hexanucleotide repeat in C9ORF72 (C9.2, C9.3, C9.4) transfected with pDsRed2Mito construct. Bar, 10μm. Figure S2. Mitochondria functionality in mutant TARDBP and C9ORF72 fibroblasts. Measurement of mitochondrial membrane potential by flow cytometry analysis of Mitotracker Red (MTR) positive fibroblasts (from 4 healthy controls, 3 TARDBP and 4 C9ORF72 mutated patients) maintained in medium with galactose and without glucose. Median ± SEM, n = 3 different DIV; One-way ANOVA with Dunnett’s multiple comparison test; **p < 0.01). Figure S3. Analysis of mitochondria membrane potential in mutant TARDBP and C9ORF72 fibroblasts in glycolitic conditions. Measurement of mitochondrial (MMP) by flow cytometry analysis of TMRM-positive fibroblasts (4 healthy controls, 3 TARDBP and 4 C9ORF72), maintained in medium with 2 g/l glucose. Median ± SEM, n = 3 different DIV; One-way ANOVA with Dunnett’s multiple comparison test). Figure S4. Analysis of mitochondria mass in mutant TARDBP and C9ORF72 fibroblasts in glycolitic conditions. Measurement of mitochondrial mass (MM) by flow cytometry analysis of Mitotracker green (MTG)-positive fibroblasts (4 healthy controls, 3 TARDBP and 4 C9ORF72), maintained in medium with 2 g/l glucose. Median ± SEM, n = 3 different DIV; One-way ANOVA with Dunnett’s multiple comparison test). Figure S5. TDP-43 sub-cellular localization in C9ORF72 fibroblasts after 48 h in galactose medium. Representative immunofluorescence images of TDP-43 (green) in primary fibroblasts from healthy controls (four) and ALS/FTD patients carrying mutation in C9ORF72 gene (four), incubated with Mitotracker Red. Bar, 10 μm. Table S1. Clinical features of healthy controls, TARDBP- and C9ORF72-mutated patients. Table S2. Primer sequences and probes for Quantitative Real time PCR. Table S3. Respiratory electron transport chain activity. Values are expressed as nmol/min/mg protein and normalised with citrate synthase activity. (PDF 609 kb

Main characteristics of patients with septic shock at ICU admission.

<p>ICU: intensive care unit. SAPS II: simplified acute physiology score II (referred to the first 24 h from admission). SOFA: sepsis-related organ failure assessment (referred to the first 24 h from admission). Norepinephrine equivalent dose was calculated as norepinephrine (µg/min)+[dopamine (µg/kg/min)÷2]+epinephrine (µg/min)+[phenylephrine (µg/min)÷10] <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096205#pone.0096205-Russell1" target="_blank">[41]</a>. Central venous oxymetry was monitored in twenty-five patients; mixed venous oxymetry was monitored in five patients. Please note that most of the patients were firstly resuscitated in the Emergency Department and then transferred to the ICU.</p

Skeletal muscle mitochondrial biochemistry during septic shock.

<p>Mitochondrial biochemistry was measured on triceps brachii muscle of ten surgical controls (white bars) and thirty patients with septic shock (<24 h from ICU admission) (black bars). Results of two patients are not available due to technical troubles. Activities of nicotinamide adenine dinucleotide dehydrogenase (NADH) (p = 0.369) (<b>A</b>), complex I (CI) (p = 0.021) (<b>B</b>), complex I and III (CI+III) (p = 0.286) (<b>C</b>), succinate dehydrogenase (SDH) (p = 0.432) (<b>D</b>), complex II and III (CII+III) (p = 0.273) (<b>E</b>) and complex IV (CIV) (p = 0.760) (<b>F</b>) are expressed as percentages of citrate synthase (CS) activity (p = 0.458) (<b>G</b>). Data are reported as means and standard deviations. *p<0.05 <i>vs.</i> controls (Student’s <i>t</i> test).</p

Platelet mitochondrial biochemistry during septic shock.

<p>Mitochondrial biochemistry was measured on platelets of ten surgical controls (white bars) and thirty patients with septic shock (<24 h from ICU admission) (black bars). Activities of nicotinamide adenine dinucleotide dehydrogenase (NADH) (p = 0.015^#) (<b>A</b>), complex I (CI) (p = 0.018^#) (<b>B</b>), complex I and III (CI+III) (p<0.001) (<b>C</b>), succinate dehydrogenase (SDH) (p = 0.086) (<b>D</b>), complex II and III (CII+III) (p = 0.672) (<b>E</b>) and complex IV (CIV) (p = 0.012^#) (<b>F</b>) are expressed as percentages of citrate synthase (CS) activity (p = 0.002) (<b>G</b>). Data are reported as means and standard deviations. *p<0.05 <i>vs.</i> controls [Student’s <i>t</i> or (^#) Wilcoxon rank sum tests]. Platelet count (182±83 <i>vs.</i> 176±63 *10³/mm³) did not differ between groups (p = 0.901).</p

Relationship between platelet and skeletal muscle mitochondrial biochemistry.

<p>Mitochondrial biochemistry was measured on platelets (Y-axis) and triceps brachii muscle (X-axis) of ten surgical controls (white dots) and thirty patients with septic shock (<24 h from ICU admission) (black dots). Results of skeletal muscle mitochondrial biochemistry of two patients are not available due to technical troubles. Activities of nicotinamide adenine dinucleotide dehydrogenase (NADH) (<b>A</b>), complex I (CI) (<b>B</b>), complex I and III (CI+III) (<b>C</b>), succinate dehydrogenase (SDH) (<b>D</b>), complex II and III (CII+III) (<b>E</b>) and complex IV (CIV) (<b>F</b>) are expressed as percentages of citrate synthase (CS) activity (<b>G</b>). r² and p values refer to Pearson product moment test.</p

Supplementary information files for NEB mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy Item

Supplementary files for article NEB mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients’ muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin‐deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin‐deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.  </p

<i>NEB</i> mutations disrupt the super-relaxed state of myosin and remodel the muscle metabolic proteome in nemaline myopathy

Nemaline myopathy (NM) is one of the most common non-dystrophic genetic muscle disorders. NM is often associated with mutations in the NEB gene. Even though the exact NEB-NM pathophysiological mechanisms remain unclear, histological analyses of patients’ muscle biopsies often reveal unexplained accumulation of glycogen and abnormally shaped mitochondria. Hence, the aim of the present study was to define the exact molecular and cellular cascade of events that would lead to potential changes in muscle energetics in NEB-NM. For that, we applied a wide range of biophysical and cell biology assays on skeletal muscle fibres from NM patients as well as untargeted proteomics analyses on isolated myofibres from a muscle-specific nebulin‐deficient mouse model. Unexpectedly, we found that the myosin stabilizing conformational state, known as super-relaxed state, was significantly impaired, inducing an increase in the energy (ATP) consumption of resting muscle fibres from NEB-NM patients when compared with controls or with other forms of genetic/rare, acquired NM. This destabilization of the myosin super-relaxed state had dynamic consequences as we observed a remodeling of the metabolic proteome in muscle fibres from nebulin‐deficient mice. Altogether, our findings explain some of the hitherto obscure hallmarks of NM, including the appearance of abnormal energy proteins and suggest potential beneficial effects of drugs targeting myosin activity/conformations for NEB-NM.</p