Peroxisome Proliferator Activated Receptor δ (PPARδ) Agonist But Not PPARα Corrects Carnitine Palmitoyl Transferase 2 Deficiency in Human Muscle Cells

International audienceType 2 carnitine palmitoyl transferase (CPT2) is involved in the transfer of long-chain fatty acid into the mitochondria. CPT2-deficient patients carry gene mutations associated with different clinical presentations, correlating with various levels of fatty acid oxidation (FAO) and residual CPT2 enzyme activity. We tested the hypothesis that pharmacological stimulation of peroxisome proliferator-activated receptors (PPAR) can stimulate FAO in CPT2-deficient muscle cells. Accordingly, we show that a 48-h treatment of CPT2-deficient myoblasts by bezafibrate restored FAO in patient cells. Specific agonists of PPARdelta (GWdelta 0742), and, to a lower extent, PPARalpha (GWalpha 7647) also stimulated FAO in control myoblasts. However, when tested in CPT2-deficient myoblasts, only the delta-agonist was able to restore FAO, whereas the alpha-agonist had no effect. GWdelta 0742 increased CPT2 mRNA levels, whereas no change in CPT2 transcripts was found in response to GWalpha 7647. Bezafibrate and GWdelta 0742 increased residual CPT2 activity and normalized long-chain acylcarnitine production by deficient cells. Finally, CPT1-B mRNA was also stimulated after PPAR agonist treatment, and this likely takes part in drug-induced increase of FAO in control muscle cells. In conclusion, this study clearly suggests that PPARs could be therapeutic targets for correction of inborn beta-oxidation defects in human muscle. Furthermore, these data also illustrate a selective control of beta-oxidation enzyme gene expression by PPARdelta, with no contribution of PPARalpha

Activation of Peroxisome Proliferator-Activated Receptor Pathway Stimulates the Mitochondrial Respiratory Chain and Can Correct Deficiencies in Patients' Cells Lacking Its Components

International audienceContext: The mitochondrial respiratory chain (RC) disorders are the largest group of inborn errors of metabolism and still remain without treatment in most cases.Objective: We tested whether bezafibrate, a drug acting as a peroxisome proliferator-activated receptor (PPAR) agonist, could stimulate RC capacities.Design: Fibroblasts or myoblasts from controls or patients deficient in complex I (CI), complex III (CIII), or complex IV (CIV) were cultured with or without bezafibrate.Main outcome measures: Enzyme activities, mRNA and protein expression, and respiration rates were measured.Results: In control cells, bezafibrate increased the CI, CIII, and CIV enzyme activities (+42 to +52%), as well as RC mRNAs (+40 to +120%) and RC protein levels (+50 to +150%). Nine of 14 patient cell lines tested exhibited a significant increase in the activity of the deficient RC complex after bezafibrate treatment (+46 to +133%), and full pharmacological correction could be achieved in seven cell lines. Similar effects were obtained using a PPARdelta agonist. These changes were related to a drug-induced increase in the mutated mRNAs and RC protein levels. Finally, the molecular mechanisms by which the PPAR pathway could induce the expression of genes encoding structural subunits or ancillary proteins of the RC apparatus, leading to stimulate the activity and protein levels of RC complex, likely involved the PPARgamma coactivator-1alpha.Conclusions: This study suggests a rationale for a possible correction of moderate RC disorders due to mutations in nuclear genes, using existing drugs, and brings new insights into the role of PPAR in the regulation of the mitochondrial RC in human cells

Inhibitors of the Neisseria meningitidis PilF ATPase provoke type IV pilus disassembly

International audienceDespite the availability of antibiotics and vaccines, Neisseria meningitidis remains a major cause of meningitis and sepsis in humans. Due to its extracellular lifestyle, bacterial adhesion to host cells constitutes an attractive therapeutic target. Here, we present a high-throughput microscopy-based approach that allowed the identification of compounds able to decrease type IV pilus-mediated interaction of bacteria with endothelial cells in the absence of bacterial or host cell toxicity. Compounds specifically inhibit the PilF ATPase enzymatic activity that powers type IV pilus extension but remain inefficient on the ATPase that promotes pilus retraction, thus leading to rapid pilus disappearance from the bacterial surface and loss of pili-mediated functions. Structure activity relationship of the most active compound identifies specific moieties required for the activity of this compound and highlights its specificity. This study therefore provides compounds targeting pilus biogenesis, thereby inhibiting bacterial adhesion, and paves the way for a novel therapeutic option for meningococcal infections

Lung development in late gestation IGF-1R^−/− mice.

<p><b>A–L</b>, Lungs prepared from IGF-1R^+/+ and IGF-1R^−/− embryos at developmental stages E14.5, E17.5 and E19.5. <b>A–F</b>, Ventral view of whole lungs. <b>G–L</b>, Rim of lung lobe. Abbreviations: AL, apical lobe; AzL, azygous lobe; CL, cardiac lobe; DL, diaphragmatic lobes; LL, left lobe. <b>M–X</b>, Lung histology of IGF-1R^+/+ versus IGF-1R^−/− embryos. H&E stained lung sections at developmental stages E14.5 (<b>M</b>–<b>P</b>), E17.5 (<b>Q</b>–<b>T</b>) and E19.5 (<b>U</b>–<b>X</b>), showing that saccular walls are thicker and acinar buds smaller in IGF-1R^−/− embryos as compared with controls of the same stage. Note that histomorphological appearance is similar when comparing E19.5 IGF-1R^−/− (<b>V</b>, <b>X</b>) with two days younger E17.5 IGF-1R^+/+ lungs (<b>Q</b>, <b>S</b>).</p

Immunohistochemistry of lung differentiation markers.

<p><b>A</b> and <b>B,</b> Representative tissue sections from IGF-1R^+/+ and IGF-1R^−/− embryos at stage E17.5 showing CD31-immunoreactivity specific for capillary endothelia. <b>C</b>, Morphometric comparison of CD31 signal between genotypes (n = 5 per group; two-tailed <i>t</i>-test). <b>D–F</b>, Capillary complexity was estimated calculating the density of capillary junctions from CD31 IHC. <b>G–J</b>, Sections from IGF-1R^+/+ and IGF-1R^−/− embryos at E17.5 and E19.5 show IHC of blood vessel-specific von Willebrand protein. Arrows (I, J) point to small blood vessels developing in saccular walls. Large blood vessels were similarly marked in all specimen. <b>K–M</b>, Representative lung histology from IGF-1R^+/+ and IGF-1R^−/− embryos at E17.5. NKX2-1 distal-to-proximal IHC signal ratio was measured in 6 IGF-1R^+/+ and 5 IGF-1R^−/− embryos. NS, not significant; Wilcoxon Mann-Whitney U test. <b>N-Q</b>, Epithelial cell-specific NKX2-1 transcription factor was detected in IGF-1R^+/+ and IGF-1R^−/− embryos at E17.5 and E19.5. <b>R–Y</b>, IHC of type 2-specific pro-SP-C at low (R-U) and high magnification (V-Y). Interestingly, for NKX2-1 and pro-SP-C, the IHC pattern of IGF-1R^−/− lungs at E19.5 resembles controls at E17.5 (panel N <i>versus</i> Q, R <i>versus</i> U, and V <i>versus</i> Y), suggesting an approximately 2-day developmental delay in IGF-1R^−/− end-gestational lungs.</p

Lung, heart, liver and kidney weight relative to body weight, in IGF-1R^+/+ and IGF-1R^−/− embryos.

<p>Values are mean ± SEM. * <i>P</i><0.05; ** <i>P</i><0.01; *** <i>P</i><0.001 compared with IGF-1R^+/+ mice. Student’s <i>t</i>-test.</p>a<p>n = 5; BW, body weight; ND, not determined.</p

IGF-1R protein levels, lung histology and respiratory function in adult IGF-1R^neo/− mice. A

<p>, Western immunoblot of IGF-1R in lung from mice with distinct combinations of mutant IGF-1R alleles. Total proteins were extracted from lung tissue from IGF-1R^neo/−, IGF-1R^+/−, IGF-1R^+/neo and IGF-1R^+/+ mice (n = 3 for each genotype), and IGF-1R^−/− embryo (negative control), and were probed with anti-IGF-1Rβ (upper panel) or anti-β-actin antibodies (lower panel). IGF-1R^neo/− mice have 22% of receptor levels present in IGF-1R^+/+ mice (quantified in B), IGF-1R^+/− have 50%, IGF-1R^+/neo mice are between 70 and 80%, and IGF-1R^−/− mice lack IGF-1R completely. <b>B</b>, IGF-1R abundance determined in lung tissue. Bar graph shows IGF-1R levels relative to β-actin from 5 IGF-1R^+/+ and 6 IGF-1R^neo/− individuals (Error bars SEM; Student’s <i>t</i>-test). Image shows 4 representative lanes from western immunoblot. <b>C</b>, Hematoxylin-eosin stained lung sections from IGF-1R^+/+ and IGF-1R^neo/− males. <b>D</b>, Alveolar airspace, <b>E</b>, alveolar boundary length density, <b>F</b>, alveolar wall thickness, in IGF-1R^+/+ (n = 4) and IGF-1R^neo/− mice (n = 4). Error bars indicate SEM; Wilcoxon Mann-Whitney U test. <b>G-I</b>, Respiratory function in adult IGF-1R^neo/− mice. Mice were challenged with 6% and 8% CO₂. <b>G</b>, Minute ventilation (V_E), <b>H</b>, tidal volume (V_T), and <b>I</b>, respiratory frequency (BR) were measured in 6 individuals per group. Differences between room air and hypercapnia were significant, but no significant differences were found between genotypes. Values labeled <i>b</i> were different from <i>a</i> (<i>P</i><0.005); Error bars indicate SEM; Wilcoxon Mann-Whitney U test.</p

Lung histomorphology and cell turnover in the absence of IGF-1R.

<p><b>A</b>, Saccular airspace and <b>B</b>, saccular wall thickness (mean ± SEM) at developmental stages E17.5 in IGF-1R^+/+ (n = 4) and IGF-1R^−/− embryos (n = 4). Wilcoxon Mann-Whitney U test. <b>C–F,</b> Extended gestation period and lung histology. H&E stain of lung tissue from embryos at 19.5 (<b>C</b>, <b>D</b>) and 21.5 days (<b>E</b>, <b>F</b>). To extend gestation period up to 21.5 days, pregnant mothers were treated with progesterone from E17.5 onwards. Note the presence of red blood cell extravasation in E21.5 lung samples from IGF-1R^+/+ and IGF-1R^−/− mice. <b>G–O,</b> Cell turnover in IGF-1R^−/− embryonic lung at E17.5. Lung histology from IGF-1R^+/+ embryos (G, J and M) and IGF-1R^−/− embryos (H, K and N) at E17.5. Bar graphs (I, L and O) show quantification (mean ± SEM; n = 3–7 individuals per group; Student’s <i>t</i>-test). <b>G–I</b>, Cells were counted using DAPI staining (blue signal). <b>J–L</b>, Cell proliferation was measured using phospho–histone H3 immunohistochemistry (brown staining). <b>M–O</b>, Apoptosis was detected using cleaved caspase-3 immunohistochemistry (brown staining). Cleaved caspase-3 (P-U) and phospho-histone H3 labeling (V-AA) at high magnification showing examples for IHC-positive epithelial (red arrows), vascular endothelial (blue) and mesenchymal cells (green), as identified by their anatomical location. Note that many of the proliferating cells are located in areas that are composed of mostly mesenchymal cells.</p