Mouse Studies to Shape Clinical Trials for Mitochondrial Diseases: High Fat Diet in Harlequin Mice

BACKGROUND: Therapeutic options in human mitochondrial oxidative phosphorylation (OXPHOS) diseases have been poorly evaluated mostly because of the scarcity of cohorts and the inter-individual variability of disease progression. Thus, while a high fat diet (HFD) is often recommended, data regarding efficacy are limited. Our objectives were 1) to determine our ability to evaluate therapeutic options in the Harlequin OXPHOS complex I (CI)-deficient mice, in the context of a mitochondrial disease with human hallmarks and 2) to assess the effects of a HFD. METHODS AND FINDINGS: Before launching long and expensive animal studies, we showed that palmitate afforded long-term death-protection in 3 CI-mutant human fibroblasts cell lines. We next demonstrated that using the Harlequin mouse, it was possible to draw solid conclusions on the efficacy of a 5-month-HFD on neurodegenerative symptoms. Moreover, we could identify a group of highly responsive animals, echoing the high variability of the disease progression in Harlequin mice. CONCLUSIONS: These results suggest that a reduced number of patients with identical genetic disease should be sufficient to reach firm conclusions as far as the potential existence of responders and non responders is recognized. They also positively prefigure HFD-trials in OXPHOS-deficient patients

Développement de méthodes analytiques pour mesurer les analogues nucléosidiques anti-VIH, leurs métabolites actifs et leur intégration dans l'ADN

PARIS-BIUSJ-Thèses (751052125) / SudocPARIS-BIUSJ-Physique recherche (751052113) / SudocSudocFranceF

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

HIGH-RESOLUTION SPECTROSCOPY ON DOUBLY DEUTERATED AMMONIA UP TO 2.6 THz

Author Institution: I. Physikalisches Institut, Universitat zu Koln, Zulpicher Str.77, 50937 Koln, Germany; Department of Radiophysics, N. Novgorod State University, RussiaHigh accuracy spectra of ND$_2$H up to frequencies of 2.6 THz have been recorded by the use of frequency multiplication techniques in combination with phase locked, high power backward wave oscillators (BWO). For the first time we used novel superlattice (SL) devices as key-element for the generation of higher order harmonics in spectroscopic applications. Odd multiplication factors up to 11 have been achieved and used to measure ground state transitions of the pure rotation and inversion-rotation spectra of ND$_2$H in the frequency range between 0.08 and 2.58 THz with accuracies of several kilohertz. Energy levels up to quantum numbers $J=18$ and $K_a=9$ have been accessed. A greatly extended experimental dataset was obtained with significantly improved accuracies. Besides a technical description of the spectrometer setup, a short presentation and analysis of the spectra will be given in the talk. The improved molecular parameters, derived in the analysis, allowed to generate predictions that can be regarded as a reliable basis for future astronomical high resolution observations throughout the microwave to terahertz regions. These predictions will be available in the Cologne Database for Molecular Spectroscopy (www.cdms.de)

Stilbenes and resveratrol metabolites improve mitochondrial fatty acid oxidation defects in human fibroblasts

International audienceBackground: Inborn enzyme defects of mitochondrial fatty acid beta-oxidation (FAO) form a large group of genetic disorders associated to variable clinical presentations ranging from life-threatening pediatric manifestations up to milder late onset phenotypes, including myopathy. Very few candidate drugs have been identified in this group of disorders. Resveratrol (RSV) is a natural polyphenol with anti-oxidant and anti-inflammatory effects, recently shown to have beneficial metabolic properties in mice models. Our study explores its possible effects on FAO and mitochondrial energy metabolism in human cells, which are still very little documented.Methods: Using cells from controls and from patients with Carnitine Palmitoyl Transferase 2 (CPT2) or Very Long Chain AcylCoA Dehydrogenase (VLCAD) deficiency we characterized the metabolic effects of RSV, RSV metabolites, and other stilbenes. We also focused on analysis of RSV uptake, and on the effects of low RSV concentrations, considering the limited bioavailability of RSV in vivo.Results: Time course of RSV accumulation in fibroblasts over 48 h of treatment were consistent with the resulting stimulation or correction of FAO capacities. At 48 h, half maximal and maximal FAO stimulations were respectively achieved for 37,5 microM (EC50) and 75 microM RSV, but we found that serum content of culture medium negatively modulated RSV uptake and FAO induction. Indeed, decreasing serum from 12% to 3% led to shift EC50 from 37,5 to 13 microM, and a 2.6-3.6-fold FAO stimulation was reached with 20 microM RSV at 3% serum, that was absent at 12% serum. Two other stilbenes often found associated with RSV, i.e. cis- RSV and piceid, also triggered significant FAO up-regulation. Resveratrol glucuro- or sulfo- conjugates had modest or no effects. In contrast, dihydro-RSV, one of the most abundant circulating RSV metabolites in human significantly stimulated FAO (1.3-2.3-fold).Conclusions: This study provides the first compared data on mitochondrial effects of resveratrol, its metabolites, and other natural compounds of the stilbene family in human cells. The results clearly indicate that several of these compounds can improve mitochondrial FAO capacities in human FAO-deficient cells

Prenyldiphosphate synthase, subunit 1 (PDSS1) and OH-benzoate polyprenyltransferase (COQ2) mutations in ubiquinone deficiency and oxidative phosphorylation disorders

Coenzyme Q(10) (CoQ(10)) plays a pivotal role in oxidative phosphorylation (OXPHOS), as it distributes electrons among the various dehydrogenases and the cytochrome segments of the respiratory chain. We have identified 2 novel inborn errors of CoQ(10) biosynthesis in 2 distinct families. In both cases, enzymologic studies showed that quinone-dependent OXPHOS activities were in the range of the lowest control values, while OXPHOS enzyme activities were normal. CoQ(10) deficiency was confirmed by restoration of normal OXPHOS activities after addition of quinone. A genome-wide search for homozygosity in family 1 identified a region of chromosome 10 encompassing the gene prenyldiphosphate synthase, subunit 1 (PDSS1), which encodes the human ortholog of the yeast COQ1 gene, a key enzyme of CoQ(10) synthesis. Sequencing of PDSS1 identified a homozygous nucleotide substitution modifying a conserved amino acid of the protein (D308E). In the second family, direct sequencing of OH-benzoate polyprenyltransferase (COQ2), the human ortholog of the yeast COQ2 gene, identified a single base pair frameshift deletion resulting in a premature stop codon (c.1198delT, N401fsX415). Transformation of yeast Δcoq1 and Δcoq2 strains by mutant yeast COQ1 and mutant human COQ2 genes, respectively, resulted in defective growth on respiratory medium, indicating that these mutations are indeed the cause of OXPHOS deficiency

Absence of cell death in control (Ctrl) cells after glucose withdrawal, palmitate or pyruvate supplementation.

<p>Values are means ± SEM from triplicate experiments; h: time from glucose withdrawal in hours.</p

Effect of bovine serum albumin (BSA), L-carnitine, (BSA+L-carnitine+palmitate) and succinate on glucose withdrawal-induced cell death in patient 1 (P1) and control (Ctrl) cells.

<p>Values are means ± SEM from triplicate experiments; h: time from glucose withdrawal in hours; L-C: L-carnitine; palm: palmitate.</p

Box-and-whisker plot of rotarod scores as a function of diet (HFD versus CD) according to age in Harlequin (Hq) and wild type (WT) animals.

<p>The results of three tests per animals performed monthly from one to six months of age are shown with the number (n) of animals indicated in each box. The vertical bars represent the range of values from the 10^th to the 90^th percentile; the boxes represent the values from the 25^th to the 75^th percentile; the horizontal lines represent the median value for each group of animals. Isolated circles represent all observations (in triplicates) below the 10^th percentile or above the 90^th percentile. *: p<0.05; **: p<0.01 (Student's two-tailed <i>t</i> test). HFD-fed WT mice exhibit significantly worse performances than the WT mice fed the CD, from 5 months of age due to their obese phenotype (A). From 4 months of age, HFD-fed Hq mice do significantly better than the Hq fed the CD with a significantly lower progression of the rotarod scores loss (B).</p

Palmitate-induced death protection in two other CI-deficient human fibroblasts.

<p>Values are means ± SEM from triplicate experiments; h: time from glucose withdrawal in hours.</p