Mitochondrial Dysfunction Plus High-Sugar Diet Provokes a Metabolic Crisis That Inhibits Growth

The Drosophila mutant tko(25t) exhibits a deficiency ofmitochondrial protein synthesis, leading to a global insufficiency of respiration and oxidative phosphorylation. This entrains an organismal phenotype of developmental delay and sensitivity to seizures induced bymechanical stress. We found that the mutant phenotype is exacerbated in a dose-dependent fashion by high dietary sugar levels. tko(25t) larvae were found to exhibit severe metabolic abnormalities that were further accentuated by high-sugar diet. These include elevated pyruvate and lactate, decreased ATP and NADPH. Dietary pyruvate or lactate supplementation phenocopied the effects of high sugar. Based on tissue-specific rescue, the crucial tissue in which this metabolic crisis initiates is the gut. It is accompanied by down-regulation of the apparatus of cytosolic protein synthesis and secretion at both the RNA and post-translational levels, including a novel regulation of S6 kinase at the protein level.Peer reviewe

Reduced Glucose Sensation Can Increase the Fitness of Saccharomyces cerevisiae Lacking Mitochondrial DNA.

Damage to the mitochondrial genome (mtDNA) can lead to diseases for which there are no clearly effective treatments. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. We found that restricting glucose or otherwise reducing the activity of the protein kinase A (PKA) pathway can lead to improved proliferation of Saccharomyces cerevisiae cells lacking mtDNA and that the transcriptional response to mtDNA loss is reduced in cells with diminished PKA activity. We have excluded many pathways and proteins from being individually responsible for the benefits provided to cells lacking mtDNA by PKA inhibition, and we found that robust import of mitochondrial polytopic membrane proteins may be required in order for cells without mtDNA to receive the full benefits of PKA reduction. Finally, we have discovered that the transcription of genes involved in arginine biosynthesis and aromatic amino acid catabolism is altered after mtDNA damage. Our results highlight the potential importance of nutrient detection and availability on the outcome of mitochondrial dysfunction

Proneural factors Ascl1 and Neurog2 contribute to neuronal subtype identities by establishing distinct chromatin landscapes

12 páginas, 7 figurasDevelopmental programs that generate the astonishing neuronal diversity of the nervous system are not completely understood and thus present a major challenge for clinical applications of guided cell differentiation strategies. Using direct neuronal programming of embryonic stem cells, we found that two main vertebrate proneural factors, Ascl1 and neurogenin 2 (Neurog2), induce different neuronal fates by binding to largely different sets of genomic sites. Their divergent binding patterns are not determined by the previous chromatin state, but are distinguished by enrichment of specific E-box sequences that reflect the binding preferences of the DNA-binding domains. The divergent Ascl1 and Neurog2 binding patterns result in distinct chromatin accessibility and enhancer activity profiles that differentially shape the binding of downstream transcription factors during neuronal differentiation. This study provides a mechanistic understanding of how transcription factors constrain terminal cell fates, and it delineates the importance of choosing the right proneural factor in neuronal reprogramming strategies.This work is supported by NICHD (R01HD079682) and Project ALS (A13-0416) to E.O.M. and by NYSTEM pre-doctoral training grant (C026880) to B.A. S.M. is supported by NIGMS (R01GM125722) and the National Science Foundation ABI Innovation Grant No. DBI1564466. Any opinions, findings and conclusions or recommendations expressed in this material are those of the authors and do not necessarily reflect the views of the National Science Foundation. M.R. is supported by NYU MSTP (T32GM007308) and Developmental Genetics T32 (T32HD007520) grants. N.F. and M.M.E. are supported by ERC Starting Grant (2011-281920). The authors would like to thank Link Tejavibulya and Apeksha Ashokkumar for their help with molecular biology; Mohammed Khalfan for his help with scRNA-seq analysis. Michael Cammer from the NYU Medical Center Microscopy Core for the ImageJ script used in calcium imaging analysis; and NYU Genomics Core facility. Finally, we would like to thank Steve Small, Nikos Konstantinidis, Pinar Onal, Orly Wapinski, Sevinç Ercan, Chris Rushlow, Claude Desplan and Mazzoni lab members for their helpful suggestions on the manuscript.Peer reviewe

Glucose inhibits proliferation of cells deleted of mtDNA.

<p>(A) Decreasing glucose concentration leads to increased proliferation of cells lacking mtDNA. Strain BY4742 (<i>WT</i>) was cultured in YEPD medium containing 2%, 0.5%, or 0.2% glucose and tested for the response to mtDNA deletion. Cells were incubated for 3 d. (B) Proliferation of <i>ρ</i>^<i>0</i> cells by Gpa2p or Gpr1p deletion is not improved further upon lowering the glucose concentration. Strains BY4742 (<i>WT</i>), CDD849 (<i>gpa2Δ</i>), and CDD850 (<i>gpr1Δ</i>) were treated as in (A), yet incubated on solid medium for 2 d.</p

Decreased PKA activity can increase proliferation of cells lacking mtDNA.

<p>(A) Overexpression of cAMP phosphodiesterase Pde2p increases the fitness of cells lacking mtDNA. Strain BY4743 (<i>WT</i>) was transformed with empty, high-copy vector pRS426 or plasmid b89 (2μ-<i>PDE2</i>). Strains were tested for their response to mtDNA loss by incubation in selective medium lacking or containing 25 μg/ml EtBr, with subsequent incubation on solid SC-Ura medium for 2 d. (B) Lack of Tpk3p increases the fitness of cells lacking mtDNA. Strains BY4742 (<i>WT</i>), CDD884 (<i>tpk1Δ</i>), CDD885 (<i>tpk2Δ</i>), CDD886 (<i>tpk3Δ</i>), CDD908 (<i>tpk1Δ tpk3Δ</i>), CDD922 (<i>tpk2Δ tpk3Δ</i>), and CDD923 (<i>tpk1Δ tpk2Δ</i>) were tested for their response to mtDNA deletion with incubation on solid YEPD medium for 2 d. (C) Cells deleted of Gpa2p or Gpr1p exhibit increased fitness after mtDNA deletion. Strains BY4742 (<i>WT</i>), CDD886 (<i>tpk3Δ</i>), CDD849 (<i>gpa2Δ</i>), and CDD850 (<i>gpr1Δ</i>) were treated as in (B).</p

Several transcription factors driving stress resistance following PKA inhibition are not individually responsible for the benefits provided by Pde2p overexpression to cells lacking mtDNA.

<p>(A) Cells lacking Gis1p and mtDNA are increased in proliferation upon Pde2p overexpression. Strains BY4742 (<i>WT</i>) and CDD801 (<i>gis1Δ</i>) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146511#pone.0146511.g001" target="_blank">Fig 1A</a>. (B) Cells lacking both Msn2p and Msn4p exhibit increased fitness following mtDNA loss upon Pde2p overexpression. Strains BY4741 (<i>WT</i>) and CDD838 (<i>msn2Δ msn4Δ</i>) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146511#pone.0146511.g001" target="_blank">Fig 1A</a>. (C) The Rim15 kinase is not required in order for Pde2p overexpression to benefit <i>ρ</i>^<i>0</i> cells. Strains CDD463 (<i>WT</i>) and CDD841 (<i>rim15Δ</i>) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146511#pone.0146511.g001" target="_blank">Fig 1A</a>. (D) A potential reduction of Hsf1p function does not prevent increased <i>ρ</i>^<i>0</i> cell fitness upon overexpression of Pde2p. Strains BY4741 (<i>WT</i>) and CDD910 (<i>hsf1-DAmP</i>) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146511#pone.0146511.g001" target="_blank">Fig 1A</a>.</p

Overexpression of Pde2p can rescue the petite-negative phenotype of several mutants defective for mitochondrial function.

<p>(A) High-copy Pde2p can allow mutants deficient in activity of the i-AAA protease to remain viable following mtDNA loss. Strains BY4741 (<i>WT</i>), CDD13 (<i>mgr1Δ</i>), and CDD15 (<i>mgr3Δ</i>) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146511#pone.0146511.g001" target="_blank">Fig 1A</a>, with additional incubation of <i>ρ</i>^<i>0</i> cells to 4 d in order to demonstrate suppression of the petite-negative phenotype. (B) PKA inhibition by Pde2p overexpression can rescue the petite-negative phenotype of mutants deficient in mitochondrial protein import and assembly. Strains BY4741 (<i>WT</i>), CDD11 (<i>mgr2Δ</i>), and CDD17 (<i>phb1Δ</i>) were treated as in (A). (C) Overexpression of Pde2p allows cells lacking mtDNA to proliferate in the absence of F₁-ATPase activity. Strains CDD463 (<i>WT</i>) and CDD215 (<i>atp2Δ</i>) were treated as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146511#pone.0146511.g001" target="_blank">Fig 1A</a>, with further incubation of <i>ρ</i>^<i>0</i> cells to 6 d.</p