Meiotic resetting of the cellular Sod1 pool is driven by protein aggregation, degradation, and transient LUTI-mediated repression

Gametogenesis requires packaging of the cellular components needed for the next generation. In budding yeast, this process includes degradation of many mitotically stable proteins, followed by their resynthesis. Here, we show that one such case-Superoxide dismutase 1 (Sod1), a protein that commonly aggregates in human ALS patients-is regulated by an integrated set of events, beginning with the formation of pre-meiotic Sod1 aggregates. This is followed by degradation of a subset of the prior Sod1 pool and clearance of Sod1 aggregates. As degradation progresses, Sod1 protein production is transiently blocked during mid-meiotic stages by transcription of an extended and poorly translated SOD1 mRNA isoform, SOD1LUTI. Expression of SOD1LUTI is induced by the Unfolded Protein Response, and it acts to repress canonical SOD1 mRNA expression. SOD1LUTI is no longer expressed following the meiotic divisions, enabling a resurgence of canonical mRNA and synthesis of new Sod1 protein such that gametes inherit a full complement of Sod1 protein. Failure to aggregate and degrade Sod1 results in reduced gamete fitness in the presence of oxidants, highlighting the importance of this regulation. Investigation of Sod1 during yeast gametogenesis, an unusual cellular context in which Sod1 levels are tightly regulated, could shed light on conserved aspects of its aggregation and degradation, with relevance to understanding Sod1's role in human disease

Tunable Transcriptional Interference at the Endogenous Alcohol Dehydrogenase Gene Locus in Drosophila melanogaster

Neighboring sequences of a gene can influence its expression. In the phenomenon known as transcriptional interference, transcription at one region in the genome can repress transcription at a nearby region in cis. Transcriptional interference occurs at a number of eukaryotic loci, including the alcohol dehydrogenase (Adh) gene in Drosophila melanogaster. Adh is regulated by two promoters, which are distinct in their developmental timing of activation. It has been shown using transgene insertion that when the promoter distal from the Adh start codon is deleted, transcription from the proximal promoter becomes de-regulated. As a result, the Adh proximal promoter, which is normally active only during the early larval stages, becomes abnormally activated in adults. Whether this type of regulation occurs in the endogenous Adh context, however, remains unclear. Here, we employed the CRISPR/Cas9 system to edit the endogenous Adh locus and found that removal of the distal promoter also resulted in the untimely expression of the proximal promoter-driven mRNA isoform in adults, albeit at lower levels than previously reported. Importantly, transcription from the distal promoter was sufficient to repress proximal transcription in larvae, and the degree of this repression was dependent on the degree of distal promoter activity. Finally, upregulation of the distal Adh transcript led to the enrichment of histone 3 lysine 36 trimethylation over the Adh proximal promoter. We conclude that the endogenous Adh locus is developmentally regulated by transcriptional interference in a tunable manner

Transaldolase inhibition impairs mitochondrial respiration and induces a starvation-like longevity response in <i>Caenorhabditis elegans</i>

<div><p>Mitochondrial dysfunction can increase oxidative stress and extend lifespan in <i>Caenorhabditis elegans</i>. Homeostatic mechanisms exist to cope with disruptions to mitochondrial function that promote cellular health and organismal longevity. Previously, we determined that decreased expression of the cytosolic pentose phosphate pathway (PPP) enzyme transaldolase activates the mitochondrial unfolded protein response (UPR^mt) and extends lifespan. Here we report that transaldolase (<i>tald-1</i>) deficiency impairs mitochondrial function <i>in vivo</i>, as evidenced by altered mitochondrial morphology, decreased respiration, and increased cellular H₂O₂ levels. Lifespan extension from knockdown of <i>tald-1</i> is associated with an oxidative stress response involving p38 and c-Jun N-terminal kinase (JNK) MAPKs and a starvation-like response regulated by the transcription factor EB (TFEB) homolog HLH-30. The latter response promotes autophagy and increases expression of the flavin-containing monooxygenase 2 (<i>fmo-2</i>). We conclude that cytosolic redox established through the PPP is a key regulator of mitochondrial function and defines a new mechanism for mitochondrial regulation of longevity.</p></div

Transaldolase deficiency causes a starvation-like response that decreases animal fat content and rewires lipid metabolism gene expression.

<p><b>(A)</b> Intestinal fat staining decreases from RNAi knockdown of <i>tald-1</i> or <i>cco-1</i>. Oil Red O (ORO) staining was performed on day 3 from hatching animals propagated at 20°C. Scale bar, 50 μm. <b>(B)</b> Quantification of ORO staining within anterior intestine (N = 2 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(C)</b> RNAi knockdown of <i>tald-1</i> causes an increase in adipose triglyceride lipase ATGL-1 protein levels. Scale bar, 200 μm. <b>(D)</b> Mean relative fluorescence of ATGL-1::GFP signal in animals grown on <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i>. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 4 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(E)</b> RNAi knockdown of <i>tald-1</i> or <i>cco-1</i> causes a decrease in stearoyl-CoA desaturase <i>fat-7p</i>::<i>gfp</i> reporter expression. Scale bar, 200 μm. <b>(F)</b> Mean relative fluorescence of <i>fat-7p</i>::<i>gfp</i> reporter animals grown on <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i>. Fluorescence is calculated relative to <i>EV(RNAi)</i> controls (N = 3 independent experiments, pooled individual worm values, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(G)</b> Gene expression of starvation-responsive lipid metabolism genes is altered in <i>tald-1(RNAi)</i> animals. Log2 fold change calculated to emphasize the increases and decreases in gene expression levels from RNAi treatments (N = 6–8 independent experiments, error bars indicate s.e.m., paired student’s t-tests with Bonferroni’s correction). <b>(H)</b> RNAi knockdown of <i>tald-1</i> does not robustly extend lifespan of BD animals. N2 fed <i>EV(RNAi)</i> (mean 18.2±0.2 days, n = 161), N2 fed <i>tald-1(RNAi)</i> (mean 20.4±0.2 days, n = 151), BD animals developed on <i>EV(RNAi)</i> (mean 20.2±0.2 days, n = 123), BD animals developed on <i>tald-1(RNAi)</i> (mean 21.5±0.3 days, n = 150). Lifespans were performed at 25°C, with one experiment shown. <b>(I)</b> RNAi knockdown of <i>cco-1</i> extends lifespan dissimilar from BD. N2 fed <i>EV(RNAi)</i> (mean 18.2±0.2 days, n = 161), N2 fed <i>cco-1(RNAi)</i> (mean 24±0.3 days, n = 156), BD animals developed on <i>EV(RNAi)</i> (mean 20.2±0.2 days, n = 123), BD animals developed on <i>cco-1(RNAi)</i> (mean 27.1±0.3 days, n = 148). Lifespans were performed at 25°C, with one experiment shown. <b>(J)</b> RNAi knockdown of <i>tald-1</i> does not require NHR-49 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 17.5±0.1 days, n = 366), N2 fed <i>tald-1(RNAi)</i> (mean 20.1±0.1 days, n = 397), <i>nhr-49(nr2041)</i> fed <i>EV(RNAi)</i> (mean 11.5±0.1 days, n = 310), <i>nhr-49(nr2041)</i> fed <i>tald-1(RNAi)</i> (mean 12.9±0.1 days, n = 333). Lifespans were performed at 25°C, with pooled data from three independent experiments shown. <b>(K)</b> RNAi knockdown of <i>cco-1</i> does not require NHR-49 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 17±0.1 days, n = 532), N2 fed <i>cco-1(RNAi)</i> (mean 22.6±0.2 days, n = 344), <i>nhr-49(nr2041)</i> fed <i>EV(RNAi)</i> (mean 11.1±0.1 days, n = 495), <i>nhr-49(nr2041)</i> fed <i>cco-1(RNAi)</i> (mean 15±0.1 days, n = 489). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>. In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Transaldolase deficiency alters mitochondrial morphology and decreases <i>in vivo</i> mitochondrial respiration.

<p><b>(A)</b> Diagram depicting the posterior intestinal cells that were visualized for mitochondrial morphology. <b>(B)</b> Intestinal mitochondrial morphology is altered by <i>tald-1(RNAi)</i> and <i>cco-1(RNAi)</i>. The top panel represents a single 0.34 μm slice imaged using confocal microscopy, with a magnified area displayed in a white dotted box to highlight morphology differences. The bottom panel consists of a max intensity projection of five z-slices to emphasize mitochondrial content in these cells. Scale bar, 10 μm. <b>(C)</b> Quantification of percent mitochondrial area per cell. (N = 2 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(D)</b> Mitochondrial morphology changes from <i>tald-1(RNAi)</i> are regulated by DRP-1. RNAi treatments include <i>EV(RNAi)</i>, <i>tald-1(RNAi)</i> [50:50 with <i>EV(RNAi)</i>], <i>drp-1(RNAi)</i> [50:50 with <i>EV(RNAi)</i>], and <i>tald-1(RNAi)</i> [50:50 with <i>drp-1(RNAi)</i>]. Scale bar, 10 μm. <b>(E)</b> Oxygen consumption rate decreases with <i>tald-1(RNAi)</i> and <i>cco-1(RNAi)</i>. OCR was measured using the Seahorse XF Analyzer and normalized to animal number (N = 6 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(F)</b> P/O ratio (the ATP produced per oxygen atom reduced), <b>(G)</b> respiratory control index (State 3:State 4 rates), <b>(H)</b> malate-driven respiration (Complex I-IV), succinate-driven respiration (Complex II-IV), and TMPD/ascorbate-driven respiration (Complex IV) were measured using the OXPHOS assay on isolated mitochondria from RNAi treated animals. Respiratory rates were measured as rate of disappearance of oxygen (nmol[O₂]) per minute per mg protein (N = 4 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). Also, in this figure, color coating of bars and lines reflect the legend in (C).</p

Lifespan extension from <i>tald-1(RNAi)</i> or <i>cco-1(RNAi)</i> requires stress-activated MAPKs.

<p><b>(A)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the JNK MAPK JNK-1. N2 fed <i>EV(RNAi)</i> (mean 17.2±0.1 days, n = 506), N2 fed <i>tald-1(RNAi)</i> (mean 20.4±0.1 days, n = 500), <i>jnk-1(gk7)</i> fed <i>EV(RNAi)</i> (mean 17±0.1 days, n = 582), <i>jnk-1(gk7)</i> fed <i>tald-1(RNAi)</i> (mean 18.1±0.1 days, n = 488). Lifespans were performed at 25°C, with pooled data from five independent experiments shown. <b>(B)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the JNK MAPK JNK-1. N2 fed <i>EV(RNAi)</i> (mean 16.9±0.1 days, n = 494), N2 fed <i>cco-1(RNAi)</i> (mean 22.7±0.2 days, n = 431), <i>jnk-1(gk7)</i> fed <i>EV(RNAi)</i> (mean 16.1±0.1 days, n = 594), <i>jnk-1(gk7)</i> fed <i>cco-1(RNAi)</i> (mean 19.9±0.2 days, n = 408). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(C)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the JNK MAPK KGB-1. N2 fed <i>EV(RNAi)</i> (mean 15±0.1 days, n = 630), N2 fed <i>tald-1(RNAi)</i> (mean 18.7±0.1 days, n = 657), <i>kgb-1(um3)</i> fed <i>EV(RNAi)</i> (mean 13.1±0.1 days, n = 580), <i>kgb-1</i> fed <i>tald-1(RNAi)</i> (mean 11.9±0.1 days, n = 600). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(D)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the JNK MAPK KGB-1. N2 fed <i>EV(RNAi)</i> (mean 15±0.1 days, n = 630), N2 fed <i>cco-1(RNAi)</i> (mean 23.2±0.2 days, n = 511), <i>kgb-1(um3)</i> fed <i>EV(RNAi)</i> (mean 13.1±0.1 days, n = 580), <i>kgb-1</i> fed <i>cco-1(RNAi)</i> (mean 15.8±0.2 days, n = 501). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(E)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the p38 MAPK PMK-1. N2 fed <i>EV(RNAi)</i> (mean 16.8±0.1 days, n = 494), N2 fed <i>tald-1(RNAi)</i> (mean 19.3±0.1 days, n = 460), <i>pmk-1(km25)</i> fed <i>EV(RNAi)</i> (mean 14.3±0.1 days, n = 514), <i>pmk-1(km25)</i> fed <i>tald-1(RNAi)</i> (mean 14±0.1 days, n = 525). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(F)</b> RNAi knockdown of <i>cco-1</i> does not require the p38 MAPK PMK-1 for lifespan extension. N2 fed <i>EV(RNAi)</i> (mean 16±0.1 days, n = 575), N2 fed <i>cco-1(RNAi)</i> (mean 22.3±0.2 days, n = 448), <i>pmk-1(km25)</i> fed <i>EV(RNAi)</i> (mean 13.8±0.1 days, n = 609), <i>pmk-1(km25)</i> fed <i>cco-1(RNAi)</i> (mean 18.7±0.1 days, n = 535). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(G)</b> RNAi knockdown of <i>tald-1</i> extends lifespan through the MAP3K NSY-1. N2 fed <i>EV(RNAi)</i> (mean 14.6±0.1 days, n = 542), N2 fed <i>tald-1(RNAi)</i> (mean 17.2±0.1 days, n = 599), <i>nsy-1(ag3)</i> fed <i>EV(RNAi)</i> (mean 14.9±0.1 days, n = 473), <i>nsy-1(ag3)</i> fed <i>tald-1(RNAi)</i> (mean 14.4±0.1 days, n = 508). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. <b>(H)</b> RNAi knockdown of <i>cco-1</i> extends lifespan partially through the MAP3K NSY-1. N2 fed <i>EV(RNAi)</i> (mean 14.6±0.1 days, n = 542), N2 fed <i>cco-1(RNAi)</i> (mean 22.5±0.2 days, n = 454), <i>nsy-1(ag3)</i> fed <i>EV(RNAi)</i> (mean 14.9±0.1 days, n = 473), <i>nsy-1(ag3)</i> fed <i>cco-1(RNAi)</i> (mean 18.5±0.2 days, n = 458). Lifespans were performed at 25°C, with pooled data from four independent experiments shown. Lifespans in this figure are indicated as mean±s.e.m. and statistical analysis is provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006695#pgen.1006695.s011" target="_blank">S1 Table</a>.</p

Redox stress is downstream of transaldolase deficiency.

<p><b>(A)</b> H₂O₂ levels increase from RNAi knockdown of <i>tald-1</i> or <i>cco-1</i> (N = 7 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(B)</b> NADPH levels decrease from RNAi knockdown of <i>tald-1</i> (N = 5+ biological replicates, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). <b>(C)</b> RNAi knockdown of <i>tald-1</i> causes sensitivity to paraquat (PQ). Percent survival of N2 worms grown on RNAi bacteria and 10 mM PQ was measured over seven days. Survival analyses were performed at 25°C (N = 6 independent experiments, error bars indicate s.e.m., student’s t-test with Bonferroni’s correction). In this figure, statistics are displayed as: * <i>p</i><0.05, ** <i>p</i><0.01, *** <i>p</i><0.001.</p

Model of transaldolase deficiency mediated longevity.

<p>Reduced activity of the pentose phosphate pathway enzyme transaldolase has several consequences, including inhibition of mitochondrial respiration, induction of a mitochondrial stress response, alterations in redox homeostasis, and activation of a starvation-like metabolic response. Lifespan extension in response to transaldolase deficiency appears to be mediated by both MAPK signaling and HLH-30 mediated induction of autophagy and activation of FMO-2.</p