VAPB/ALS8 MSP Ligands Regulate Striated Muscle Energy Metabolism Critical for Adult Survival in <i>Caenorhabditis elegans</i>

<div><p>Mutations in VAPB/ALS8 are associated with amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), two motor neuron diseases that often include alterations in energy metabolism. We have shown that <i>C. elegans</i> and Drosophila neurons secrete a cleavage product of VAPB, the N-terminal major sperm protein domain (vMSP). Secreted vMSPs signal through Roundabout and Lar-like receptors expressed on striated muscle. The muscle signaling pathway localizes mitochondria to myofilaments, alters their fission/fusion balance, and promotes energy production. Here, we show that neuronal loss of the <i>C. elegans</i> VAPB homolog triggers metabolic alterations that appear to compensate for muscle mitochondrial dysfunction. When vMSP levels drop, cytoskeletal or mitochondrial abnormalities in muscle induce elevated DAF-16, the Forkhead Box O (FoxO) homolog, transcription factor activity. DAF-16 promotes muscle triacylglycerol accumulation, increases ATP levels in adults, and extends lifespan, despite reduced muscle mitochondria electron transport chain activity. Finally, <i>Vapb</i> knock-out mice exhibit abnormal muscular triacylglycerol levels and FoxO target gene transcriptional responses to fasting and refeeding. Our data indicate that impaired vMSP signaling to striated muscle alters FoxO activity, which affects energy metabolism. Abnormalities in energy metabolism of ALS patients may thus constitute a compensatory mechanism counterbalancing skeletal muscle mitochondrial dysfunction.</p></div

Effect of Arp2/3 inactivation on muscle fat levels.

<p>DIC and fluorescent images of muscle in live 3-day-old hermaphrodite worms fed Bodipy-FAs. <i>arx-2</i> encodes the Arp2 component of the Arp2/3 complex. Arrowheads indicate Bodipy-FA-stained fat droplets. Bar, 5 µm.</p

Effect of <i>Vapb</i> ablation on fasting/refeeding energy metabolism in mice.

<p>(A) TAG concentration in GA muscle and liver of wild-type (+/+) and <i>Vapb</i> knock-out (−/−) mice after 24-hour fasting (red) or 24 hours fasting followed by 6 hours of refeeding (blue). (B and C) Quantitative RT-PCR of indicated genes in liver (B) and TA muscle (C) of wild-type (+/+) and <i>Vapb</i> knock-out (−/−) mice after 24-hour fasting (red) or 24 hours fasting followed by 6 hours of refeeding (blue). Relative mRNA levels are shown on the Y-axis. #, <i>P</i><0.05 compared to fed mice of the same genotype. <i>*</i>, <i>P</i><0.05 compared to +/+ under the same condition.</p

Effect of tissue-specific <i>vpr-1</i> expression on fat levels.

<p>(A) DIC images of muscle in live wild-type and <i>vpr-1(tm1411)</i> mutant hermaphrodites expressing wild-type VPR-1 or VPR-1(P56S) under indicated tissue-specific promoters. Arrowheads indicate lipid-like droplets. Bar, 5 µm. (B) Sudan Black B staining images of <i>vpr-1</i> mutants expressing <i>vpr-1</i> under the <i>unc-119</i> pan-neuronal promoter. Arrows indicate muscle fat droplets. Anterior is to the left in all panels. Wild-type controls (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003738#pgen.1003738.s003" target="_blank">Figure S3</a>) are similar to transgenic <i>vpr-1(tm1411)</i> mutants expressing <i>unc119p::vpr-1</i>. Low magnification bars, 50 µm; high magnification bars, 25 µm.</p

DAF-16 localization and activity in wild-type and mutant worms.

<p>(A) Transgenic strains expressing DAF-16::GFP under its endogenous promoter. Transgenic controls raised at 20°C are similar to those raised at 20°C then shifted to 35°C for 30 minutes (see panel B for quantification). Close up images of boxed areas are shown. Anterior is to the left in all panels. Low magnification bar, 50 µm; high magnification bar, 25 µm. (B) Quantification of DAF-16::GFP localization in control (n = 157) and <i>vpr-1(tm1411)</i> mutants (n = 49). (−), incubation under normal growth condition; (+), incubation at 35°C for 30 minutes. (C) Magnified images showing transgenic lines expressing GFP under the <i>sod-3</i> promoter. <i>arx-2</i> encodes Arp2. Arrows indicate vulva muscle region. Anterior is to the left in all panels. Bar, 50 µm.</p

Effect of DAF-16 inactivation on muscle mitochondria.

<p>(A) Muscle mitochondrial tubules in indicated genotypes visualized using mitoGFP. Arrowheads indicate fat droplets. Asterisks indicate nucleus. Bar, 5 µm. (B) MitoTracker CMXRos staining of wild-type and mutant muscle. Asterisks indicate nucleus. Bar, 5 µm. (C and D) Oxygen consumption rates of wild-type and mutant hermaphrodites. Measured consumption rates were normalized by protein content (C) or number of worms (D). Error bars represent SD. *, <i>P</i><0.001 compared to wild type. Oxygen consumption rate of wild-type and <i>vpr-1(tm1411)</i> mutants includes published data <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003738#pgen.1003738-Han2" target="_blank">[15]</a> measured together with <i>vpr-1(tm1411) daf-16(mu86)</i> mutants.</p

Fat levels in body wall muscle of wild-type and <i>vpr-1</i> mutant worms.

<p>(A) DIC images of muscle in live adult hermaphrodites. Arrowheads indicate lipid-like droplets. Bar, 5 µm. (B) Transmission electron micrographs of body wall muscle cytoplasm in wild-type and <i>vpr-1(tm1411)</i> mutant hermaphrodites. Light blue color demarcates muscle boundary. L, Lipid-like droplet. Bar, 0.5 µm. (C) Fluorescent images of muscle in live adult hermaphrodites fed Bodipy-FAs. Close-up images of boxed areas are shown below. Arrowheads indicate examples of Bodipy-FA-stained droplets. Anterior is to the left in all panels. Bars, 50 µm. (D) High magnification images of muscle showing Bodipy-FA-stained fluorescent droplets and droplets observed by DIC microscopy. Bar, 5 µm. (E) Comparison of total ion chromatograms of wild-type and <i>vpr-1(tm1411)</i> mutant adults extracts for 18∶0 TAG (Neutral Loss 284) and phosphatidylethanolamine (Neutral Loss 141).</p

Effect of DAF-16 inactivation on ATP level and lifespan.

<p>(A) ATP concentration in wild-type and <i>vpr-1(tm1411)</i> mutant adult extracts. *, <i>P</i><0.001 compared to wild type. Error bars represent SD. ATP concentration of wild-type and <i>vpr-1(tm1411)</i> mutants at 1-day-old adults include published data <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003738#pgen.1003738-Han2" target="_blank">[15]</a> measured together with <i>vpr-1(tm1411) daf-16(mu86)</i> mutants. (B) Lifespan measurements of indicated genotypes. The lifespan of <i>daf-16(mu86)</i> mutants (not shown) was similar to the wild type, as previously shown.</p