Neuronal CRTC-1 Governs Systemic Mitochondrial Metabolism and Lifespan via a Catecholamine Signal

SummaryLow energy states delay aging in multiple species, yet mechanisms coordinating energetics and longevity across tissues remain poorly defined. The conserved energy sensor AMP-activated protein kinase (AMPK) and its corresponding phosphatase calcineurin modulate longevity via the CREB regulated transcriptional coactivator (CRTC)-1 in C. elegans. We show that CRTC-1 specifically uncouples AMPK/calcineurin-mediated effects on lifespan from pleiotropic side effects by reprogramming mitochondrial and metabolic function. This pro-longevity metabolic state is regulated cell nonautonomously by CRTC-1 in the nervous system. Neuronal CRTC-1/CREB regulates peripheral metabolism antagonistically with the functional PPARα ortholog, NHR-49, drives mitochondrial fragmentation in distal tissues, and suppresses the effects of AMPK on systemic mitochondrial metabolism and longevity via a cell-nonautonomous catecholamine signal. These results demonstrate that while both local and distal mechanisms combine to modulate aging, distal regulation overrides local contribution. Targeting central perception of energetic state is therefore a potential strategy to promote healthy aging

Characterization of the Proteostasis Roles of Glycerol Accumulation, Protein Degradation and Protein Synthesis during Osmotic Stress in C. elegans

Exposure of C. elegans to hypertonic stress-induced water loss causes rapid and widespread cellular protein damage. Survival in hypertonic environments depends critically on the ability of worm cells to detect and degrade misfolded and aggregated proteins. Acclimation of C. elegans to mild hypertonic stress suppresses protein damage and increases survival under more extreme hypertonic conditions. Suppression of protein damage in acclimated worms could be due to 1) accumulation of the chemical chaperone glycerol, 2) upregulation of protein degradation activity, and/or 3) increases in molecular chaperoning capacity of the cell. Glycerol and other chemical chaperones are widely thought to protect proteins from hypertonicity-induced damage. However, protein damage is unaffected by gene mutations that inhibit glycerol accumulation or that cause dramatic constitutive elevation of glycerol levels. Pharmacological or RNAi inhibition of proteasome and lyosome function and measurements of cellular protein degradation activity demonstrated that upregulation of protein degradation mechanisms plays no role in acclimation. Thus, changes in molecular chaperone capacity must be responsible for suppressing protein damage in acclimated worms. Transcriptional changes in chaperone expression have not been detected in C. elegans exposed to hypertonic stress. However, acclimation to mild hypertonicity inhibits protein synthesis 50–70%, which is expected to increase chaperone availability for coping with damage to existing proteins. Consistent with this idea, we found that RNAi silencing of essential translational components or acute exposure to cycloheximide results in a 50–80% suppression of hypertonicity-induced aggregation of polyglutamine-YFP (Q35::YFP). Dietary changes that increase protein production also increase Q35::YFP aggregation 70–180%. Our results demonstrate directly for the first time that inhibition of protein translation protects extant proteins from damage brought about by an environmental stressor, demonstrate important differences in aging- versus stress-induced protein damage, and challenge the widely held view that chemical chaperones are accumulated during hypertonic stress to protect protein structure/function

Effect of acclimation to mild hypertonic stress on protein degradation activity.

<p><i>A:</i> Effect of treatment of control and acclimated worms with vehicle only (1% DMSO) or 20 mM chloroquine (CQ) and 100 µM MG-132 on spontaneous aggregation of Q35::YFP. (<i>n</i> = 9–15). *P<0.003 compared to vehicle-treated control worms. **P<0.0001 compared to drug-treated control worms. <i>B:</i> Effect of RNAi silencing of Hos genes on spontaneous Q35::YFP aggregation in control and acclimated worms. Animals were fed bacteria expressing nonspecific (control) dsRNA or dsRNA targeting proteasome (<i>pas-6</i> and <i>rpn-3</i>) and lysosome (<i>vha-13</i>) components, or a putative lysosomal serine carboxypeptidase (F13D12.6). (n = 16–51). *P<0.001 compared to control or acclimated worms fed a nonspecific dsRNA. **P<0.03 compared to unacclimated <i>vha-13(RNAi)</i> worms. <i>C:</i> Percent of red mutant ubiquitin (UbG76V) tagged Dendra2 remaining in body wall muscle cells 24 h after photoconversion in control and 200 mM NaCl acclimated worms exposed to control or hypertonic growth media. Control and acclimated animals were exposed to 400 mM and 600 mM NaCl, respectively. (<i>n</i> = 3–8). *P<0.01 compared to unstressed worms. <i>D:</i> Percent change in ³⁵S-methionine labeled total protein levels in control and acclimated worms treated with 500 µg/ml of cycloheximide for 6 h to inhibit protein synthesis. (<i>n</i> = 3).</p

Effect of elevated glycerol levels on hypertonic stress-induced aggregation of endogenous proteins.

<p><i>A:</i> Effect of increasing NaCl concentrations on motility in control, acclimated, <i>osm-11</i> and acclimated <i>gpdh-1; gpdh-2</i> worms. <i>gpdh-1; gpdh-2</i> mutants lack functional GPDH-1 and GPDH-2 enzymes resulting in greatly reduced glycerol accumulation under hypertonic stress conditions <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034153#pone.0034153-Lamitina2" target="_blank">[12]</a>. (<i>n</i> = 5–18 experiments with 15–60 worms/experiment). <i>B:</i> Left panel, relative insoluble protein in acclimated <i>gpdh-1; gpdh-2</i> worms maintained in either 200 mM NaCl or exposed to 500 mM NaCl for 4 h. Insoluble protein was quantified as a fraction of total protein and is plotted relative to that observed in worms maintained on 200 mM NaCl. (<i>n</i> = 3 experiments with 4000–5000 worms/experiment). Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from acclimated <i>gpdh-1; gpdh-2</i> worms maintained in 200 mM NaCl or exposed to 500 mM NaCl. <i>C:</i> Left panel, relative insoluble protein in <i>osm-11</i> worms grown under control conditions (51 mM NaCl) or exposed to 700 mM NaCl for 4 h. Insoluble protein was quantified and plotted in the same manner as described in <i>B</i>. (<i>n</i> = 3 samples of 4000–5000 worms/sample). *P<0.03 compared to animals maintained on 51 mM NaCl. Right panel, examples of SDS-PAGE gels of total and detergent insoluble (insol.) proteins isolated from <i>osm-11</i> worms exposed to 51 or 700 mM NaCl.</p

Effect of elevated glycerol levels on aging-induced aggregation of Q35::YFP.

<p><i>A:</i> Whole worm glycerol levels in controls worms, worms acclimated to 200 mM NaCl and <i>osm-11</i> mutant animals. (<i>n</i> = 4 samples of ∼4000 worms/sample). <i>B:</i> Time course of aging-induced Q35::YFP aggregation in control, acclimated and <i>osm-11</i> mutant animals. (<i>n</i> = 7 experiments with 10–15 worms/experiment).</p

Effect of elevated glycerol levels on the properties of age-induced Q35::YFP aggregates.

<p><i>A:</i> Fluorescence micrographs of aggregate morphology in body wall muscle cells of control worms, worms acclimated to 200 mM NaCl and <i>osm-11</i> mutant animals. Images were taken from 7-day old adult worms. Scale bar is 10 µm. <i>B:</i> Time course of bleaching and fluorescence recovery in aggregates of young (4-day old) and old (10-day old) adult control, acclimated and <i>osm-11</i> worms (<i>n</i> = 3). <i>C:</i> Aggregate toxicity in control, acclimated and <i>osm-11</i> worms. Toxicity is measured as reductions in motility, which is mediated by body wall muscle cells. (<i>n</i> = 5–12).</p

Effect of acute hypertonic stress on ³⁵S-methionine incorporation into total protein.

<p>Worms were transferred to 200 mM NaCl agar plates at time 0 and ³⁵S-methionine incorporation into total protein was quantified 20 min and 1, 12 and 48 h after transfer. Values are expressed relative to unstressed control worms (i.e., time 0). (<i>n</i> = 3). *P<0.05 compared to control worms.</p

Synthetic Ligands of Cannabinoid Receptors Affect Dauer Formation in the Nematode Caenorhabditis elegans

Under adverse environmental conditions the nematode Caenorhabditis elegans can enter an alternate developmental stage called the dauer larva. To identify lipophilic signaling molecules that influence this process, we screened a library of bioactive lipids and found that AM251, an antagonist of the human cannabinoid (CB) receptor, suppresses dauer entry in daf-2 insulin receptor mutants. AM251 acted synergistically with glucose supplementation indicating that the metabolic status of the animal influenced the activity of this compound. Similarly, loss of function mutations in the energy-sensing AMP-activated kinase subunit, aak-2, enhanced the dauer-suppressing effects of AM251, while constitutive activation of aak-2 in neurons was sufficient to inhibit AM251 activity. Chemical epistasis experiments indicated that AM251 acts via G-protein signaling and requires the TGF-β ligand DAF-7, the insulin peptides DAF-28 and INS-6, and a functional ASI neuron to promote reproductive growth. AM251 also required the presence of the SER-5 serotonin receptor, but in vitro experiments suggest that this may not be via a direct interaction. Interestingly, we found that other antagonists of mammalian CB receptors also suppress dauer entry, while the nonselective CB receptor agonist, O-2545, not only inhibited the activity of AM251, but also was able to promote dauer entry when administered alone. Since worms do not have obvious orthologs of CB receptors, the effects of synthetic CBs on neuroendocrine signaling in C. elegans are likely to be mediated via another, as yet unknown, receptor mechanism. However, we cannot exclude the existence of a noncanonical CB receptor in C. elegans