High-throughput profiling of caenorhabditis elegans starvation-responsive microRNAs

MicroRNAs (miRNAs) are non-coding RNAs of ~22 nucleotides in length that regulate gene expression by interfering with the stability and translation of mRNAs. Their expression is regulated during development, under a wide variety of stress conditions and in several pathological processes. In nature, animals often face feast or famine conditions. We observed that subjecting early L4 larvae from Caenorhabditis elegans to a 12-hr starvation period produced worms that are thinner and shorter than well-fed animals, with a decreased lipid accumulation, diminished progeny, reduced gonad size, and an increased lifespan. Our objective was to identify which of the 302 known miRNAs of C. elegans changed their expression under starvation conditions as compared to well-fed worms by means of deep sequencing in early L4 larvae. Our results indicate that 13 miRNAs (miR-34-3p, the family of miR-35-3p to miR-41-3p, miR-39-5p, miR-41-5p, miR-240-5p, miR-246-3p and miR-4813-5p) were upregulated, while 2 miRNAs (let-7-3p and miR-85-5p) were downregulated in 12-hr starved vs. well-fed early L4 larvae. Some of the predicted targets of the miRNAs that changed their expression in starvation conditions are involved in metabolic or developmental process. In particular, miRNAs of the miR-35 family were upregulated 6-20 fold upon starvation. Additionally, we showed that the expression of gld-1, important in oogenesis, a validated target of miR-35-3p, was downregulated when the expression of miR-35-3p was upregulated. The expression of another reported target, the cell cycle regulator lin-23, was unchanged during starvation. This study represents a starting point for a more comprehensive understanding of the role of miRNAs during starvation in C. elegans

Steroid hormone signalling links reproduction to lifespan in dietary-restricted <em>Caenorhabditis elegans</em>.

Dietary restriction (DR) increases healthspan and longevity in many species, including primates, but it is often accompanied by impaired reproductive function. Whether signals associated with the reproductive system contribute to or are required for DR effects on lifespan has not been established. Here we show that expression of the cytochrome P450 DAF-9/CYP450 and production of the steroid hormone &Delta;(7)-dafachronic acid (DA) are increased in C. elegans subjected to DR. DA signalling through the non-canonical nuclear hormone receptor NHR-8/NHR and the nutrient-responsive kinase let-363/mTOR is essential for DR-mediated longevity. Steroid signalling also affects germline plasticity in response to nutrient deprivation and this is required to achieve lifespan extension. These data demonstrate that steroid signalling links germline physiology to lifespan when nutrients are limited, and establish a central role for let-363/mTOR in integrating signals derived from nutrients and steroid hormones

Fast separation and quantification of steroid hormones &Delta;4- and &Delta;7-dafachronic acid in <em>Caenorhabditis elegans</em>.

Separation of isomeric molecular species, e.g. double bond position isomers, is a challenging task for liquid chromatography. The two steroid hormones &Delta;4- and &Delta;7-dafachronic acid (DA) represent such an isomeric pair. DAs are 3-ketosteroids found in the nematode Caenorhabditis elegans and generated from cholesterol. &Delta;4- and &Delta;7-DA have important biological activities and are produced by two different biological pathways in C. elegans. Here we have described a fast separation method for these two isomers using a 1.3&mu;m core-shell particle in less than 10min together with a simple MeOH extraction. Using this method we were able to independently quantify &Delta;4- and &Delta;7-DA in C. elegans independently from each other and limits of detection of about 5ng/ml for each isomer were achieved with a good day-to-day reproducibility. As proof-of-principle the method has been applied to the quantification of DAs in worms fed ad libitum or under bacterial deprivation

The homeodomain-interacting protein kinase HPK-1 preserves protein homeostasis and longevity through master regulatory control of the HSF-1 chaperone network and TORC1-restricted autophagy in <i>Caenorhabditis elegans</i>

<div><p>An extensive proteostatic network comprised of molecular chaperones and protein clearance mechanisms functions collectively to preserve the integrity and resiliency of the proteome. The efficacy of this network deteriorates during aging, coinciding with many clinical manifestations, including protein aggregation diseases of the nervous system. A decline in proteostasis can be delayed through the activation of cytoprotective transcriptional responses, which are sensitive to environmental stress and internal metabolic and physiological cues. The homeodomain-interacting protein kinase (<i>hipk</i>) family members are conserved transcriptional co-factors that have been implicated in both genotoxic and metabolic stress responses from yeast to mammals. We demonstrate that constitutive expression of the sole <i>Caenorhabditis elegans</i> Hipk homolog, <i>hpk-1</i>, is sufficient to delay aging, preserve proteostasis, and promote stress resistance, while loss of <i>hpk-1</i> is deleterious to these phenotypes. We show that HPK-1 preserves proteostasis and extends longevity through distinct but complementary genetic pathways defined by the heat shock transcription factor (HSF-1), and the target of rapamycin complex 1 (TORC1). We demonstrate that HPK-1 antagonizes sumoylation of HSF-1, a post-translational modification associated with reduced transcriptional activity in mammals. We show that inhibition of sumoylation by RNAi enhances HSF-1-dependent transcriptional induction of chaperones in response to heat shock. We find that <i>hpk-1</i> is required for HSF-1 to induce molecular chaperones after thermal stress and enhances hormetic extension of longevity. We also show that HPK-1 is required in conjunction with HSF-1 for maintenance of proteostasis in the absence of thermal stress, protecting against the formation of polyglutamine (Q35::YFP) protein aggregates and associated locomotory toxicity. These functions of HPK-1/HSF-1 undergo rapid down-regulation once animals reach reproductive maturity. We show that HPK-1 fortifies proteostasis and extends longevity by an additional independent mechanism: induction of autophagy. HPK-1 is necessary for induction of autophagosome formation and autophagy gene expression in response to dietary restriction (DR) or inactivation of TORC1. The autophagy-stimulating transcription factors <i>pha-4</i>/FoxA and <i>mxl-2</i>/Mlx, but not <i>hlh-30</i>/TFEB or the nuclear hormone receptor <i>nhr-62</i>, are necessary for extended longevity resulting from HPK-1 overexpression. HPK-1 expression is itself induced by transcriptional mechanisms after nutritional stress, and post-transcriptional mechanisms in response to thermal stress. Collectively our results position HPK-1 at a central regulatory node upstream of the greater proteostatic network, acting at the transcriptional level by promoting protein folding via chaperone expression, and protein turnover via expression of autophagy genes. HPK-1 therefore provides a promising intervention point for pharmacological agents targeting the protein homeostasis system as a means of preserving robust longevity.</p></div

<i>pha-4</i> and <i>mxl-2</i> intersect with <i>hpk-1</i> in the maintenance of proteostasis.

<p>(A-B) Loss of <i>pha-4</i> (red traces) does not increase foci formation (A) or the onset of paralysis (B) in the absence of <i>hpk-1</i> (open circles/squares). (C-D) Loss of <i>mxl-2</i> (blue traces) does not increase foci formation (C) or the onset of paralysis (D) in the absence of <i>hpk-1</i> (open circles/squares). (E-F) Loss of <i>hlh-30</i> (green traces) does not increase foci formation (E) but delays the onset of paralysis in the absence of <i>hpk-1</i> (F) (open circles/squares). For foci formation, data are the mean and standard error of the mean (S.E.M.) of at least 15 animals from one representative trial; three independent experiments were performed. ***, **, and * indicate p-values of <0.001, <0.01, and <0.05, respectively. For paralysis, data is representative of one of two trials performed with the same conditions. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s014" target="_blank">S3 Table</a> for additional details.</p

HPK-1 colocalizes with HSF-1 in <i>C</i>. <i>elegans</i> neurons.

<p>(A-C) HPK-1 and HSF-1 colocalize in neurons under basal conditions. Representative image of transgenic animal co-expressing <i>Phpk-1</i>::<i>HPK-1</i>::<i>tdtomato</i>,<i>Phsf-1</i>::<i>HSF-1</i>::<i>GFP</i>: (A) red fluorescence, (B) green fluorescence, and (C) overlay.</p

HPK-1 but not HSF-1 is essential for autophagosome formation.

<p>(A-F) Inactivation of <i>hpk-1</i> but not <i>hsf-1</i> disrupts autophagosome formation after bacterial deprivation (BD) as visualized by puncta formation for the autophagosomal reporter <i>Plgg-1</i>::<i>LGG-1</i>::<i>GFP</i> (Atg8p/MAP-LC3) [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.ref018" target="_blank">18</a>]. (G) Quantification of LGG-1:GFP foci in L3 stage animals under <i>ad libitum</i> (AL) and bacterial deprivation (BD) conditions. BD was imposed by removal from bacterial food for 6 hours prior to scoring puncta formation. Plotted are the mean number of LGG-1::GFP puncta/seam cell visualized +/-S.D. *** indicates a p-value of <0.001 (Student’s t-test). Summary data provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1007038#pgen.1007038.s015" target="_blank">S4 Table</a>.</p

Heat shock induction of <i>hsp-16</i>.<i>2</i> is enhanced by <i>smo-1(RNAi)</i>.

<p>(A-D) DIC and GFP overlay for <i>Phsp-16</i>.<i>2</i>::<i>GFP</i> worms on empty vector (A, B) or <i>smo-1(RNAi</i>) (C, D) with (+HS) and without (-HS) heat shock. Scale bar = 100μm. (E) Western blot for HSP-16.2, GFP and β-actin from <i>hsp-16</i>.<i>2p</i>::<i>GFP</i> worms grown on empty vector (EV) without heat shock (no HS) or with heat shock (EV), <i>GFP(RNAi)</i>, <i>hsf-1(RNAi)</i> or <i>smo-1(RNAi)</i>. Fold-increases for HSP-16.2/actin on <i>smo-1(RNAi)</i> relative to EV in three independent replicates were 2.4, 4.7, and 1.8.</p

HPK-1 is essential for the transcriptional activation of autophagy.

<p>(A) <i>hpk-1</i> is necessary for the induction the autophagy genes <i>atg-18</i> and <i>bec-1</i> (Beclin1) in response to inactivation of TORC1 by <i>daf-15(RNAi)</i> (** indicates p<0.01, Student’s t-test). (B) In contrast, decreased TORC1 signaling represses the expression of the translation initiation factor genes <i>ifg-1</i> and <i>iftb-1</i> independently from <i>hpk-1</i>. (C) Similarly, TORC1 inhibition mildly induces <i>hsp-16</i>.<i>2</i> and <i>hsp-70</i> independently from <i>hpk-1</i>. Columns labeled <i>hpk-1</i> indicate <i>hpk-1(pk1393)</i>. Expression levels are presented as fold change +/- S.D. normalized to <i>cdc-42</i> and averaged across four independent experiments.</p

HPK-1 delays aging and maintains proteostasis by potentiating TORC1 mediated autophagy and blocking HSF-1 inactivation through sumoylation.

<p>Model of HPK-1 functions in longevity control: HPK-1 functions as a central hub to maintain proteostasis by preventing sumoylation and inactivation of HSF-1 and by stimulating the expression of autophagy genes by <i>pha-4</i> and <i>mxl-2</i>. TORC1 inhibits <i>hpk-1</i> expression to limit the induction of autophagy genes under basal conditions. Under nutrient stress, TORC1 is inactivated resulting in increased <i>hpk-1</i> expression, which promotes autophagy gene expression through PHA-4/FoxA and MXL-2/Mlx. Thermal stress increases HPK-1 protein levels to reduce the threshold of activation of the heat shock response, and HPK-1 promotes longevity through modulation of HSF-1 activity under normal growth conditions.</p