Hydralazine targets cAMP-dependent protein kinase leading to sirtuin1/5 activation and lifespan extension in C. elegans.

Therapeutic activation of mitochondrial function has been suggested as an effective strategy to combat aging. Hydralazine is an FDA-approved drug used in the treatment of hypertension, heart failure and cancer. Hydralazine has been recently shown to promote lifespan in C. elegans, rotifer and yeast through a mechanism which has remained elusive. Here we report cAMP-dependent protein kinase (PKA) as the direct target of hydralazine. Using in vitro and in vivo models, we demonstrate a mechanism in which binding and stabilization of a catalytic subunit of PKA by hydralazine lead to improved mitochondrial function and metabolic homeostasis via the SIRT1/SIRT5 axis, which underlies hydralazine's prolongevity and stress resistance benefits. Hydralazine also protects mitochondrial metabolism and function resulting in restoration of health and lifespan in C. elegans under high glucose and other stress conditions. Our data also provide new insights into the mechanism(s) that explain various other known beneficial effects of hydralazine.

Author Correction: Small-molecule TFEB pathway agonists that ameliorate metabolic syndrome in mice and extend C. elegans lifespan.

The originally published version of this Article contained an error in the spelling of the author Nathaniel W. Oswald, which was incorrectly given as Nathaniel W. Olswald. This has now been corrected in both the PDF and HTML versions of the Article.

Hydralazine induces stress resistance and extends C. elegans lifespan by activating the NRF2/SKN-1 signalling pathway.

Nuclear factor (erythroid-derived 2)-like 2 and its Caenorhabditis elegans ortholog, SKN-1, are transcription factors that have a pivotal role in the oxidative stress response, cellular homeostasis, and organismal lifespan. Similar to other defense systems, the NRF2-mediated stress response is compromised in aging and neurodegenerative diseases. Here, we report that the FDA approved drug hydralazine is a bona fide activator of the NRF2/SKN-1 signaling pathway. We demonstrate that hydralazine extends healthy lifespan (~25%) in wild type and tauopathy model C. elegans at least as effectively as other anti-aging compounds, such as curcumin and metformin. We show that hydralazine-mediated lifespan extension is SKN-1 dependent, with a mechanism most likely mimicking calorie restriction. Using both in vitro and in vivo models, we go on to demonstrate that hydralazine has neuroprotective properties against endogenous and exogenous stressors. Our data suggest that hydralazine may be a viable candidate for the treatment of age-related disorders.

Small-molecule TFEB pathway agonists that ameliorate metabolic syndrome in mice and extend C. elegans lifespan.

Drugs that mirror the cellular effects of starvation mimics are considered promising therapeutics for common metabolic disorders, such as obesity, liver steatosis, and for ageing. Starvation, or caloric restriction, is known to activate the transcription factor EB (TFEB), a master regulator of lipid metabolism and lysosomal biogenesis and function. Here, we report a nanotechnology-enabled high-throughput screen to identify small-molecule agonists of TFEB and discover three novel compounds that promote autophagolysosomal activity. The three lead compounds include the clinically approved drug, digoxin; the marine-derived natural product, ikarugamycin; and the synthetic compound, alexidine dihydrochloride, which is known to act on a mitochondrial target. Mode of action studies reveal that these compounds activate TFEB via three distinct Ca2+-dependent mechanisms. Formulation of these compounds in liver-tropic biodegradable, biocompatible nanoparticles confers hepatoprotection against diet-induced steatosis in murine models and extends lifespan of Caenorhabditis elegans. These results support the therapeutic potential of small-molecule TFEB activators for the treatment of metabolic and age-related disorders.

Infection of Caenorhabditis elegans with Vesicular Stomatitis Virus via Microinjection.

Over the past 15 years, the free-living nematode, Caenorhabditis elegans has become an important model system for exploring eukaryotic innate immunity to bacterial and fungal pathogens. More recently, infection models using either natural or non-natural nematode viruses have also been established in C. elegans. These models offer new opportunities to use the nematode to understand eukaryotic antiviral defense mechanisms. Here we report protocols for the infection of C. elegans with a non-natural viral pathogen, vesicular stomatitis virus (VSV) through microinjection. We also describe how recombinant VSV strains encoding fluorescent or luciferase reporter genes can be used in conjunction with simple fluorescence-, survival-, and luminescence-based assays to identify host genetic backgrounds with differential susceptibilities to virus infection.

Reciprocal signaling by Wnt and Notch specifies a muscle precursor in the C. elegans embryo.

The MS blastomere produces one-third of the body wall muscles (BWMs) in the C. elegans embryo. MS-derived BWMs require two distinct cell-cell interactions, the first inhibitory and the second, two cell cycles later, required to overcome this inhibition. The inductive interaction is not required if the inhibitory signal is absent. Although the Notch receptor GLP-1 was implicated in both interactions, the molecular nature of the two signals was unknown. We now show that zygotically expressed MOM-2 (Wnt) is responsible for both interactions. Both the inhibitory and the activating interactions require precise spatiotemporal expression of zygotic MOM-2, which is dependent upon two distinct Notch signals. In a Notch mutant defective only in the inductive interaction, MS-derived BWMs can be restored by preventing zygotic MOM-2 expression, which removes the inhibitory signal. Our results suggest that the inhibitory interaction ensures the differential lineage specification of MS and its sister blastomere, whereas the inductive interaction promotes the expression of muscle-specifying genes by modulating TCF and ß-catenin levels. These results highlight the complexity of cell fate specification by cell-cell interactions in a rapidly dividing embryo.

The Maternal-to-Zygotic Transition in C. elegans.

In Caenorhabditis elegans, the first zygotic transcription can be detected in the 4-cell stage C. elegans embryo, a little over 2h after fertilization. However, early development until the onset of gastrulation at approximately the 28-cell stage takes place normally even in the absence of zygotic transcription. Therefore, posttranslational and posttranscriptional regulation of the maternal proteins and mRNAs, respectively, that are loaded into the developing oocytes is sufficient to direct development prior to gastrulation. Protein phosphorylation is extensively used throughout the C. elegans maternal-to-zygotic transition (MZT): (1) for maternal protein activation, (2) for coordination of the meiotic and mitotic cell cycle, (3) to mark specific proteins for degradation, and/or (4) to switch the biochemical activity of specific proteins. Maternally loaded mRNAs are regulated primarily by a set of maternal RNA-binding proteins (RBPs), each of which binds to sometimes overlapping target sequences within the mRNA 3'UTRs and either promotes or inhibits translation. Most maternal transcripts are uniformly distributed throughout the embryo but specific transcripts are translated only in certain blastomeres. This control is achieved by the asymmetric distribution of the maternal RBPs, such that the blastomere-specific constellation of RBPs present, and their relative levels, determines the translational readout for their target transcripts. In certain well-studied cases, such as the specification of the sole endodermal precursor in the 8-cell embryo, the maternal transcripts and proteins along with their directly targeted zygotic genes have been identified.

ß-Catenin-related protein WRM-1 is a multifunctional regulatory subunit of the LIT-1 MAPK complex.

Vertebrate ß-catenin has two functions, as a structural component of the adherens junction in cell adhesion and as the T-cell factor (TCF) transcriptional coactivator in canonical Wnt (wingless-related integration site) signaling. These two functions are split between three of the four ß-catenin-related proteins present in the round worm Caenorhabditis elegans. The fourth ß-catenin-related protein, WRM-1, exhibits neither of these functions. Instead, WRM-1 binds the MAPK loss of intestine 1 (LIT-1), and these two proteins have been shown to be essential for the transcription of Wnt target genes by phosphorylating and regulating the nuclear level of the sole worm TCF protein. We showed previously that WRM-1 binds to worm TCF and functions as the substrate-binding subunit for LIT-1. In this study, we show that phosphorylation of T220 in the activation loop is essential for LIT-1 kinase activity in vivo and in vitro. T220 can be phosphorylated either through LIT-1 autophosphorylation or directly by the upstream MAP3K MOM-4. Our data support a model in which WRM-1, which can undergo homotypic interaction, binds LIT-1 and thereby generates a kinase complex in which LIT-1 molecules are situated in a conformation enabling autophosphorylation as well as promoting phosphorylation of the T220 residue by MOM-4. In addition, we show that WRM-1 is essential for the translocation of the LIT-1 kinase complex to the nucleus, the site of its TCF substrate. To our knowledge, this is the first report of a MAP3K directly activating a MAPK by phosphorylation within the activation loop. This study should help uncover novel and as yet underappreciated functions of vertebrate ß-catenin.

Translational control of the oogenic program by components of OMA ribonucleoprotein particles in Caenorhabditis elegans.

The oocytes of most sexually reproducing animals arrest in meiotic prophase I. Oocyte growth, which occurs during this period of arrest, enables oocytes to acquire the cytoplasmic components needed to produce healthy progeny and to gain competence to complete meiosis. In the nematode Caenorhabditis elegans, the major sperm protein hormone promotes meiotic resumption (also called meiotic maturation) and the cytoplasmic flows that drive oocyte growth. Prior work established that two related TIS11 zinc-finger RNA-binding proteins, OMA-1 and OMA-2, are redundantly required for normal oocyte growth and meiotic maturation. We affinity purified OMA-1 and identified associated mRNAs and proteins using genome-wide expression data and mass spectrometry, respectively. As a class, mRNAs enriched in OMA-1 ribonucleoprotein particles (OMA RNPs) have reproductive functions. Several of these mRNAs were tested and found to be targets of OMA-1/2-mediated translational repression, dependent on sequences in their 3'-untranslated regions (3'-UTRs). Consistent with a major role for OMA-1 and OMA-2 in regulating translation, OMA-1-associated proteins include translational repressors and activators, and some of these proteins bind directly to OMA-1 in yeast two-hybrid assays, including OMA-2. We show that the highly conserved TRIM-NHL protein LIN-41 is an OMA-1-associated protein, which also represses the translation of several OMA-1/2 target mRNAs. In the accompanying article in this issue, we show that LIN-41 prevents meiotic maturation and promotes oocyte growth in opposition to OMA-1/2. Taken together, these data support a model in which the conserved regulators of mRNA translation LIN-41 and OMA-1/2 coordinately control oocyte growth and the proper spatial and temporal execution of the meiotic maturation decision.

Uncoupling different characteristics of the C. elegans E lineage from differentiation of intestinal markers.

In the 4-cell C. elegans embryo, a signal from P2 to its anterior sister, EMS, specifies the posterior daughter of EMS, E, as the sole founder cell for intestine. The P2-to-EMS signal restricts high level zygotic expression of the redundant GATA transcription factors, END-1 and END-3, to only the E lineage. Expression of END-1 or END-3 in early blastomeres is sufficient to drive intestinal differentiation. We show here that a number of E lineage characteristics, which are also regulated through P2-EMS signaling, can be uncoupled from intestine development, and each with a different sensitivity to specific perturbations of the P2-EMS signal. For example, we show that the extended cell cycle in Ea and Ep depends on the P2-induced high level expression of the cell cycle regulator, WEE-1.1, in E. A mutation in wee-1.1 results in shortened Ea and Ep cell cycles, but has no effect upon intestinal differentiation or embryogenesis. Furthermore, it has been shown previously that the total number of E lineage cell divisions is regulated by a mechanism dependent upon E being specified as the intestinal founder cell. We now show, however, that cell division counting can be uncoupled from intestine differentiation in the E lineage. Many mutations in P2-EMS signal genes exhibit nonfully-penetrant defects in intestinal differentiation. When embryos with those mutations generate intestinal cells, they often make too many intestinal cells. In addition, at the level of individual embryos, expression of end-1 and end-3, and another very early E-specific zygotic gene, sdz-23, exhibit stochastic and discordant defects in P2-EMS signaling mutants. We show here that sdz-23 is expressed close to wildtype levels in embryos deleted of both end-1 and end-3. sdz-23 does not appear to function in intestine development, raising the intriguing possibility that the P2-EMS interaction has downstream molecular consequences within the E lineage independent of end-1/3 and intestinal development.

Regulation of maternal Wnt mRNA translation in C. elegans embryos.

The restricted spatiotemporal translation of maternal mRNAs, which is crucial for correct cell fate specification in early C. elegans embryos, is regulated primarily through the 3'UTR. Although genetic screens have identified many maternally expressed cell fate-controlling RNA-binding proteins (RBPs), their in vivo targets and the mechanism(s) by which they regulate these targets are less clear. These RBPs are translated in oocytes and localize to one or a few blastomeres in a spatially and temporally dynamic fashion unique for each protein and each blastomere. Here, we characterize the translational regulation of maternally supplied mom-2 mRNA, which encodes a Wnt ligand essential for two separate cell-cell interactions in early embryos. A GFP reporter that includes only the mom-2 3'UTR is translationally repressed properly in oocytes and early embryos, and then correctly translated only in the known Wnt signaling cells. We show that the spatiotemporal translation pattern of this reporter is regulated combinatorially by a set of nine maternally supplied RBPs. These nine proteins all directly bind the mom-2 3'UTR in vitro and function as positive or negative regulators of mom-2 translation in vivo. The net translational readout for the mom-2 3'UTR reporter is determined by competitive binding between positive- and negative-acting RBPs for the 3'UTR, along with the distinct spatiotemporal localization patterns of these regulators. We propose that the 3'UTR of maternal mRNAs contains a combinatorial code that determines the topography of associated RBPs, integrating positive and negative translational inputs.

The oocyte-to-embryo transition.

The oocyte-to-embryo transition refers to the process whereby a fully grown, relatively quiescent oocyte undergoes maturation, fertilization, and is converted into a developmentally active, mitotically dividing embryo, arguably one of the most dramatic transitions in biology. This transition occurs very rapidly in Caenorhabditis elegans, with fertilization of a new oocyte occurring every 23 min and the first mitotic division occurring 45 min later. Molecular events regulating this transition must be very precisely timed. This chapter reviews our current understanding of the coordinated temporal regulation of different events during this transition. We divide the oocyte-to-embryo transition into a number of component processes, which are coordinated primarily through the MBK-2 kinase, whose activation is intimately tied to completion of meiosis, and the OMA-1/OMA-2 proteins, whose expression and functions span multiple processes during this transition. The oocyte-to-embryo transition occurs in the absence of de novo transcription, and all the factors required for the process, whether mRNA or protein, are already present within the oocyte. Therefore, all regulation of this transition is posttranscriptional. The combination of asymmetric partitioning of maternal factors, protein modification-mediated functional switching, protein degradation, and highly regulated translational repression ensure a smooth oocyte-to-embryo transition. We will highlight protein degradation and translational repression, two posttranscriptional processes which play particularly critical roles in this transition.

Multiple RNA-binding proteins function combinatorially to control the soma-restricted expression pattern of the E3 ligase subunit ZIF-1.

In C. elegans embryos, transcriptional repression in germline blastomeres requires PIE-1 protein. Germline blastomere-specific localization of PIE-1 depends, in part, upon regulated degradation of PIE-1 in somatic cells. We and others have shown that the temporal and spatial regulation of PIE-1 degradation is controlled by translation of the substrate-binding subunit, ZIF-1, of an E3 ligase. We now show that ZIF-1 expression in embryos is regulated by five maternally-supplied RNA-binding proteins. POS-1, MEX-3, and SPN-4 function as repressors of ZIF-1 expression, whereas MEX-5 and MEX-6 antagonize this repression. All five proteins bind directly to the zif-1 3' UTR in vitro. We show that, in vivo, POS-1 and MEX-5/6 have antagonistic roles in ZIF-1 expression. In vitro, they bind to a common region of the zif-1 3' UTR, with MEX-5 binding impeding that by POS-1. The region of the zif-1 3' UTR bound by MEX-5/6 also partially overlaps with that bound by MEX-3, consistent with their antagonistic functions on ZIF-1 expression in vivo. Whereas both MEX-3 and SPN-4 repress ZIF-1 expression, neither protein alone appears to be sufficient, suggesting that they function together in ZIF-1 repression. We propose that MEX-3 and SPN-4 repress ZIF-1 expression exclusively in 1- and 2-cell embryos, the only period during embryogenesis when these two proteins co-localize. As the embryo divides, ZIF-1 continues to be repressed in germline blastomeres by POS-1, a germline blastomere-specific protein. MEX-5/6 antagonize repression by POS-1 and MEX-3, enabling ZIF-1 expression in somatic blastomeres. We propose that ZIF-1 expression results from a net summation of complex positive and negative translational regulation by 3' UTR-binding proteins, with expression in a specific blastomere dependent upon the precise combination of these proteins in that cell.

Our evolving view of Wnt signaling in C. elegans: If two's company and three's a crowd, is four really necessary?

In this commentary, we discuss how our recent paper by Yang et al. contributes a new wrinkle to the already somewhat curious Wnt signaling pathway in C. elegans. We begin with a historical perspective on the Wnt pathway in the worm, followed by a summary of the key salient point from Yang et al., 2011, namely demonstration of mutually inhibitory binding of a ß-catenin SYS-1 to the N-terminus and another ß-catenin WRM-1 to the C-terminus of the TCF protein POP-1, and a plausible structural explanation for these differential binding specificities. The mutually inhibitory binding creates one population of POP-1 that is bound by WRM-1, phosphorylated by the NLK kinase and exported from the nucleus, and another bound by coactivator SYS-1 that remains in the nucleus. We speculate on the evolutionary history of the four ß-catenins in C. elegans and suggest a possible link between multiple ß-catenin gene duplications and the requirement to reduce nuclear POP-1 levels to activate Wnt target genes.

Distinct and mutually inhibitory binding by two divergent ß-catenins coordinates TCF levels and activity in C. elegans.

Wnt target gene activation in C. elegans requires simultaneous elevation of ß-catenin/SYS-1 and reduction of TCF/POP-1 nuclear levels within the same signal-responsive cell. SYS-1 binds to the conserved N-terminal ß-catenin-binding domain (CBD) of POP-1 and functions as a transcriptional co-activator. Phosphorylation of POP-1 by LIT-1, the C. elegans Nemo-like kinase homolog, promotes POP-1 nuclear export and is the main mechanism by which POP-1 nuclear levels are lowered. We present a mechanism whereby SYS-1 and POP-1 nuclear levels are regulated in opposite directions, despite the fact that the two proteins physically interact. We show that the C terminus of POP-1 is essential for LIT-1 phosphorylation and is specifically bound by the diverged ß-catenin WRM-1. WRM-1 does not bind to the CBD of POP-1, nor does SYS-1 bind to the C-terminal domain. Furthermore, binding of WRM-1 to the POP-1 C terminus is mutually inhibitory with SYS-1 binding at the CBD. Computer modeling provides a structural explanation for the specificity in WRM-1 and SYS-1 binding to POP-1. Finally, WRM-1 exhibits two independent and distinct molecular functions that are novel for ß-catenins: WRM-1 serves both as the substrate-binding subunit and an obligate regulatory subunit for the LIT-1 kinase. Mutual inhibitory binding would result in two populations of POP-1: one bound by WRM-1 that is LIT-1 phosphorylated and exported from the nucleus, and another, bound by SYS-1, that remains in the nucleus and transcriptionally activates Wnt target genes. These studies could provide novel insights into cancers arising from aberrant Wnt activation.

Functional analyses of vertebrate TCF proteins in C. elegans embryos.

In the canonical Wnt pathway, signaling results in the stabilization and increased levels of ß-catenin in responding cells. ß-catenin then enters the nucleus, functioning as a coactivator for the Wnt effector, TCF/LEF protein. In the absence of Wnt signaling, TCF is complexed with corepressors, together repressing Wnt target genes. In C. elegans, Wnt signaling specifies the E blastomere to become the endoderm precursor. Activation of endoderm genes in E requires not only an increase in ß-catenin level, but a concomitant decrease in the nuclear level of POP-1, the sole C. elegans TCF. A decrease in nuclear POP-1 levels requires Wnt-induced phosphorylation of POP-1 and 14-3-3 protein-mediated nuclear export. Nuclear POP-1 levels remain high in the sister cell of E, MS, where POP-1 represses the expression of endoderm genes. Here we express three vertebrate TCF proteins (human TCF4, mouse LEF1 and Xenopus TCF3) in C. elegans embryos and compare their localization, repression and activation functions to POP-1. All three TCFs are localized to the nucleus in C. elegans embryos, but none undergoes Wnt-induced nuclear export. Although unable to undergo Wnt-induced nuclear export, human TCF4, but not mouse LEF1 or Xenopus TCF3, can repress endoderm genes in MS, in a manner very similar to POP-1. This repressive activity requires that human TCF4 recognizes specific promoter sequences upstream of endoderm genes and interacts with C. elegans corepressors. Domain swapping identified two regions of POP-1 that are sufficient to confer nuclear asymmetry to human TCF4 when swapped with its corresponding domains. Despite undergoing Wnt-induced nuclear export, the human TCF4/POP-1 chimeric protein continues to function as a repressor for endoderm genes in E, a result we attribute to the inability of hTCF4 to bind to C. elegans ß-catenin. Our results reveal a higher degree of species specificity among TCF proteins for coactivator interactions than for corepressor interactions, and uncover a basic difference between how POP-1 and human TCF4 steady state nuclear levels are regulated.

zif-1 translational repression defines a second, mutually exclusive OMA function in germline transcriptional repression.

Specification of primordial germ cells requires global repression of transcription. In C. elegans, primordial germ cells are generated through four rounds of asymmetric divisions, starting from the zygote P0, each producing a transcriptionally repressed germline blastomere (P1-P4). Repression in P2-P4 requires PIE-1, which is provided maternally in oocytes and segregated to all germline blastomeres. We have shown previously that OMA-1 and OMA-2 repress global transcription in P0 and P1 by sequestering TAF-4, an essential component of TFIID. Soon after the first mitotic cycle, OMA proteins undergo developmentally regulated degradation. Here, we show that OMA proteins also repress transcription in P2-P4 indirectly, through a completely different mechanism that operates in oocytes. OMA proteins bind to both the 3' UTR of the zif-1 transcript and the eIF4E-binding protein, SPN-2, repressing translation of zif-1 mRNA in oocytes. zif-1 encodes the substrate-binding subunit of the E3 ligase for PIE-1 degradation. Inhibition of zif-1 translation in oocytes ensures high PIE-1 levels in oocytes and germline blastomeres. The two OMA protein functions are strictly regulated in both space and time by MBK-2, a kinase activated following fertilization. Phosphorylation by MBK-2 facilitates the binding of OMA proteins to TAF-4 and simultaneously inactivates their function in repressing zif-1 translation. Phosphorylation of OMA proteins displaces SPN-2 from the zif-1 3' UTR, releasing translational repression. We propose that MBK-2 phosphorylation serves as a developmental switch, converting OMA proteins from specific translational repressors in oocytes to global transcriptional repressors in embryos, together effectively repressing transcription in all germline blastomeres.

An eIF4E-binding protein regulates katanin protein levels in C. elegans embryos.

In Caenorhabditis elegans, the MEI-1-katanin microtubule-severing complex is required for meiosis, but must be down-regulated during the transition to embryogenesis to prevent defects in mitosis. A cullin-dependent degradation pathway for MEI-1 protein has been well documented. In this paper, we report that translational repression may also play a role in MEI-1 down-regulation. Reduction of spn-2 function results in spindle orientation defects due to ectopic MEI-1 expression during embryonic mitosis. MEL-26, which is both required for MEI-1 degradation and is itself a target of the cullin degradation pathway, is present at normal levels in spn-2 mutant embryos, suggesting that the degradation pathway is functional. Cloning of spn-2 reveals that it encodes an eIF4E-binding protein that localizes to the cytoplasm and to ribonucleoprotein particles called P granules. SPN-2 binds to the RNA-binding protein OMA-1, which in turn binds to the mei-1 3' untranslated region. Thus, our results suggest that SPN-2 functions as an eIF4E-binding protein to negatively regulate translation of mei-1.

Global transcriptional repression in C. elegans germline precursors by regulated sequestration of TAF-4.

In C. elegans, four asymmetric divisions, beginning with the zygote (P0), generate transcriptionally repressed germline blastomeres (P1-P4) and somatic sisters that become transcriptionally active. The protein PIE-1 represses transcription in the later germline blastomeres but not in the earlier germline blastomeres P0 and P1. We show here that OMA-1 and OMA-2, previously shown to regulate oocyte maturation, repress transcription in P0 and P1 by binding to and sequestering in the cytoplasm TAF-4, a component critical for assembly of TFIID and the pol II preinitiation complex. OMA-1/2 binding to TAF-4 is developmentally regulated, requiring phosphorylation by the DYRK kinase MBK-2, which is activated at meiosis II after fertilization. OMA-1/2 are normally degraded after the first mitosis, but ectopic expression of wild-type OMA-1 is sufficient to repress transcription in both somatic and later germline blastomeres. We propose that phosphorylation by MBK-2 serves as a developmental switch, converting OMA-1/2 from oocyte to embryo regulators.