Daf-16 mediated repression of cytosolic ribosomal protein genes facilitates a hypoxia sensitive to hypoxia resistant transformation in long-lived germline mutants.

In C. elegans, germline ablation leads to long life span and stress resistance. It has been reported that mutations that block oogenesis or an upstream step in germline development confer strong resistance to hypoxia. We demonstrate here that the hypoxia resistance of sterile mutants is dependent on developmental stage and age. In just a 12-hour period, sterile animals transform from hypoxia sensitive L4 larvae into hypoxia resistant adults. Since this transformation occurs in animals with no germline, the physiological programs that determine hypoxia sensitivity in germline mutants occur independently of germline signals and instead rely on signals from somatic tissues. Furthermore, we found two distinct mechanisms of hypoxia resistance in germline deficient animals. First, a DAF-16/FoxO independent mechanism that occurs in all hypoxia resistant sterile adults and, second, a DAF-16/FoxO dependent mechanism that confers an added layer of resistance, or "super-resistance", to animals with no germline as they age past day 1 of adulthood. RNAseq data showed that genes involved in both cytosolic and mitochondrial protein translation are repressed in sterile adults and further repressed only in germline deficient mutants as they age. Importantly, mutation of daf-16 specifically blocked the repression of cytosolic ribosomal protein genes, but not mitochondrial ribosomal protein genes, implicating DAF-16/FoxO mediated repression of cytosolic ribosomal protein genes as a mechanism of hypoxia super-resistance. Consistent with this hypothesis, the hypoxia super-resistance of aging germline deficient adults was also suppressed by dual mutation of ncl-1 and larp-1, two regulators of protein translation and ribosomal protein abundance. These studies provide novel insight into a profound physiological transformation that takes place in germline mutants during development, showing that some of the unique physiological properties of these long-lived animals are derived from developmentally dependent DAF-16/FoxO mediated repression of genes involved in cytosolic protein translation.

Role of oxygen consumption in hypoxia protection by translation factor depletion.

The reduction of protein synthesis has been associated with resistance to hypoxic cell death. Which components of the translation machinery control hypoxic sensitivity and the precise mechanism has not been systematically investigated, although a reduction in oxygen consumption has been widely assumed to be the mechanism. Using genetic reagents in Caenorhabditis elegans, we examined the effect on organismal survival after hypoxia of knockdown of 10 factors functioning at the three principal steps in translation. Reduction-of-function of all 10 translation factors significantly increased hypoxic survival to varying degrees, not fully accounted for by the level of translational suppression. Measurement of oxygen consumption showed that strong hypoxia resistance was possible without a significant decrease in oxygen consumption. Hypoxic sensitivity had no correlation with lifespan or reactive oxygen species sensitivity, two phenotypes associated with reduced translation. Resistance to tunicamycin, which produces misfolded protein toxicity, was the only phenotype that significantly correlated with hypoxic sensitivity. Translation factor knockdown was also hypoxia protective for mouse primary neurons. These data show that translation factor knockdown is hypoxia protective in both C. elegans and mouse neurons and that oxygen consumption does not necessarily determine survival; rather, mitigation of misfolded protein toxicity is more strongly associated with hypoxic protection.

Hypoxia-induced mitochondrial stress granules.

Perturbations of mitochondrial proteostasis have been associated with aging, neurodegenerative diseases, and recently with hypoxic injury. While examining hypoxia-induced mitochondrial protein aggregation in C. elegans, we found that sublethal hypoxia, sodium azide, or heat shock-induced abundant ethidium bromide staining mitochondrial granules that preceded evidence of protein aggregation. Genetic manipulations that reduce cellular and organismal hypoxic death block the formation of these mitochondrial stress granules (mitoSG). Knockdown of mitochondrial nucleoid proteins also blocked the formation of mitoSG by a mechanism distinct from the mitochondrial unfolded protein response. Lack of the major mitochondrial matrix protease LONP-1 resulted in the constitutive formation of mitoSG without external stress. Ethidium bromide-staining RNA-containing mitochondrial granules were also observed in rat cardiomyocytes treated with sodium azide, a hypoxia mimetic. Mitochondrial stress granules are an early mitochondrial pathology controlled by LONP and the nucleoid, preceding hypoxia-induced protein aggregation.

Severe Polyethylene Glycol Allergy Considerations for Perioperative Management: A Case Report.

Patients with severe polyethylene glycol (PEG) allergies face broad challenges, especially when presenting to the hospital for surgery, as PEG is used often as an excipient in medications and in medical supplies. Although rare, this allergy is increasingly reported and likely underdiagnosed. We present a patient with known past anaphylactic reaction to PEG and a detailed account of her perioperative course. More broadly, we provide recommendations and resources for the safe management of similar patients with a severe PEG allergy.

A gain-of-function mutation in adenylate cyclase confers isoflurane resistance in Caenorhabditis elegans.

BACKGROUND: Volatile general anesthetics inhibit neurotransmitter release by a mechanism not fully understood. Genetic evidence in Caenorhabditis elegans has shown that a major mechanism of action of volatile anesthetics acting at clinical concentrations in this animal is presynaptic inhibition of neurotransmission. To define additional components of this presynaptic volatile anesthetic mechanism, C. elegans mutants isolated as phenotypic suppressors of a mutation in syntaxin, an essential component of the neurotransmitter release machinery, were screened for anesthetic sensitivity phenotypes. METHODS: Sensitivity to isoflurane concentrations was measured in locomotion assays on adult C. elegans. Sensitivity to the acetylcholinesterase inhibitor aldicarb was used as an assay for the global level of C. elegans acetylcholine release. Comparisons of isoflurane sensitivity (measured by the EC50) were made by simultaneous curve-fitting and F test. RESULTS: Among the syntaxin suppressor mutants, js127 was the most isoflurane resistant, with an EC50 more than 3-fold that of wild type. Genetic mapping, sequencing, and transformation phenocopy showed that js127 was an allele of acy-1, which encodes an adenylate cyclase expressed throughout the C. elegans nervous system and in muscle. js127 behaved as a gain-of-function mutation in acy-1 and had increased concentrations of cyclic adenosine monophosphate. Testing of single and double mutants along with selective tissue expression of the js127 mutation revealed that acy-1 acts in neurons within a Gαs-PKA-UNC-13-dependent pathway to regulate behavior and isoflurane sensitivity. CONCLUSIONS: Activation of neuronal adenylate cyclase antagonizes isoflurane inhibition of locomotion in C. elegans.

Effect of the mitochondrial unfolded protein response on hypoxic death and mitochondrial protein aggregation.

Mitochondria are the main oxygen consumers in cells and as such are the primary organelle affected by hypoxia. All hypoxia pathology presumably derives from the initial mitochondrial dysfunction. An early event in hypoxic pathology in C. elegans is disruption of mitochondrial proteostasis with induction of the mitochondrial unfolded protein response (UPRmt) and mitochondrial protein aggregation. Here in C. elegans, we screen through RNAis and mutants that confer either strong resistance to hypoxic cell death or strong induction of the UPRmt to determine the relationship between hypoxic cell death, UPRmt activation, and hypoxia-induced mitochondrial protein aggregation (HIMPA). We find that resistance to hypoxic cell death invariantly mitigated HIMPA. We also find that UPRmt activation invariantly mitigated HIMPA. However, UPRmt activation was neither necessary nor sufficient for resistance to hypoxic death and vice versa. We conclude that UPRmt is not necessarily hypoxia protective against cell death but does protect from mitochondrial protein aggregation, one of the early hypoxic pathologies in C. elegans.

Coordinate Regulation of Ribosome and tRNA Biogenesis Controls Hypoxic Injury and Translation.

The translation machinery is composed of a myriad of proteins and RNAs whose levels must be coordinated to efficiently produce proteins without wasting energy or substrate. However, protein synthesis is clearly not always perfectly tuned to its environment, as disruption of translation machinery components can lengthen lifespan and stress survival. While much has been learned from bacteria and yeast about translational regulation, much less is known in metazoans. In a screen for mutations protecting C. elegans from hypoxic stress, we isolated multiple genes impacting protein synthesis: a ribosomal RNA helicase gene, tRNA biosynthesis genes, and a gene controlling amino acid availability. To define better the mechanisms by which these genes impact protein synthesis, we performed a second screen for suppressors of the conditional developmental arrest phenotype of the RNA helicase mutant and identified genes involved in ribosome biogenesis. Surprisingly, these suppressor mutations restored normal hypoxic sensitivity and protein synthesis to the tRNA biogenesis mutants, but not to the mutant reducing amino acid uptake. Proteomic analysis demonstrated that reduced tRNA biosynthetic activity produces a selective homeostatic reduction in ribosomal subunits, thereby offering a mechanism for the suppression results. Our study uncovers an unrecognized higher-order-translation regulatory mechanism in a metazoan whereby ribosome biogenesis genes communicate with genes controlling tRNA abundance matching the global rate of protein synthesis with available resources.

Hypoxic preconditioning requires the apoptosis protein CED-4 in C. elegans.

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.

Systematic identification of gene activities promoting hypoxic death.

The sensitivity of an organism to hypoxic injury varies widely across species and among cell types. However, a systematic description of the determinants of metazoan hypoxic sensitivity is lacking. Toward this end, we screened a whole-genome RNAi library for genes that promote hypoxic sensitivity in Caenorhabditis elegans. RNAi knockdown of 198 genes conferred an invariant hypoxia-resistant phenotype (Hyp-r). Eighty-six per cent of these hyp genes had strong homologs in other organisms, 73 with human reciprocal orthologs. The hyp genes were distributed among multiple functional categories. Transcription factors, chromatin modifying enzymes, and intracellular signaling proteins were highly represented. RNAi knockdown of about half of the genes produced no apparent deleterious phenotypes. The hyp genes had significant overlap with previously identified life span extending genes. Testing of the RNAi's in a mutant background defective in somatic RNAi machinery showed that most genes function in somatic cells to control hypoxic sensitivity. DNA microarray analysis identified a subset of the hyp genes that may be hypoxia regulated. siRNA knockdown of human orthologs of the hyp genes conferred hypoxia resistance to transformed human cells for 40% of the genes tested, indicating extensive evolutionary conservation of the hypoxic regulatory activities. The results of the screen provide the first systematic picture of the genetic determinants of hypoxic sensitivity. The number and diversity of genes indicates a surprisingly nonredundant genetic network promoting hypoxic sensitivity.

The sodium-activated potassium channel is encoded by a member of the Slo gene family.

Na(+)-activated potassium channels (K(Na)) have been identified in cardiomyocytes and neurons where they may provide protection against ischemia. We now report that K(Na) is encoded by the rSlo2 gene (also called Slack), the mammalian ortholog of slo-2 in C. elegans. rSlo2, heterologously expressed, shares many properties of native K(Na) including activation by intracellular Na(+), high conductance, and prominent subconductance states. In addition to activation by Na(+), we report that rSLO-2 channels are cooperatively activated by intracellular Cl(-), similar to C. elegans SLO-2 channels. Since intracellular Na(+) and Cl(-) both rise in oxygen-deprived cells, coactivation may more effectively trigger the activity of rSLO-2 channels in ischemia. In C. elegans, mutational and physiological analysis revealed that the SLO-2 current is a major component of the delayed rectifier. We demonstrate in C. elegans that slo-2 mutants are hypersensitive to hypoxia, suggesting a conserved role for the slo-2 gene subfamily.

Volatile anesthetic preconditioning present in the invertebrate Caenorhabditis elegans.

BACKGROUND: Volatile anesthetics (VAs) have been found to induce a delayed protective response called preconditioning to subsequent hypoxic/ischemic injury. VA preconditioning has been primarily studied in canine and rodent heart. A more genetically tractable model of VA preconditioning would be extremely useful. Here, the authors report the development of the nematode Caenorhabditis elegans as a model of VA preconditioning. METHODS: Wild-type and mutant C. elegans were exposed to isoflurane, halothane, or air under otherwise identical conditions. After varying recovery periods, the animals were challenged with hypoxic, azide, or hyperthermic incubations. After recovery from these incubations, mortality was scored. RESULTS: Isoflurane- and halothane-preconditioned animals had significantly reduced mortality to all three types of injuries compared with air controls. Concentrations as low as 1 vol% isoflurane (0.64 mm) and halothane (0.71 mm) induced significant protection. The onset and duration of protection after anesthetic were 6 and 9 h, respectively. A mutation that blocks inhibition of neurotransmitter release by isoflurane did not attenuate the preconditioning effect. A loss-of-function mutation of the Apaf-1 homolog CED-4 blocked the preconditioning effect of isoflurane, but mutation of the downstream caspase CED-3 did not. CONCLUSIONS: Volatile anesthetic preconditioning extends beyond the vertebrate subphylum. This markedly broadens the scope of VA preconditioning and suggests that its mechanisms are widespread across species and is a fundamental and evolutionarily conserved cellular response. C. elegans offers a means to dissect genetically the mechanism for VA preconditioning as illustrated by the novel finding of the requirement for the Apaf-1 homolog CED-4.

An evolutionarily conserved presynaptic protein is required for isoflurane sensitivity in Caenorhabditis elegans.

BACKGROUND: Volatile general anesthetics inhibit neurotransmitter release by an unknown mechanism. A mutation in the presynaptic soluble NSF attachment protein receptor (SNARE) protein syntaxin 1A was previously shown to antagonize the anesthetic isoflurane in Caenorhabditis elegans. The mechanism underlying this antagonism may identify presynaptic anesthetic targets relevant to human anesthesia. METHODS: Sensitivity to isoflurane concentrations in the human clinical range was measured in locomotion assays on adult C. elegans. Sensitivity to the acetylcholinesterase inhibitor aldicarb was used as an assay for the global level of C. elegans neurotransmitter release. Comparisons of isoflurane sensitivity (measured by the EC50) were made by simultaneous curve fitting and F test as described by Waud. RESULTS: Expression of a truncated syntaxin fragment (residues 1-106) antagonized isoflurane sensitivity in C. elegans. This portion of syntaxin interacts with the presynaptic protein UNC-13, suggesting the hypothesis that truncated syntaxin binds to UNC-13 and antagonizes an inhibitory effect of isoflurane on UNC-13 function. Consistent with this hypothesis, overexpression of UNC-13 suppressed the isoflurane resistance of the truncated syntaxins, and unc-13 loss-of-function mutants were highly isoflurane resistant. Normal anesthetic sensitivity was restored by full-length UNC-13, by a shortened form of UNC-13 lacking a C2 domain, but not by a membrane-targeted UNC-13 that might bypass isoflurane inhibition of membrane translocation of UNC-13. Isoflurane was found to inhibit synaptic localization of UNC-13. CONCLUSIONS: These data show that UNC-13, an evolutionarily conserved protein that promotes neurotransmitter release, is necessary for isoflurane sensitivity in C. elegans and suggest that its vertebrate homologs may be a component of the general anesthetic mechanism.

A screen for protective drugs against delayed hypoxic injury.

Despite longstanding efforts to develop cytoprotective drugs against ischemia/reperfusion (IR) injuries, there remains no effective therapeutics to treat hypoxic injury. The failure of traditional strategies at solving this problem suggests the need for novel and unbiased approaches that can lead to previously unsuspected targets and lead compounds. Towards this end, we report here a unique small molecule screen in the nematode C. elegans for compounds that improve recovery when applied after the hypoxic insult, using a C. elegans strain engineered to have delayed cell non-autonomous death. In a screen of 2000 compounds, six were found to produce significant protection of C. elegans from delayed death. Four of the compounds were tested in an ex vivo mouse heart ischemia/reperfusion model and two, meclocycline and 3-amino-1,2,4-triazole, significantly reduced infarction size. Our work demonstrates the feasibility of this novel C. elegans screen to discover hypoxia protective drugs that are also protective in a mammalian model of hypoxic injury.

Ageing and hypoxia cause protein aggregation in mitochondria.

Aggregation of cytosolic proteins is a pathological finding in disease states, including ageing and neurodegenerative diseases. We have previously reported that hypoxia induces protein misfolding in Caenorhabditis elegans mitochondria, and electron micrographs suggested protein aggregates. Here, we seek to determine whether mitochondrial proteins actually aggregate after hypoxia and other cellular stresses. To enrich for mitochondrial proteins that might aggregate, we performed a proteomics analysis on purified C. elegans mitochondria to identify relatively insoluble proteins under normal conditions (110 proteins identified) or after sublethal hypoxia (65 proteins). A GFP-tagged mitochondrial protein (UCR-11 - a complex III electron transport chain protein) in the normally insoluble set was found to form widespread aggregates in mitochondria after hypoxia. Five other GFP-tagged mitochondrial proteins in the normally insoluble set similarly form hypoxia-induced aggregates. Two GFP-tagged mitochondrial proteins from the soluble set as well as a mitochondrial-targeted GFP did not form aggregates. Ageing also resulted in aggregates. The number of hypoxia-induced aggregates was regulated by the mitochondrial unfolded protein response (UPRmt) master transcriptional regulator ATFS-1, which has been shown to be hypoxia protective. An atfs-1(loss-of-function) mutant and RNAi construct reduced the number of aggregates while an atfs-1(gain-of-function) mutant increased aggregates. Our work demonstrates that mitochondrial protein aggregation occurs with hypoxic injury and ageing in C. elegans. The UPRmt regulates aggregation and may protect from hypoxia by promoting aggregation of misfolded proteins.

Ethanol targets: a BK channel cocktail in C. elegans.

A genetic screen for resistance to ethanol intoxication in Caenorhabditis elegans isolated mutants of the gene slo-1. slo-1 encodes the pore-forming subunit of a large-conductance Ca(2+)-activated K(+) channel previously shown to limit excitatory neurotransmitter release in C. elegans. Electrophysiological data recorded in vivo are consistent with a model in which ethanol potentiation of SLO-1 produces intoxication in C. elegans by reducing excitatory neurotransmitter release.

A Caenorhabditis elegans pheromone antagonizes volatile anesthetic action through a go-coupled pathway.

Volatile anesthetics (VAs) disrupt nervous system function by an ill-defined mechanism with no known specific antagonists. During the course of characterizing the response of the nematode C. elegans to VAs, we discovered that a C. elegans pheromone antagonizes the VA halothane. Acute exposure to pheromone rendered wild-type C. elegans resistant to clinical concentrations of halothane, increasing the EC(50) from 0.43 +/- 0.03 to 0.90 +/- 0.02. C. elegans mutants that disrupt the function of sensory neurons required for the action of the previously characterized dauer pheromone blocked pheromone-induced resistance (Pir) to halothane. Pheromone preparations from loss-of-function mutants of daf-22, a gene required for dauer pheromone production, lacked the halothane-resistance activity, suggesting that dauer and Pir pheromone are identical. However, the pathways for pheromone's effects on dauer formation and VA action were not identical. Not all mutations that alter dauer formation affected the Pir phenotype. Further, mutations in genes not known to be involved in dauer formation completely blocked Pir, including those altering signaling through the G proteins Goalpha and Gqalpha. A model in which sensory neurons transduce the pheromone activity through antagonistic Go and Gq pathways, modulating VA action against neurotransmitter release machinery, is proposed.

Resistance to volatile anesthetics by mutations enhancing excitatory neurotransmitter release in Caenorhabditis elegans.

The molecular mechanisms whereby volatile general anesthetics (VAs) disrupt behavior remain undefined. In Caenorhabditis elegans mutations in the gene unc-64, which encodes the presynaptic protein syntaxin 1A, produce large allele-specific differences in VA sensitivity. UNC-64 syntaxin normally functions to mediate fusion of neurotransmitter vesicles with the presynaptic membrane. The precise role of syntaxin in the VA mechanism is as yet unclear, but a variety of results suggests that a protein interacting with syntaxin to regulate neurotransmitter release is essential for VA action in C. elegans. To identify additional proteins that function with syntaxin to control neurotransmitter release and VA action, we screened for suppressors of the phenotypes produced by unc-64 reduction of function. Loss-of-function mutations in slo-1, which encodes a Ca(2+)-activated K+ channel, and in unc-43, which encodes CaM-kinase II, and a gain-of-function mutation in egl-30, which encodes Gqalpha, were isolated as syntaxin suppressors. The slo-1 and egl-30 mutations conferred resistance to VAs, but unc-43 mutations did not. The effects of slo-1 and egl-30 on VA sensitivity can be explained by their actions upstream or parallel to syntaxin to increase the level of excitatory neurotransmitter release. These results strengthen the link between transmitter release and VA action.

Mitochondrial Proteostatic Collapse Leads to Hypoxic Injury.

Hypoxic injury is a key pathological event in a variety of diseases. Despite the clinical importance of hypoxia, modulation of hypoxic injury mechanisms for therapeutic benefit has not been achieved, suggesting that critical features of hypoxic injury have not been identified or fully understood. Because mitochondria are the main respiratory organelles of the cell, they have been the focus of much research into hypoxic injury. Previous research has focused on mitochondria as effectors of hypoxic injury, primarily in the context of apoptosis activation and calcium regulation; however, little is known about how mitochondria themselves are injured by hypoxia. Maintenance of protein folding is essential for normal mitochondrial function, whereas failure to maintain protein homeostasis (proteostasis) appears to be a component of aging and a variety of diseases. Previously, it has been demonstrated that mitochondria possess their own unfolded protein response that is activated in response to mitochondrial protein folding stress, a response that is best understood in C. elegans. Because hypoxia has been shown to disrupt ATP production and translation of nuclear encoded proteins--both of which are shown to disrupt mitochondrial proteostasis in other contexts-we hypothesized that failure to maintain mitochondrial proteostasis may play a role in hypoxic injury. Utilizing C. elegans models of global, focal, and cell non-autonomous hypoxic injury, we have found evidence of mitochondrial protein misfolding post-hypoxia and have found that manipulation of the mitochondrial protein folding environment is an effective hypoxia protective strategy.