Mechanistic target of rapamycin (mTOR) functions in mTOR complex 1 (mTORC1) with raptor to match metazoan metabolism to available nutrients to regulate multiple cellular, physiological, and pathological processes. Hypoxic cellular injury is influenced by the mTORC1 pathway, but whether its activity promotes or prevents injury is unclear, and which mTORC1-regulated mechanisms control hypoxic injury are obscure. Here, we report the discovery of a hypoxia-resistant, temperature-sensitive raptor mutant in an unbiased forward mutagenesis screen in C. elegans. This raptor mutant is both hypoxia resistant and long lived at intermediate temperatures, while unable to develop at higher temperatures. Temperature-shift experiments show that the conditional hypoxia resistance can be induced in the raptor mutant immediately prior to the hypoxic insult. At these intermediate temperatures, the raptor mutation selectively reduces protein synthesis without affecting autophagy, and epistasis experiments implicate mTOR-targeted translation regulators as components of the hypoxia resistance mechanism. Using the conditional developmental arrest phenotype in a selection for suppressors of raptor loss of function, we isolated multiple second-site raptor missense mutants, whose mutated residue is predicted to interact with RagA, a raptor-binding protein. These suppressor mutations restore normal protein synthesis, hypoxic sensitivity, and lifespan and thereby implicate raptor-RagA interactions as critical to these biological processes.

Most of science involves making observations, forming hypotheses, and testing those hypotheses, to form valid conclusions. However, a distinct, longstanding, and very productive scientific approach does not follow this paradigm; rather, it begins with a screen through a random collection of drugs or genetic variations for a particular effect or phenotype. Subsequently, the identity of the drug or gene is determined, and only then are hypotheses formed and the more standard scientific method employed. This alternative approach is called forward screening and includes methods such as genetic mutant screens, small molecule screens, metabolomics, proteomics, and transcriptomics. This review explains the rational for forward screening approaches and uses examples of screens for mutants with altered anesthetic sensitivities and for novel anesthetics to illustrate the methods and impact of the approach. Forward screening approaches are becoming even more powerful with advances in bioinformatics aided by artificial intelligence.

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.

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.

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.

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.

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.

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.

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.

Gain-of-function mutations in the mouse nicotinamide mononucleotide adenylyltransferase type 1 (Nmnat1) produce two remarkable phenotypes: protection against traumatic axonal degeneration and reduced hypoxic brain injury. Despite intensive efforts, the mechanism of Nmnat1 cytoprotection remains elusive. To develop a new model to define this mechanism, we heterologously expressed a mouse Nmnat1 non-nuclear-localized gain-of-function mutant gene (m-nonN-Nmnat1) in the nematode Caenorhabditis elegans and show that it provides protection from both hypoxia-induced animal death and taxol-induced axonal pathology. Additionally, we find that m-nonN-Nmnat1 significantly lengthens C. elegans lifespan. Using the hypoxia-protective phenotype in C. elegans, we performed a candidate screen for genetic suppressors of m-nonN-Nmnat1 cytoprotection. Loss of function in two genes, haf-1 and dve-1, encoding mitochondrial unfolded protein response (mitoUPR) factors were identified as suppressors. M-nonN-Nmnat1 induced a transcriptional reporter of the mitoUPR gene hsp-6 and provided protection from the mitochondrial proteostasis toxin ethidium bromide. M-nonN-Nmnat1 was also protective against axonal degeneration in C. elegans induced by the chemotherapy drug taxol. Taxol markedly reduced basal expression of a mitoUPR reporter; the expression was restored by m-nonN-Nmnat1. Taken together, these data implicate the mitoUPR as a mechanism whereby Nmnat1 protects from hypoxic and axonal 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 [1] and calcium regulation [2]; however, little is known about how mitochondria themselves are injured by hypoxia. Maintenance of protein folding is essential for normal mitochondrial function [3], whereas failure to maintain protein homeostasis (proteostasis) appears to be a component of aging [4, 5] and a variety of diseases [6, 7]. Previously, it has been demonstrated that mitochondria possess their own unfolded protein response [8-10] 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 [11] both of which are shown to disrupt mitochondrial proteostasis in other contexts [3, 12] we hypothesized that failure to maintain mitochondrial proteostasis may play a role in hypoxic injury. Utilizing C. elegans models of global [13, 14], focal [15], 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.

After hypoxia, cells may die immediately or have a protracted course, living or dying depending on an incompletely understood set of cell autonomous and nonautonomous factors. In stroke, for example, some neurons are thought to die from direct hypoxic injury by cell autonomous primary mechanisms, whereas other so called innocent bystander neurons die from factors released from the primarily injured cells. A major limitation in identifying these factors is the inability of current in vivo models to selectively target a set of cells for hypoxic injury so that the primarily injured cells and the innocent bystanders are clearly delineated. In order to develop such a model, we generated transgenic Caenorhabditis elegans strains where 2–3% of somatic cells were made selectively sensitive to hypoxia. This was accomplished by cell type-specific wild-type rescue in either pharyngeal myocytes or GABAergic neurons of a hypoxia resistance-producing translation factor mutation. Surprisingly, hypoxic targeting of these relatively small subsets of non-essential cells produced widespread innocent bystander cell injury, behavioral dysfunction and eventual organismal death. The hypoxic injury phenotypes of the myocyte or neuron sensitized strains were virtually identical. Using this model, we show that the C. elegans insulin receptor/FOXO transcription factor pathway improves survival when activated only after hypoxic injury and blocks innocent bystander death.

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.

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.

Prolonged cellular hypoxia results in energy failure and ultimately cell death. However, less-severe hypoxia can induce a cytoprotective response termed hypoxic preconditioning (HP). The unfolded protein response pathway (UPR) has been known for some time to respond to hypoxia and regulate hypoxic sensitivity; however, the role of the UPR, if any, in HP essentially has been unexplored. We have shown previously that a sublethal hypoxic exposure of the nematode Caenorhabditis elegans induces a protein chaperone component of the UPR (L. L. Anderson, X. Mao, B. A. Scott, and C. M. Crowder, Science 323:630-633, 2009). Here, we show that HP induces the UPR and that the pharmacological induction of misfolded proteins is itself sufficient to stimulate a delayed protective response to hypoxic injury that requires the UPR pathway proteins IRE-1, XBP-1, and ATF-6. HP also required IRE-1 but not XBP-1 or ATF-6; instead, GCN-2, which is known to suppress translation and induce an adaptive transcriptional response under conditions of UPR activation or amino acid deprivation, was required for HP. The phosphorylation of the translation factor eIF2α, an established mechanism of GCN-2-mediated translational suppression, was not necessary for HP. These data suggest a model where hypoxia-induced misfolded proteins trigger the activation of IRE-1, which along with GCN-2 controls an adaptive response that is essential to HP.

Hypoxia is important in a wide range of biological processes, such as animal hibernation and cell survival, and is particularly relevant in many diseases. The sensitivity of cells and organisms to hypoxic injury varies widely, but the molecular basis for this variation is incompletely understood. Using forward genetic screens in Caenorhabditis elegans , we isolated a hypoxia-resistant reduction-of-function mutant of rrt-1 that encodes an arginyl–transfer RNA (tRNA) synthetase, an enzyme essential for protein translation. Knockdown of rrt-1 , and of most other genes encoding aminoacyl-tRNA synthetases, rescued animals from hypoxia-induced death, and the level of hypoxia resistance was inversely correlated with translation rate. The unfolded protein response was induced by hypoxia and was required for the hypoxia resistance of the reduction-of-function mutant of rrt-1 . Thus, translational suppression produces hypoxia resistance, in part by reducing unfolded protein toxicity.

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.

Organisms and their cells vary greatly in their tolerance of low oxygen environments (hypoxia). A delineation of the determinants of hypoxia tolerance is incomplete, despite intense interest for its implications in diseases such as stroke and myocardial infarction. The insulin/IGF-1 receptor (IGFR) signaling pathway controls survival of Caenorhabditis elegans from a variety of stressors including aging, hyperthermia, and hypoxia. daf-2 encodes a C. elegans IGFR homolog whose primary signaling pathway modulates the activity of the FOXO transcription factor DAF-16. DAF-16 regulates the transcription of a large number of genes, some of which have been shown to control aging. To identify genes that selectively regulate hypoxic sensitivity, we compared the whole-organismal transcriptomes of three daf-2 reduction-of-function alleles, all of which are hypoxia resistant, thermotolerant, and long lived, but differ in their rank of severities for these phenotypes. The transcript levels of 172 genes were increased in the most hypoxia resistant daf-2 allele, e1370, relative to the other alleles whereas transcripts from only 10 genes were decreased in abundance. RNAi knockdown of 6 of the 10 genes produced a significant increase in organismal survival after hypoxic exposure as would be expected if down regulation of these genes by the e1370 mutation was responsible for hypoxia resistance. However, RNAi knockdown of these genes did not prolong lifespan. These genes definitively separate the mechanisms of hypoxic sensitivity and lifespan and identify biological strategies to survive hypoxic injury.