The FOXO Transcription Factor DAF-16 Bypasses ire-1 Requirement to Promote Endoplasmic Reticulum Homeostasis

SummaryThe unfolded protein response (UPR) allows cells to adjust the capacity of the endoplasmic reticulum (ER) to the load of ER-associated tasks. We show that activation of the Caenorhabditis elegans transcription factor DAF-16 and its human homolog FOXO3 restore secretory protein metabolism when the UPR is dysfunctional. We show that DAF-16 establishes alternative ER-associated degradation systems that degrade misfolded proteins independently of the ER stress sensor ire-1 and the ER-associated E3 ubiquitin ligase complex sel-11/sel-1. This is achieved by enabling autophagy-mediated degradation and by increasing the levels of skr-5, a component of an ER-associated ubiquitin ligase complex. These degradation systems can act together with the conserved UPR to improve ER homeostasis and ER stress resistance, beyond wild-type levels. Because there is no sensor in the ER that activates DAF-16 in response to intrinsic ER stress, natural or artificial interventions that activate DAF-16 may be useful therapeutic approaches to maintain ER homeostasis

Stimulation of the Sphenopalatine Ganglion Induces Reperfusion and Blood-Brain Barrier Protection in the Photothrombotic Stroke Model

The treatment of stroke remains a challenge. Animal studies showing that electrical stimulation of the sphenopalatine ganglion (SPG) exerts beneficial effects in the treatment of stroke have led to the initiation of clinical studies. However, the detailed effects of SPG stimulation on the injured brain are not known.The effect of acute SPG stimulation was studied by direct vascular imaging, fluorescent angiography and laser Doppler flowmetry in the sensory motor cortex of the anaesthetized rat. Focal cerebral ischemia was induced by the rose bengal (RB) photothrombosis method. In chronic experiments, SPG stimulation, starting 15 min or 24 h after photothrombosis, was given for 3 h per day on four consecutive days. Structural damage was assessed using histological and immunohistochemical methods. Cortical functions were assessed by quantitative analysis of epidural electro-corticographic (ECoG) activity continuously recorded in behaving animals.Stimulation induced intensity- and duration-dependent vasodilation and increased cerebral blood flow in both healthy and photothrombotic brains. In SPG-stimulated rats both blood brain-barrier (BBB) opening, pathological brain activity and lesion volume were attenuated compared to untreated stroke animals, with no apparent difference in the glial response surrounding the necrotic lesion.SPG-stimulation in rats induces vasodilation of cortical arterioles, partial reperfusion of the ischemic lesion, and normalization of brain functions with reduced BBB dysfunction and stroke volume. These findings support the potential therapeutic effect of SPG stimulation in focal cerebral ischemia even when applied 24 h after stroke onset and thus may extend the therapeutic window of currently administered stroke medications

Fatal Prion Disease in a Mouse Model of Genetic E200K Creutzfeldt-Jakob Disease

Genetic prion diseases are late onset fatal neurodegenerative disorders linked to pathogenic mutations in the prion protein-encoding gene, PRNP. The most prevalent of these is the substitution of Glutamate for Lysine at codon 200 (E200K), causing genetic Creutzfeldt-Jakob disease (gCJD) in several clusters, including Jews of Libyan origin. Investigating the pathogenesis of genetic CJD, as well as developing prophylactic treatments for young asymptomatic carriers of this and other PrP mutations, may well depend upon the availability of appropriate animal models in which long term treatments can be evaluated for efficacy and toxicity. Here we present the first effective mouse model for E200KCJD, which expresses chimeric mouse/human (TgMHu2M) E199KPrP on both a null and a wt PrP background, as is the case for heterozygous patients and carriers. Mice from both lines suffered from distinct neurological symptoms as early as 5–6 month of age and deteriorated to death several months thereafter. Histopathological examination of the brain and spinal cord revealed early gliosis and age-related intraneuronal deposition of disease-associated PrP similarly to human E200K gCJD. Concomitantly we detected aggregated, proteinase K resistant, truncated and oxidized PrP forms on immunoblots. Inoculation of brain extracts from TgMHu2ME199K mice readily induced, the first time for any mutant prion transgenic model, a distinct fatal prion disease in wt mice. We believe that these mice may serve as an ideal platform for the investigation of the pathogenesis of genetic prion disease and thus for the monitoring of anti-prion treatments

Pricing of reusable resources under ambiguous distributions of demand and service time with emerging applications

Monopolistic pricing models for revenue management are widely used in practice to set prices of multiple products with uncertain demand arrivals. The literature often assumes deterministic time of serving each demand and that the distribution of uncertainty is fully known. In this paper, we consider a new class of revenue management problems inspired by emerging applications such as cloud computing and city parking, where we dynamically determine prices for multiple products sharing limited resource and aim to maximize the expected revenue over a finite horizon. Random demand of each product arrives in each period, modeled by a function of the arrival time, product type, and price. Unlike the traditional monopolistic pricing, here each demand stays in the system for uncertain time. Both demand and service time follow ambiguous distributions, and we formulate robust deterministic approximation models to construct efficient heuristic fixed-price pricing policies. We conduct numerical studies by testing cloud computing service pricing instances based on data published by the Amazon Web Services (AWS) and demonstrate the efficacy of our approach for managing revenue and risk under various distributions of demand and service time

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7.

In C. elegans, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named irg-7, as a novel mediator of longevity in germlineless animals. We consider irg-7 to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, irg-7 activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks

Innate immunity mediated longevity and longevity induced by germ cell removal converge on the C-type lectin domain protein IRG-7

<div><p>In <i>C</i>. <i>elegans</i>, removal of the germline triggers molecular events in the neighboring intestine, which sends an anti-aging signal to the rest of the animal. In this study, we identified an innate immunity related gene, named <i>irg-7</i>, as a novel mediator of longevity in germlineless animals. We consider <i>irg-7</i> to be an integral downstream component of the germline longevity pathway because its expression increases upon germ cell removal and its depletion interferes with the activation of the longevity-promoting transcription factors DAF-16 and DAF-12 in germlineless animals. Furthermore, <i>irg-7</i> activation by itself sensitizes the animals' innate immune response and extends the lifespan of animals exposed to live bacteria. This lifespan-extending pathogen resistance relies on the somatic gonad as well as on many genes previously associated with the reproductive longevity pathway. This suggests that these genes are also relevant in animals with an intact gonad, and can affect their resistance to pathogens. Altogether, this study demonstrates the tight association between germline homeostasis and the immune response of animals, and raises the possibility that the reproductive system can act as a signaling center to divert resources towards defending against putative pathogen attacks.</p></div

The transcription factor DAF-12 is activated in <i>irg-7(zc6)</i> mutants.

<p>(A-B) The <i>irg-7(zc6)</i> mutation is sufficient to increase the expression of the <i>Pcdr-6</i>::<i>gfp</i> reporter (Student's t-test, p<0.0001) in a <i>daf-12(+)</i> background but not in a <i>daf-12(-)</i> background (Student's t-test, p = 0.075). Asterisks mark Student's t-test values of P<0.001 compared to the fluorescence in wild-type animals. 30–35 animals analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) The <i>irg-7(zc6)</i> mutation is not sufficient to increase the expression of the DAF-16 reporter <i>Psod-3</i>::<i>gfp</i> (Student's t-test, P = 0.067). (D) The <i>irg-7(zc6)</i> mutation is not sufficient for the accumulation of DAF-16::GFP translational reporter in the intestine.</p

<i>irg-7(zc6)</i> is a gain of function mutation in F40F4.6.

<p>(A) <i>F40F4</i>.<i>6</i> RNAi significantly shortened the lifespan of <i>irg-7(zc6)</i> mutants (Mantel-Cox, P<0.0001) but did not affect the lifespan of otherwise wild-type animals (Mantel-Cox,P = 0.9). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (B) Representative fluorescence micrographs (100-fold magnification) of day-1 adults harboring an integrated <i>Phsp-4</i>::<i>gfp</i> transgene. <i>F40F4</i>.<i>6</i> RNAi significantly reduced the expression of the <i>Phsp-4</i>::<i>gfp</i> in <i>zc6</i> mutants. Asterisks mark Student's T-test values of P<0.001 compared to fluorescence on control RNAi. 40 animals were analyzed per genotype. Error bars represent SE of 3 independent biological replicates. (C) Expression of fosmid WRM0619aB08 (which includes F40F4.6) significantly extended the lifespan of wild-type animals (Mantel-Cox, P<0.001), whereas expression of the partially overlapping fosmid WRM0635bF05 (which does not include F40F4.6) did not extend the lifespan of wild-type animals (Mantel-Cox, P = 0.4). Mean lifespans are indicated within each graph. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s004" target="_blank">S2 Table</a></b> for more lifespan data. (D) Expression of an NLS-RFP reporter fused to the putative promoter upstream of the F40F4.6 gene drives expression in the posterior cells of the intestine. Fluorescence of this reporter increased upon exposure of L4 animals to Photorhabdus luminescens subsp Hb bacteria (HB) or Enterococcus faecalis bacteria (EF). Asterisks mark Student's T-test values of P<0.001 compared to wild-type fluorescence on OP50 bacteria. (E) Schematic representation of the major domains in the F40F4.6 protein. The region deleted by the <i>zc6</i> mutation is indicated by a dashed line. This region includes an EGF domain. Note that the deletion preserves the ORF of the original gene. See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s002" target="_blank">S2 Fig</a> for sequence data.</p

The longevitiy induced by germ cell removal and by the <i>irg-7(zc6)</i> mutation extend lifespan by a shared mechanism.

<p>(A) Bar graph presenting the percentage of lifespan extension conferred by the <i>irg-7(zc6)</i> mutation in the indicated genetic backgrounds. Each bar is the average of at least three independent lifespans. Error bars represent SE of at least 3 independent biological replicates. Asterisks mark bars in which all the lifespan experiments were increased significantly by the <i>irg-7(zc6)</i> mutation (P<0.05). Colored boxes indicate longevity pathways associated with each longevity gene: the reproductive longevity pathway induced by germ cell removal (Red), the insulin/IGF1 signaling pathway (Blue), the dietary restriction pathway (Orange) and the mitochondrial respiration pathway (Green). Note that the dietary restriction pathway is only partly dependent of <i>daf-</i>16 (marked with an Orange triangle), depending on the dietary regimen used. Note that the longevity of <i>irg-7(zc6) gof</i> mutants requires many genes implicated in the longevity of germline-less animals with the exception of <i>nhr-80</i>, which is not required for <i>irg-7(zc6)</i> longevity although it is required for the longevity of germlineless animals. See <b><a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s008" target="_blank">S6 Table</a></b> for more lifespan data. (B-C) The <i>irg-7(zc6) gof</i> mutation extended the lifespan of wild-type animals with an intact reproductive system but shortened the lifespan of animals that lack a somatic gonad (B). It did not further extend the lifespan of germline-less animals (C). Mean lifespan and Mantel-Cox P-values are indicated within each graph. Mantel Cox P-value for the wild type vs. <i>irg-7(zc6)</i> single mutant comparison is in green; Mantel Cox P-value for the mutant vs. the mutant; <i>irg-7(zc6)</i> double mutant comparison is in black. <b>See <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006577#pgen.1006577.s008" target="_blank">S6 Table</a></b> for additional lifespan data.</p