Quantitative Image Analysis Reveals Distinct Structural Transitions during Aging in Caenorhabditis elegans Tissues

Aging is associated with functional and structural declines in many body systems, even in the absence of underlying disease. In particular, skeletal muscles experience severe declines during aging, a phenomenon termed sarcopenia. Despite the high incidence and severity of sarcopenia, little is known about contributing factors and development. Many studies focus on functional aspects of aging-related tissue decline, while structural details remain understudied. Traditional approaches for quantifying structural changes have assessed individual markers at discrete intervals. Such approaches are inadequate for the complex changes associated with aging. An alternative is to consider changes in overall morphology rather than in specific markers. We have used this approach to quantitatively track tissue architecture during adulthood and aging in the C. elegans pharynx, the neuromuscular feeding organ. Using pattern recognition to analyze aged-grouped pharynx images, we identified discrete step-wise transitions between distinct morphologies. The morphology state transitions were maintained in mutants with pharynx neurotransmission defects, although the pace of the transitions was altered. Longitudinal measurements of pharynx function identified a predictive relationship between mid-life pharynx morphology and function at later ages. These studies demonstrate for the first time that adult tissues undergo distinct structural transitions reflecting postdevelopmental events. The processes that underlie these architectural changes may contribute to increased disease risk during aging, and may be targets for factors that alter the aging rate. This work further demonstrates that pattern analysis of an image series offers a novel and generally accessible approach for quantifying morphological changes and identifying structural biomarkers

Co-Regulation of the DAF-16 Target Gene, cyp-35B1/dod-13, by HSF-1 in C. elegans Dauer Larvae and daf-2 Insulin Pathway Mutants

Insulin/IGF-I-like signaling (IIS) has both cell autonomous and non-autonomous functions. In some cases, targets through which IIS regulates cell-autonomous functions, such as cell growth and metabolism, have been identified. In contrast, targets for many non-autonomous IIS functions, such as C. elegans dauer morphogenesis, remain elusive. Here, we report the use of genomic and genetic approaches to identify potential non-autonomous targets of C. elegans IIS. First, we used transcriptional microarrays to identify target genes regulated non-autonomously by IIS in the intestine or in neurons. C. elegans IIS controls expression of a number of stress response genes, which were differentially regulated by tissue-restricted IIS. In particular, expression of sod-3, a MnSOD enzyme, was not regulated by tissue-restricted IIS on the microarrays, while expression of hsp-16 genes was rescued back to wildtype by tissue restricted IIS. One IIS target regulated non-autonomously by age-1 was cyp-35B1/dod-13, encoding a cytochrome P450. Genetic analysis of the cyp-35B1 promoter showed both DAF-16 and HSF-1 are direct regulators. Based on these findings, we propose that hsf-1 may participate in the pathways mediating non-autonomous activities of age-1 in C. elegans

Strongyloides stercoralis age-1: A Potential Regulator of Infective Larval Development in a Parasitic Nematode

Infective third-stage larvae (L3i) of the human parasite Strongyloides stercoralis share many morphological, developmental, and behavioral attributes with Caenorhabditis elegans dauer larvae. The ‘dauer hypothesis’ predicts that the same molecular genetic mechanisms control both dauer larval development in C. elegans and L3i morphogenesis in S. stercoralis. In C. elegans, the phosphatidylinositol-3 (PI3) kinase catalytic subunit AGE-1 functions in the insulin/IGF-1 signaling (IIS) pathway to regulate formation of dauer larvae. Here we identify and characterize Ss-age-1, the S. stercoralis homolog of the gene encoding C. elegans AGE-1. Our analysis of the Ss-age-1 genomic region revealed three exons encoding a predicted protein of 1,209 amino acids, which clustered with C. elegans AGE-1 in phylogenetic analysis. We examined temporal patterns of expression in the S. stercoralis life cycle by reverse transcription quantitative PCR and observed low levels of Ss-age-1 transcripts in all stages. To compare anatomical patterns of expression between the two species, we used Ss-age-1 or Ce-age-1 promoter::enhanced green fluorescent protein reporter constructs expressed in transgenic animals for each species. We observed conservation of expression in amphidial neurons, which play a critical role in developmental regulation of both dauer larvae and L3i. Application of the PI3 kinase inhibitor LY294002 suppressed L3i in vitro activation in a dose-dependent fashion, with 100 µM resulting in a 90% decrease (odds ratio: 0.10, 95% confidence interval: 0.08–0.13) in the odds of resumption of feeding for treated L3i in comparison to the control. Together, these data support the hypothesis that Ss-age-1 regulates the development of S. stercoralis L3i via an IIS pathway in a manner similar to that observed in C. elegans dauer larvae. Understanding the mechanisms by which infective larvae are formed and activated may lead to novel control measures and treatments for strongyloidiasis and other soil-transmitted helminthiases

A Decline in p38 MAPK Signaling Underlies Immunosenescence in Caenorhabditis elegans

The decline in immune function with aging, known as immunosenescence, has been implicated in evolutionarily diverse species, but the underlying molecular mechanisms are not understood. During aging in Caenorhabditis elegans, intestinal tissue deterioration and the increased intestinal proliferation of bacteria are observed, but how innate immunity changes during C. elegans aging has not been defined. Here we show that C. elegans exhibits increased susceptibility to bacterial infection with age, and we establish that aging is associated with a decline in the activity of the conserved PMK-1 p38 mitogen-activated protein kinase pathway, which regulates innate immunity in C. elegans. Our data define the phenomenon of innate immunosenescence in C. elegans in terms of the age-dependent dynamics of the PMK-1 innate immune signaling pathway, and they suggest that a cycle of intestinal tissue aging, immunosenescence, and bacterial proliferation leads to death in aging C. elegans

Caenorhabditis elegans Battling Starvation Stress: Low Levels of Ethanol Prolong Lifespan in L1 Larvae

The nematode Caenorhabditis elegans arrests development at the first larval stage if food is not present upon hatching. Larvae in this stage provide an excellent model for studying stress responses during development. We found that supplementing starved larvae with ethanol markedly extends their lifespan within this L1 diapause. The effects of ethanol-induced lifespan extension can be observed when the ethanol is added to the medium at any time between 0 and 10 days after hatching. The lowest ethanol concentration that extended lifespan was 1 mM (0.005%); higher concentrations to 68 mM (0.4%) did not result in increased survival. In spite of their extended survival, larvae did not progress to the L2 stage. Supplementing starved cultures with n-propanol and n-butanol also extended lifespan, but methanol and isopropanol had no measurable effect. Mass spectrometry analysis of nematode fatty acids and amino acids revealed that L1 larvae can incorporate atoms from ethanol into both types of molecules. Based on these data, we suggest that ethanol supplementation may extend the lifespan of L1 larvae by either serving as a carbon and energy source and/or by inducing a stress response

Activation of Akt Signaling Reduces the Prevalence and Intensity of Malaria Parasite Infection and Lifespan in Anopheles stephensi Mosquitoes

Malaria (Plasmodium spp.) kills nearly one million people annually and this number will likely increase as drug and insecticide resistance reduces the effectiveness of current control strategies. The most important human malaria parasite, Plasmodium falciparum, undergoes a complex developmental cycle in the mosquito that takes approximately two weeks and begins with the invasion of the mosquito midgut. Here, we demonstrate that increased Akt signaling in the mosquito midgut disrupts parasite development and concurrently reduces the duration that mosquitoes are infective to humans. Specifically, we found that increased Akt signaling in the midgut of heterozygous Anopheles stephensi reduced the number of infected mosquitoes by 60–99%. Of those mosquitoes that were infected, we observed a 75–99% reduction in parasite load. In homozygous mosquitoes with increased Akt signaling parasite infection was completely blocked. The increase in midgut-specific Akt signaling also led to an 18–20% reduction in the average mosquito lifespan. Thus, activation of Akt signaling reduced the number of infected mosquitoes, the number of malaria parasites per infected mosquito, and the duration of mosquito infectivity

Honey Bee PTEN – Description, Developmental Knockdown, and Tissue-Specific Expression of Splice-Variants Correlated with Alternative Social Phenotypes

Phosphatase and TENsin (PTEN) homolog is a negative regulator that takes part in IIS (insulin/insulin-like signaling) and Egfr (epidermal growth factor receptor) activation in Drosophila melanogaster. IIS and Egfr signaling events are also involved in the developmental process of queen and worker differentiation in honey bees (Apis mellifera). Here, we characterized the bee PTEN gene homologue for the first time and begin to explore its potential function during bee development and adult life.Honey bee PTEN is alternatively spliced, resulting in three splice variants. Next, we show that the expression of PTEN can be down-regulated by RNA interference (RNAi) in the larval stage, when female caste fate is determined. Relative to controls, we observed that RNAi efficacy is dependent on the amount of PTEN dsRNA that is delivered to larvae. For larvae fed queen or worker diets containing a high amount of PTEN dsRNA, PTEN knockdown was significant at a whole-body level but lethal. A lower dosage did not result in a significant gene down-regulation. Finally, we compared same-aged adult workers with different behavior: nursing vs. foraging. We show that between nurses and foragers, PTEN isoforms were differentially expressed within brain, ovary and fat body tissues. All isoforms were expressed at higher levels in the brain and ovaries of the foragers. In fat body, isoform B was expressed at higher level in the nurse bees.Our results suggest that PTEN plays a central role during growth and development in queen- and worker-destined honey bees. In adult workers, moreover, tissue-specific patterns of PTEN isoform expression are correlated with differences in complex division of labor between same-aged individuals. Therefore, we propose that knowledge on the roles of IIS and Egfr activity in developmental and behavioral control may increase through studies of how PTEN functions can impact bee social phenotypes

In Vitro Cellular Adaptations of Indicators of Longevity in Response to Treatment with Serum Collected from Humans on Calorie Restricted Diets

Calorie restriction (CR) produces several health benefits and increases lifespan in many species. Studies suggest that alternate-day fasting (ADF) and exercise can also provide these benefits. Whether CR results in lifespan extension in humans is not known and a direct investigation is not feasible. However, phenotypes observed in CR animals when compared to ad libitum fed (AL) animals, including increased stress resistance and changes in protein expression, can be simulated in cells cultured with media supplemented with blood serum from CR and AL animals. Two pilot studies were undertaken to examine the effects of ADF and CR on indicators of health and longevity in humans. In this study, we used sera collected from those studies to culture human hepatoma cells and assessed the effects on growth, stress resistance and gene expression. Cells cultured in serum collected at the end of the dieting period were compared to cells cultured in serum collected at baseline (before the dieting period). Cells cultured in serum from ADF participants, showed a 20% increase in Sirt1 protein which correlated with reduced triglyceride levels. ADF serum also induced a 9% decrease in proliferation and a 25% increase in heat resistance. Cells cultured in serum from CR participants induced an increase in Sirt1 protein levels by 17% and a 30% increase in PGC-1α mRNA levels. This first in vitro study utilizing human serum to examine effects on markers of health and longevity in cultured cells resulted in increased stress resistance and an up-regulation of genes proposed to be indicators of increased longevity. The use of this in vitro technique may be helpful for predicting the potential of CR, ADF and other dietary manipulations to affect markers of longevity in humans

The Functions of Myosin II and Myosin V Homologs in Tip Growth and Septation in Aspergillus nidulans

Because of the industrial and medical importance of members of the fungal genus Aspergillus, there is considerable interest in the functions of cytoskeletal components in growth and secretion in these organisms. We have analyzed the genome of Aspergillus nidulans and found that there are two previously unstudied myosin genes, a myosin II homolog, myoB (product = MyoB) and a myosin V homolog, myoE (product = MyoE). Deletions of either cause significant growth defects. MyoB localizes in strings that coalesce into contractile rings at forming septa. It is critical for septation and normal deposition of chitin but not for hyphal extension. MyoE localizes to the Spitzenkörper and to moving puncta in the cytoplasm. Time-lapse imaging of SynA, a v-SNARE, reveals that in myoE deletion strains vesicles no longer localize to the Spitzenkörper. Tip morphology is slightly abnormal and branching occurs more frequently than in controls. Tip extension is slower than in controls, but because hyphal diameter is greater, growth (increase in volume/time) is only slightly reduced. Concentration of vesicles into the Spitzenkörper before incorporation into the plasma membrane is, thus, not required for hyphal growth but facilitates faster tip extension and a more normal hyphal shape

Pseudomonas aeruginosa Suppresses Host Immunity by Activating the DAF-2 Insulin-Like Signaling Pathway in Caenorhabditis elegans

Some pathogens have evolved mechanisms to overcome host immune defenses by inhibiting host defense signaling pathways and suppressing the expression of host defense effectors. We present evidence that Pseudomonas aeruginosa is able to suppress the expression of a subset of immune defense genes in the animal host Caenorhabditis elegans by activating the DAF-2/DAF-16 insulin-like signaling pathway. The DAF-2/DAF-16 pathway is important for the regulation of many aspects of organismal physiology, including metabolism, stress response, longevity, and immune function. We show that intestinal expression of DAF-16 is required for resistance to P. aeruginosa and that the suppression of immune defense genes is dependent on the insulin-like receptor DAF-2 and the FOXO transcription factor DAF-16. By visualizing the subcellular localization of DAF-16::GFP fusion protein in live animals during infection, we show that P. aeruginosa–mediated downregulation of a subset of immune genes is associated with the ability to translocate DAF-16 from the nuclei of intestinal cells. Suppression of DAF-16 is mediated by an insulin-like peptide, INS-7, which functions upstream of DAF-2. Both the inhibition of DAF-16 and downregulation of DAF-16–regulated genes, such as thn-2, lys-7, and spp-1, require the P. aeruginosa two-component response regulator GacA and the quorum-sensing regulators LasR and RhlR and are not observed during infection with Salmonella typhimurium or Enterococcus faecalis. Our results reveal a new mechanism by which P. aeruginosa suppresses host immune defense