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Investigation of Life Span Extension by Torin-2 in the nematode Caenorhabditis elegans.

Name
Andrey
Surname
Zheltukhin
Scientific organization
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russia
Academic degree
none
Position
young researcher
Scientific discipline
Life Sciences & Medicine
Topic
Investigation of Life Span Extension by Torin-2 in the nematode Caenorhabditis elegans.
Abstract
In this study we investigated effects of chemical inhibitor of TOR kinase torin-2 in the C. elegans model. We found that torin-2 is an efficient TOR inhibitor that directly inhibits TOR kinase affecting prs6 phosphorylation and inducing the autophagy, thus significantly extends the average lifespan.
Keywords
TOR, aging, rapamycin, torin2
Summary

Investigation of Life Span Extension by Torin-2 in the nematode Caenorhabditis elegans.


Zheltukhin A.O.1,  Kornev A. B. 2 Chumakov P.M. 1
(1Engelhardt Institute of Molecular Biology of the Russian Academy of Science, Moscow, Russia, 2Institute of Problems of Chemical Physics of the Russian Academy of Sciences, Chernogolovka, Russia. e-mail: aozheltukhin@gmail.com )

 

Serine-threonine specific protein kinase TOR is implicated as a major determinant driving the aging process, while its inhibition has a life-extension effect in different organisms, including flies, nematodes and  mice.   In the nematode model of Caenorhabditis elegans an inhibition of let-363/CeTor  gene results in a significant increase in lifespan.  Rapamycin, a natural small molecule inhibitor of TOR, has also potent life-extension activity in different organisms.  In the present study we tested effects of the second-generation chemical inhibitor of TOR kinase torin-2 in the C. elegans model.  Normal lifespan of the nematodes that have constant number of postmitotic cells is around 20 days.   Sixty individual worms at the L4 stage were maintained in the nutrient agar medium supplemented with increasing concentrations of torin-2, up to 0.2 nM/ml. Another TOR inhibitor rapamycin was used as a control, along with TOR inhibition by RNAi, by feeding with bacteria carrying fragments of the let-363/CeTor gene. We found that torin-2 at the effective dose if 25nM/ml extends the average lifespan by 19,77%, rapamycin at the effective dose of 100 microM - by 20.15%, food deprivation - by 24.82%, as compared to the untreated control. The maximum lifespan was increased by 22.22% by torin-2, rapamycin and RNAi, and by 25.02% by the food deprivation.  To study mechanisms of the torin-2 effects we monitored the ontogenesis, phosphorylation of the TOR-kinase substrate protein rsp6 and the autophagy.  For the latter we used a genetic construct expressing the LC3 analog fused with GFP (LGG::GFP) that helps visualizing and counting autophagosomes.  Animals were treated with as described above for 16 hours. Western blots with fractionated lysates of the nematodes developed with antibodies specific for GFP has revealed that torin-2 at 25 nM/ml increased the autophagy by 10%, at 100 nM/ml - by 20%, food deprivation - by 430%, RNAi - by 170%. Surprisingly, rapamycin at 100 microM/ml did not increase the autophagy. Western blots have demonstrated the reduction of rps6 protein phosphorylation by 20% with 25 nM/ml torin02, by 30% with 100 nM/ml torin-2, by 40% by food deprivation and by 60% by the RNAi.  No reduction of prs6 phosphorylation was detected by rapamycin at 100 microM/ml.  Torin-2 was also capable of 1.5-fold retardation of ontogenesis at 1 microM/ml, and a complete shut-of at 10 microM/ml, while rapamycin had barely detectable effect at 5000 microM/ml, and distinct non-specific toxic effects at higher doses.  

Conclusion: Torin-2 is an efficient TOR inhibitor in the nematode model that directly inhibits TOR kinase affecting prs6 phosphorylation and inducing the autophagy. We also conclude that the effects of rapamycin described for other animal models might be different in nematodes, although the exact differences require additional studies.