Knockdown of Indy/CeNac2 extends Caenorhabditis elegans life span by inducing AMPK/aak-2

Abstract

Reducing the expression of the Indy (Acronym for 'I'm Not Dead, Yet') gene in lower organisms promotes longevity and leads to a phenotype that resembles various aspects of caloric restriction. In C. elegans, the available data on life span extension is controversial. Therefore, the aim of this study was to determine the role of the C. elegans INDY homolog CeNAC2 in life span regulation and to delineate possible molecular mechanisms. siRNA against Indy/CeNAC2 was used to reduce expression of Indy/CeNAC2. Mean life span was assessed in four independent experiments, as well as whole body fat content and AMPK activation. Moreover, the effect of Indy/CeNAC2 knockdown in C. elegans with inactivating variants of AMPK (TG38) was studied. Knockdown of Indy/CeNAC2 increased life span by 22±3 % compared to control siRNA treated C. elegans, together with a decrease in whole body fat content by ~50%. Indy/CeNAC2 reduction also increased the activation of the intracellular energy sensor AMPK/aak2. In worms without functional AMPK/aak2, life span was not extended when Indy/CeNAC2 was reduced. Inhibition of glycolysis with deoxyglucose, an intervention known to increase AMPK/aak2 activity and life span, did not promote longevity when Indy/CeNAC2 was knocked down. Together, these data indicate that reducing the expression of Indy/CeNAC2 increases life span in C. elegans, an effect mediated at least in part by AMPK/aak2.

Keywords: AMPK; C.elegans; CeNAC-2; Indy; Slc13A5; aak-2; mIndy.

Figure 1

(A) Representative survival curves for N2 (WT), fed with E. coli GC363 harboring the empty vector L4400 (empty vector control) versus E. coli GC363 harboring INDY/CeNAC2-specific siRNA. INDY/CeNAC2-leads to a significant life span extension (P < 0.001). Life span experiments were repeated four times in independent experiments, figure shows the second experiment, other data in Table 1.(B) Oil Red O staining of N2 wild type fed with E.coli GC363 harboring the empty vector L4400 (empty vector control) versus E.coli GC363 harboring INDY/CeNAC2-specific siRNA (C). N2 wild type worms showed more intensive staining and higher total area stained compared to RNAi treatment (D), Densitometric analysis of B and C, **p = 0.005. (E) Immunoblot analysis of different C. elegans strains (N2, TG) and fasted mouse liver lysate (C) as positive control for phosphorylation (activation) of AMPK/ aak-2 after knockdown of INDY/CeNAC2 (N2/I). Down regulation of INDY/CeNAC2 leads to activation of AMPK / aak-2 compared to control fed wildtype N2. Negative control TG38 (TG) with missing phosphorylation site for AMPK-Thr172 shows no activation when fed with empty vector control neither when treated with RNAi against INDY/CeNAC2 (G/i). Control (c) = liver lysates from fasted mice.

Figure 2

(A) Survival curves for TG38 AMPK/akk-2(gt33), fed with E. coli GC363 harboring the empty vector L4400 (empty vector control) versus E. coli GC363 harboring INDY/CeNAC2-specific siRNA and CB1370 daf-2(e1370) fed with empty vector control. (B) Survival curves for N2 (WT), fed with E.coli GC363 harboring the empty vector L4400 (empty vector control) versus E.coli GC363 harboring INDY/CeNAC2 siRNA with 2-Deoxy-D-Glucose (DOG+) in the media.