Molecular mechanisms underlying the extreme longevity of C. elegans age-1 PI3K mutants

Genetic studies using C. elegans as a model system have led to the discovery of majority of genes affecting lifespan. One of the most extensively studied pathway affecting longevity in C. elegans as well as in yeast, mammals and drosophila is the Insulin/IGF-1 signaling (IIS) pathway. A key player in the C. elegans IIS pathway is the PI3Kcs homologue AGE-1 protein. The age-1 mutant, defective in its PI3Kcs homologue AGE-1 protein is the focus of this study. Strains homozygous for the nonsense mutation in age-1 gene produce progeny that show a nearly 10-fold extension of both median and maximum adult lifespan relative to the wild type worms. These long lived worms show higher resistance to oxidative and electrophilic stresses relative to wild type worms having a functional age-1 gene. We compared age-1 alleles (mg44 and m333) with age-1 mutant allele hx546 (weaker in longevity phenotype) and with wild type worms using expression microarray analysis. The age-1(mg44) worms showed a global transcriptional suppression and gene silencing differing markedly from its weaker hx546 cohort implicating novel life extending mechanisms. A subset of genes found significant by SAM analysis, were reassessed using real time PCR. The majority of these genes showed transcriptional suppression in age-1(mg44) mutants with an additional mutation in daf-16 reverting the expression levels to approximately same values as wild types. RNAi knockdown of these age-1 suppressed genes in wild type worms conferred oxidative stress protection in most cases. Seven of these genes, including five reported for the first time, led to an increase in lifespan when knocked down in wild type worms. AGE-1 in C. elegans phosphorylates PIP2 to PIP 3 which is a bioactive phospholipid responsible for recruiting numerous protein kinases on the membrane and in their activation. Lack of PI3K activity in age-1 mutants markedly suppressed the transcript levels of insulin like signaling members and various other components known to interact with IIS. Kinase activity and phosphorylated protein levels in F2 age-1(mg44) mutants were found to be decreased significantly. Most but not all of these changes again were dependent on daf-16 in that a daf-16 mutation in age-1 background reverted most of the phenotypes to wild type levels. In vitro peptide array assays also showed DAF-16 dependent suppressed kinase activity in age-1(mg44) worms with differential phosphorylation of DAF-16 protein in age-1(mg44) worms. Respirometery analysis comparing F2 age-1(mg44) worms with wild type cohorts did not show a significant change in the metabolic output of F2 age-1(mg44) worms but indicated a metabolic shift towards lipid metabolism. Sudan black staining and GCMS analysis also showed an overall increase in storage lipids in F2 age-1(mg44) worms. F1 homozygous age-1( mg44) worms having the same genetic makeup as F2 age-1( mg44) worms fail to show longevity, stress tolerance or kinase suppression as extreme as F2 generation age-1(mg44) worms. We showed by immunofluorescence and by in vivo PIP 3 assay that PIP3 levels in F1 age-1( mg44) worms are significantly lower than wild type worms and F2 age-1(mg44) worms have undetectable PIP3 levels. Such low levels of PIP3 in F2 age-1( mg44) worms due to the lack of maternal carryover of age-1 mRNA, AGE-1 or PIP3, could be the reason of such a dramatic phenotype. F1 age-1(mg44) worms receiving maternal carryover from their heterozygous mothers fail to show extreme longevity. When wild type adult worms are treated with phosphatidylinositol phosphate analogs (PIAs) the worms show enhanced stress tolerance and lifespan phenotype which is not observed in the weak age-1 hx5546 mutants indicating a mechanism of action similar to age-1 mutation. These analogs were initially designed to inhibit AKT activity and are considered potential anti-cancer agents. We believe that highly specific analogs of PIP2 and PIP3 targeting proteins like AGE-1 and AKT can have a high therapeutic potential and can be utilized for human applications such as targeted drug development.