Axenic Culture of Caenorhabditis elegans Alters Lysosomal/Proteasomal Balance and Increases Neuropeptide Expression.

Axenically cultured C. elegans show many characteristic traits of worms subjected to dietary restriction, such as slowed development, reduced fertility, and increased stress resistance. Hence, the term axenic dietary restriction (ADR) is often applied. ADR dramatically extends the worm lifespan compared to other DR regimens such as bacterial dilution. However, the underlying molecular mechanisms still remain unclear. The primary goal of this study is to comprehensively investigate transcriptional alterations that occur when worms are subjected to ADR and to estimate the molecular and physiological changes that may underlie ADR-induced longevity. One of the most enriched clusters of up-regulated genes under ADR conditions is linked to lysosomal activity, while proteasomal genes are significantly down-regulated. The up-regulation of genes specifically involved in amino acid metabolism is likely a response to the high peptide levels found in axenic culture medium. Genes related to the integrity and function of muscles and the extracellular matrix are also up-regulated. Consistent down-regulation of genes involved in DNA replication and repair may reflect the reduced fertility phenotype of ADR worms. Neuropeptide genes are found to be largely up-regulated, suggesting a possible involvement of neuroendocrinal signaling in ADR-induced longevity. In conclusion, axenically cultured worms seem to rely on increased amino acid catabolism, relocate protein breakdown from the cytosol to the lysosomes, and do not invest in DNA maintenance but rather retain muscle integrity and the extracellular matrix. All these changes may be coordinated by peptidergic signaling.

CBP-1 Acts in GABAergic Neurons to Double Life Span in Axenically Cultured Caenorhabditis elegans.

When cultured in axenic medium, Caenorhabditis elegans shows the largest life-span extension compared with other dietary restriction regimens. However, the underlying molecular mechanism still remains elusive. The gene cbp-1, encoding the worm ortholog of p300/CBP (CREB-binding protein), is one of the very few key genes known to be essential for life span doubling under axenic dietary restriction (ADR). By using tissue-specific RNAi, we found that cbp-1 expression in the germline is essential for fertility, whereas this gene functions specifically in the GABAergic neurons to support the full life span-doubling effect of ADR. Surprisingly, GABA itself is not required for ADR-induced longevity, suggesting a role of neuropeptide signaling. In addition, chemotaxis assays illustrate that neuronal inactivation of CBP-1 affects the animals' food sensing behavior. Together, our results show that the strong life-span extension in axenic medium is under strict control of GABAergic neurons and may be linked to food sensing.

Life-Span Extension by Axenic Dietary Restriction Is Independent of the Mitochondrial Unfolded Protein Response and Mitohormesis in Caenorhabditis elegans.

In Caenorhabditis elegans, a broad range of dietary restriction regimens extend life span to different degrees by separate or partially overlapping molecular pathways. One of these regimens, axenic dietary restriction, doubles the worm's life span but currently, almost nothing is known about the underlying molecular mechanism. Previous studies suggest that mitochondrial stress responses such as the mitochondrial unfolded protein response (UPRmt) or mitohormesis may play a vital role in axenic dietary restriction-induced longevity. Here, we provide solid evidence that axenic dietary restriction treatment specifically induces an UPRmt response in C elegans but this induction is not required for axenic dietary restriction-mediated longevity. We also show that reactive oxygen species-mediated mitohormesis is not involved in this phenotype. Hence, changes in mitochondrial physiology and induction of a mitochondrial stress response are not necessarily causal to large increases in life span.

FOXO/DAF-16 Activation Slows Down Turnover of the Majority of Proteins in C. elegans.

Most aging hypotheses assume the accumulation of damage, resulting in gradual physiological decline and, ultimately, death. Avoiding protein damage accumulation by enhanced turnover should slow down the aging process and extend the lifespan. However, lowering translational efficiency extends rather than shortens the lifespan in C. elegans. We studied turnover of individual proteins in the long-lived daf-2 mutant by combining SILeNCe (stable isotope labeling by nitrogen in Caenorhabditiselegans) and mass spectrometry. Intriguingly, the majority of proteins displayed prolonged half-lives in daf-2, whereas others remained unchanged, signifying that longevity is not supported by high protein turnover. This slowdown was most prominent for translation-related and mitochondrial proteins. In contrast, the high turnover of lysosomal hydrolases and very low turnover of cytoskeletal proteins remained largely unchanged. The slowdown of protein dynamics and decreased abundance of the translational machinery may point to the importance of anabolic attenuation in lifespan extension, as suggested by the hyperfunction theory.

Mitochondrial efficiency is increased in axenically cultured Caenorhabditis elegans.

Culturing Caenorhabditis elegans in axenic medium leads to a twofold increase in lifespan and considering the similar phenotypical traits with dietary restricted animals, it is referred to as axenic dietary restriction (ADR). The free radical theory of aging has suggested a pivotal role for mitochondria in the aging process and previous findings established that culture in axenic medium increases metabolic rate. We asked whether axenic culture induces changes in mitochondrial functionality of C. elegans. We show that ADR induces increased electron transport chain (ETC) capacity, enhanced coupling efficiency and reduced leakiness of the mitochondria of young adult worms but not a decrease of ROS production capacity and in vivo H2O2 levels. The age-dependent increase in leak respiration and decrease in coupling efficiency is repressed under ADR conditions. Although ADR mitochondria experience a decrease in ETC capacity with age, they succeed to maintain highly efficient and well-coupled function compared to fully fed controls. This might be mediated by combination of a limited increase in supercomplex abundance and decreased individual CIV abundance, facilitating electron transport and ultimately leading to increased mitochondrial efficiency.