Identification of Novel Genes Involved in Sarcopenia Through RNAi Screening in Caenorhabditis elegans

SARCOPENIA is the progressive loss of muscle mass with advancing age characterized by a decline in muscle quantity and quality (1,2). The medical importance of sarcopenia lies in the resulting reduction in strength, which serves as a risk factor for some of the impairments in mobility and functioning seen in older people (3). The decline in strength also appears to act as a risk factor for mortality (4). Although the cause of sarcopenia is not known, there is evidence from human and vertebrate animal studies for several mechanisms including disuse atrophy associated with inactivity, loss of α-motor neurons, hormonal changes, inflammatory effects, altered caloric intake, and changes muscle cell physiology (5,6). Despite this work, the primary cause of sarcopenia is unclear, and the current treatment options are rather limited. For example, a recent promising trial of testosterone was halted due to an increase in adverse events in treated participants (7). Hence, the development of new model systems could advance research in this field and hopefully lead to new treatments. The nematode Caenorhabditis elegans has emerged as a powerful model system in multiple areas of biology. Worms are used to investigate the genetic and molecular functions of many human disease-related genes in biologic processes such as cell death and cell fate determination or to study pathological processes such as neurodegeneration and microbial pathogenesis (8). Furthermore, there are several experimental advantages to using C elegans such as a completely sequenced genome that shares significant homology with that of mammals, the ability to easily and inexpensively cultivate animals in the lab, the short life span of 2–3 weeks, and the ability to manipulate the genome by generating mutant and transgenic animals. Caenorhabditis elegans also offers the ability to complement standard mechanistic experiments with unbiased forward genetic screens. Finally, a major strength of the worm system is the ability to use RNA interference (RNAi) to manipulate gene expression and function by feeding the worm Escherichia coli bacteria producing the desired double-stranded RNA as food (9). Like humans, C elegans also develops sarcopenia during aging (10). These muscle losses start in midlife, magnify with increasing age, and are associated with reductions in mobility (10,11). These changes in mobility are not due to neuropathology during aging as microscopic examination of worms have shown that the nervous system remains intact in old worms, whereas the myofibrils of old worms become progressively disorganized and lose myosin filaments (10,11). In addition to myofibril loss, there are also changes in nuclear architecture with aging (10). Mutations in the daf-2/insulin-like signaling pathway in worms result in up to 100% increases in life span (12). This pathway comprises the insulin/insulin-like growth factor-1 receptor DAF-2 that signals through a conserved PI3-kinase/AKT pathway to phosphorylate and inhibit DAF-16, a forkhead family transcription factor (12). In daf-2 mutants, daf-16 is nuclear localized and activates a significant number of other target genes (12). In addition to increased longevity, daf-2 mutants also show increased resistance to oxidative stress, hypoxia, heat stress, heavy metals, and bacterial pathogens (12). Worms with mutations in the daf-2 pathway are also resistant to the development of sarcopenia and associated declines in mobility during aging (10,11,13). Additionally, genes in the epidermal growth factor pathway and the Rag GTPase (raga-1) pathway also act to preserve mobility and muscle function (14,15). Together, these findings suggest that sarcopenia is a malleable phenotype and likely amenable to study. This preservation of muscle mass and mobility in worms with mutations affecting the daf-2 pathway suggests that activation of daf-2 target genes either directly or indirectly affects the resistance of muscle to the development of sarcopenia. We hypothesized that specific genes acting downstream of daf-2 may be required for the delay in sarcopenia. To test this hypothesis, we investigated the effect of knockdown of a total of 43 genes, most of which have been shown to affect this pathway (16), to screen for genes, which might be required to preserve mobility and muscle function in daf-2 mutant worms.