Yohei Matsunaga

Genes down-regulated in spaceflight are involved in the control of longevity in Caenorhabditis elegans

How microgravitational space environments affect aging is not well understood. We observed that, in Caenorhabditis elegans, spaceflight suppressed the formation of transgenically expressed polyglutamine aggregates, which normally accumulate with increasing age. Moreover, the inactivation of each of seven genes that were down-regulated in space extended lifespan on the ground. These genes encode proteins that are likely related to neuronal or endocrine signaling: acetylcholine receptor, acetylcholine transporter, choline acetyltransferase, rhodopsin-like receptor, glutamate-gated chloride channel, shaker family of potassium channel, and insulin-like peptide. Most of them mediated lifespan control through the key longevity-regulating transcription factors DAF-16 or SKN-1 or through dietary-restriction signaling, singly or in combination. These results suggest that aging in C. elegans is slowed through neuronal and endocrine response to space environmental cues.

Physiological function, expression pattern, and transcriptional regulation of a Caenorhabditis elegans insulin-like peptide, INS-18.

In Caenorhabditis elegans, insulin/insulin-like growth factor (IGF)-1 signaling (IIS) is an important pathway that controls larval diapause and adult lifespan. The IIS pathway is modulated by many insulin-like peptides (ILPs) through the DAF-2 receptor, the sole insulin/IGF-1 receptor-like protein in C. elegans. We previously identified the ILP, INS-18, and predicted its tertiary structure to be similar to the crystal structures of human insulin and IGF-1. In this study, the physiological function of INS-18 was first examined by gene disruption and overexpression, and we identified INS-18 as a DAF-2 antagonist required for larval diapause and longevity. Analysis of the INS-18 expression pattern using a reporter gene showed it to be expressed in nerve cells, including hermaphrodite-specific neurons (HSNs) at the adult stage. Other ILP expressions have not been previously observed in HSNs, and we believe that INS-18 expression in these cells may contribute to longevity by regulating reproduction. Loss of the DAF-16 transcription factor located downstream of the IIS pathway completely blocked ins-18 expression. We propose a positive feedback model for the regulation of ins-18 expression in which an antagonist binding to the DAF-2 receptor increases ins-18 gene expression, thus leading to increased INS-18 protein levels and increased DAF-2 receptor binding. Thus, this study provides a new insight into the hormonal regulation of insulin, an important and widespread process in the animal kingdom.

Vitamin B12 deficiency in Caenorhabditis elegans results in loss of fertility, extended life cycle, and reduced lifespan

Vitamin B12 (B12) deficiency has been linked to developmental disorders, metabolic abnormalities, and neuropathy; however, the mechanisms involved remain poorly understood. Caenorhabditis elegans grown under B12‐deficient conditions for five generations develop severe B12 deficiency associated with various phenotypes that include decreased egg‐laying capacity (infertility), prolonged life cycle (growth retardation), and reduced lifespan. These phenotypes resemble the consequences of B12 deficiency in mammals, and can be induced in C. elegans in only 15 days. Thus, C. elegans is a suitable animal model for studying the biological processes induced by vitamin deficiency.

Indoles from commensal bacteria extend healthspan

Significance Increases in human life expectancy over the next century will be accompanied by increased frailty and massive and unsustainable health care costs. Developing means to extend the time that individuals remain healthy and free of age-related infirmities, called healthspan, has therefore become a critical goal of aging research. We show that small molecules produced by the microbiota and related to indole extend healthspan in geriatric worms, flies, and mice, without attendant effects on lifespan. Indoles act via the aryl hydrocarbon receptor and cause animals to retain a youthful gene expression profile. Indoles may represent a new class of therapeutics that improve the way we age as opposed to simply extending how long we live. Multiple studies have identified conserved genetic pathways and small molecules associated with extension of lifespan in diverse organisms. However, extending lifespan does not result in concomitant extension in healthspan, defined as the proportion of time that an animal remains healthy and free of age-related infirmities. Rather, mutations that extend lifespan often reduce healthspan and increase frailty. The question arises as to whether factors or mechanisms exist that uncouple these processes and extend healthspan and reduce frailty independent of lifespan. We show that indoles from commensal microbiota extend healthspan of diverse organisms, including Caenorhabditis elegans, Drosophila melanogaster, and mice, but have a negligible effect on maximal lifespan. Effects of indoles on healthspan in worms and flies depend upon the aryl hydrocarbon receptor (AHR), a conserved detector of xenobiotic small molecules. In C. elegans, indole induces a gene expression profile in aged animals reminiscent of that seen in the young, but which is distinct from that associated with normal aging. Moreover, in older animals, indole induces genes associated with oogenesis and, accordingly, extends fecundity and reproductive span. Together, these data suggest that small molecules related to indole and derived from commensal microbiota act in diverse phyla via conserved molecular pathways to promote healthy aging. These data raise the possibility of developing therapeutics based on microbiota-derived indole or its derivatives to extend healthspan and reduce frailty in humans.

A Caenorhabditis elegans Insulin-Like Peptide, INS-17: Its Physiological Function and Expression Pattern

The insulin/insulin-like growth factor-1 signaling pathway of Caenorhabditis elegans regulates larval diapause and adult lifespan through the sole insulin receptor-like protein, DAF-2. In the present study, the physiological function and expression pattern of INS-17, one of the C. elegans insulin-like peptides, were examined by disruption and overexpression of the gene, and by the use of a reporter gene. INS-17 might function as a DAF-2 antagonist in the regulation of larval diapause, but not of the adult lifespan. The reporter protein was intensively expressed during larval diapause. It showed a drastic decrease in amount after larval diapause, which matches well the physiological function of INS-17.

Muscle contraction phenotypic analysis enabled by optogenetics reveals functional relationships of sarcomere components in Caenorhabditis elegans

The sarcomere, the fundamental unit of muscle contraction, is a highly-ordered complex of hundreds of proteins. Despite decades of genetics work, the functional relationships and the roles of those sarcomeric proteins in animal behaviors remain unclear. In this paper, we demonstrate that optogenetic activation of the motor neurons that induce muscle contraction can facilitate quantitative studies of muscle kinetics in C. elegans. To increase the throughput of the study, we trapped multiple worms in parallel in a microfluidic device and illuminated for photoactivation of channelrhodopsin-2 to induce contractions in body wall muscles. Using image processing, the change in body size was quantified over time. A total of five parameters including rate constants for contraction and relaxation were extracted from the optogenetic assay as descriptors of sarcomere functions. To potentially relate the genes encoding the sarcomeric proteins functionally, a hierarchical clustering analysis was conducted on the basis of those parameters. Because it assesses physiological output different from conventional assays, this method provides a complement to the phenotypic analysis of C. elegans muscle mutants currently performed in many labs; the clusters may provide new insights and drive new hypotheses for functional relationships among the many sarcomere components.

Twitchin kinase interacts with MAPKAP kinase 2 in Caenorhabditis elegans striated muscle.

Titin-like giant polypeptides of muscle have protein kinase domains near their C-termini. These kinases are autoinhibited by portions of their own sequences. A putative activator for Caenorhabditis elegans twitchin kinase, MAK-1 (MAPKAP kinase 2), is expressed in nematode striated muscle, partially colocalizes with twitchin in sarcomeres, and binds to and phosphorylates twitchin kinase in vitro.

An altered method of feeding RNAi that knocks down multiple genes simultaneously in the nematode Caenorhabditis elegans.

In reverse genetics, RNA interference (RNAi) which is substitutable for gene-disruption, is an outstanding method for knockdown of a gene’s function. In Caenorhabditis elegans, feeding RNAi is most convenient, but this RNAi is not suitable for knockdown of multiple genes. Hence, we attempted to establish an efficient method of feeding RNAi for multiple knockdown. We produced bacteria yielding three distinct double-stranded RNAs bound to one another, and fed those bacteria to C. elegans. Quantitative RT-PCR and observation of phenotypes indicated that our method is much more efficient than the traditional one. Our method is useful for investigating genes’ functions in C. elegans.

Diapause is associated with a change in the polarity of secretion of insulin-like peptides

The insulin/IGF-1 signalling (IIS) pathway plays an important role in the regulation of larval diapause, the long-lived growth arrest state called dauer arrest, in Caenorhabditis elegans. In this nematode, 40 insulin-like peptides (ILPs) have been identified as putative ligands of the IIS pathway; however, it remains unknown how ILPs modulate larval diapause. Here we show that the secretory polarity of INS-35 and INS-7, which suppress larval diapause, is changed in the intestinal epithelial cells at larval diapause. These ILPs are secreted from the intestine into the body cavity during larval stages. In contrast, they are secreted into the intestinal lumen and degraded during dauer arrest, only to be secreted into the body cavity again when the worms return to developmental growth. The process that determines the secretory polarity of INS-35 and INS-7, thus, has an important role in the modulation of larval diapause.

Suppressor Mutations Suggest a Surface on PAT-4 (Integrin-linked Kinase) That Interacts with UNC-112 (Kindlin)

Background: Interaction between PAT-4 (ILK) and UNC-112 (kindlin) is required for localization of each protein to integrin adhesions. Results: PAT-4 missense mutations were identified that restore binding of a mutant UNC-112 protein and its proper localization. Conclusion: Residues mutated in the suppressor mutants cluster in two regions on the surface of a homology model of PAT-4. Significance: An interaction surface between PAT-4 and UNC-112 is suggested. Caenorhabditis elegans striated muscle cells attach to basement membrane and transmit the force of muscle contraction through integrin adhesion complexes. The cytoplasmic tail of β-integrin (PAT-3) is associated with a conserved four-protein complex that includes UNC-112 (kindlin), PAT-4 (integrin-linked kinase), PAT-6 (α-parvin/actopaxin), and UNC-97 (PINCH). The proper localization of UNC-112 to muscle integrin adhesion sites requires PAT-4. A recent report (Qadota, H., Moerman, D. G., and Benian, G. M. (2012) A molecular mechanism for the requirement of PAT-4 (integrin-linked kinase (ILK)) for the localization of UNC-112 (kindlin) to integrin adhesion sites. J. Biol. Chem. 287, 28537–28551) suggests a possible molecular mechanism for this requirement: that UNC-112 exists in closed inactive and open active conformations, and conversion to the open active form is promoted by binding to PAT-4 (ILK). Previously, we also reported identification of a single missense mutation in UNC-112, D382V, which abolishes both binding to PAT-4 and normal localization to integrin adhesion sites in vivo. In this report, we describe isolation and characterization of PAT-4 missense mutations that permit binding with UNC-112 D382V and place nine affected residues on a homology model of PAT-4. These nine residues cluster in two regions on the surface of PAT-4, do not overlap the likely binding surface for PAT-6 (α-parvin), and therefore may reside along the interaction surface of PAT-4 for UNC-112 (kindlin). We also show that one of these PAT-4 mutations restores the ability of UNC-112 D382V to localize to integrin adhesions and participate in complex formation.