New Possibilities for Evaluating the Development of Age-Related Pathologies Using the Dynamical Network Biomarkers Theory.

Aging is the slowest process in a living organism. During this process, mortality rate increases exponentially due to the accumulation of damage at the cellular level. Cellular senescence is a well-established hallmark of aging, as well as a promising target for preventing aging and age-related diseases. However, mapping the senescent cells in tissues is extremely challenging, as their low abundance, lack of specific markers, and variability arise from heterogeneity. Hence, methodologies for identifying or predicting the development of senescent cells are necessary for achieving healthy aging. A new wave of bioinformatic methodologies based on mathematics/physics theories have been proposed to be applied to aging biology, which is altering the way we approach our understand of aging. Here, we discuss the dynamical network biomarkers (DNB) theory, which allows for the prediction of state transition in complex systems such as living organisms, as well as usage of Raman spectroscopy that offers a non-invasive and label-free imaging, and provide a perspective on potential applications for the study of aging.

Musashi expression in intestinal stem cells attenuates radiation-induced decline in intestinal permeability and survival in Drosophila.

Exposure to genotoxic stress by environmental agents or treatments, such as radiation therapy, can diminish healthspan and accelerate aging. We have developed a Drosophila melanogaster model to study the molecular effects of radiation-induced damage and repair. Utilizing a quantitative intestinal permeability assay, we performed an unbiased GWAS screen (using 156 strains from the Drosophila Genetic Reference Panel) to search for natural genetic variants that regulate radiation-induced gut permeability in adult D. melanogaster. From this screen, we identified an RNA binding protein, Musashi (msi), as one of the possible genes associated with changes in intestinal permeability upon radiation. The overexpression of msi promoted intestinal stem cell proliferation, which increased survival after irradiation and rescued radiation-induced intestinal permeability. In summary, we have established D. melanogaster as an expedient model system to study the effects of radiation-induced damage to the intestine in adults and have identified msi as a potential therapeutic target.

Dietary restriction improves intestinal cellular fitness to enhance gut barrier function and lifespan in D. melanogaster.

Loss of gut integrity is linked to various human diseases including inflammatory bowel disease. However, the mechanisms that lead to loss of barrier function remain poorly understood. Using D. melanogaster, we demonstrate that dietary restriction (DR) slows the age-related decline in intestinal integrity by enhancing enterocyte cellular fitness through up-regulation of dMyc in the intestinal epithelium. Reduction of dMyc in enterocytes induced cell death, which leads to increased gut permeability and reduced lifespan upon DR. Genetic mosaic and epistasis analyses suggest that cell competition, whereby neighboring cells eliminate unfit cells by apoptosis, mediates cell death in enterocytes with reduced levels of dMyc. We observed that enterocyte apoptosis was necessary for the increased gut permeability and shortened lifespan upon loss of dMyc. Furthermore, moderate activation of dMyc in the post-mitotic enteroblasts and enterocytes was sufficient to extend health-span on rich nutrient diets. We propose that dMyc acts as a barometer of enterocyte cell fitness impacting intestinal barrier function in response to changes in diet and age.

Proteasome activity determines pupation timing through the degradation speed of timer molecule Blimp-1.

The transcriptional repressor Blimp-1 is a labile protein. This characteristic is key for determining pupation timing because the timing of the disappearance of Blimp-1 affects pupation timing by regulating the expression of its target ßftz-f1. However, the molecular mechanisms that regulate the protein turnover of Blimp-1 are still unclear. Here, we demonstrate that Blimp-1 is regulated by the ubiquitin proteasome system. We show that Blimp-1 degradation is inhibited by proteasome inhibitor MG132. Pupation timing was delayed in mutants of 26S proteasome subunits as well as FBXO11, which recruits target proteins to the 26S proteasome as a component of the SCF ubiquitin ligase complex by slowing down the degradation speed of Blimp-1. Delay in pupation timing in the FBXO11 mutant was suppressed by the induction of ßFTZ-F1. Furthermore, fat-body-specific knockdown of proteasomal activity was sufficient to induce a delay in pupation timing. These results suggest that Blimp-1 is degraded by the 26S proteasome and is recruited by FBXO11 in the fat body, which is important for determining pupation timing.

A biological timer in the fat body comprising Blimp-1, ßFtz-f1 and Shade regulates pupation timing in Drosophila melanogaster.

During the development of multicellular organisms, many events occur with precise timing. In Drosophila melanogaster, pupation occurs about 12 h after puparium formation and its timing is believed to be determined by the release of a steroid hormone, ecdysone (E), from the prothoracic gland. Here, we demonstrate that the ecdysone-20-monooxygenase Shade determines pupation timing by converting E to 20-hydroxyecdysone (20E) in the fat body, which is the organ that senses nutritional status. The timing of shade expression is determined by its transcriptional activator ßFtz-f1. The ßftz-f1 gene is activated after a decline in the expression of its transcriptional repressor Blimp-1, which is temporally expressed around puparium formation in response to a high titer of 20E. The expression level and stability of Blimp-1 is critical for the precise timing of pupation. Thus, we propose that Blimp-1 molecules function like sand in an hourglass in this precise developmental timer system. Furthermore, our data suggest that a biological advantage results from both the use of a transcriptional repressor for time determination and the association of developmental timing with nutritional status of the organism.

Peripheral Circadian Clocks Mediate Dietary Restriction-Dependent Changes in Lifespan and Fat Metabolism in Drosophila.

Endogenous circadian clocks orchestrate several metabolic and signaling pathways that are known to modulate lifespan, suggesting clocks as potential targets for manipulation of metabolism and lifespan. We report here that the core circadian clock genes, timeless (tim) and period (per), are required for the metabolic and lifespan responses to DR in Drosophila. Consistent with the involvement of a circadian mechanism, DR enhances the amplitude of cycling of most circadian clock genes, including tim, in peripheral tissues. Mass-spectrometry-based lipidomic analysis suggests a role of tim in cycling of specific medium chain triglycerides under DR. Furthermore, overexpression of tim in peripheral tissues improves its oscillatory amplitude and extends lifespan under ad libitum conditions. Importantly, effects of tim on lifespan appear to be mediated through enhanced fat turnover. These findings identify a critical role for specific clock genes in modulating the effects of nutrient manipulation on fat metabolism and aging.

Autocrine regulation of ecdysone synthesis by ß3-octopamine receptor in the prothoracic gland is essential for Drosophila metamorphosis.

In Drosophila, pulsed production of the steroid hormone ecdysone plays a pivotal role in developmental transitions such as metamorphosis. Ecdysone production is regulated in the prothoracic gland (PG) by prothoracicotropic hormone (PTTH) and insulin-like peptides (Ilps). Here, we show that monoaminergic autocrine regulation of ecdysone biosynthesis in the PG is essential for metamorphosis. PG-specific knockdown of a monoamine G protein-coupled receptor, ß3-octopamine receptor (Octß3R), resulted in arrested metamorphosis due to lack of ecdysone. Knockdown of tyramine biosynthesis genes expressed in the PG caused similar defects in ecdysone production and metamorphosis. Moreover, PTTH and Ilps signaling were impaired by Octß3R knockdown in the PG, and activation of these signaling pathways rescued the defect in metamorphosis. Thus, monoaminergic autocrine signaling in the PG regulates ecdysone biogenesis in a coordinated fashion on activation by PTTH and Ilps. We propose that monoaminergic autocrine signaling acts downstream of a body size checkpoint that allows metamorphosis to occur when nutrients are sufficiently abundant.

The binding of multiple nuclear receptors to a single regulatory region is important for the proper expression of EDG84A in Drosophila melanogaster.

Nuclear receptor transcription factor family members share target sequence similarity; however, little is known about how these factors exert their specific regulatory control. Here, we examine the mechanism regulating the expression of the Drosophila EDG84A gene, a target gene of the orphan nuclear receptor ßFTZ-F1, as a model to study the cooperative behavior among nuclear receptors. We show that the three nuclear receptors ßFTZ-F1, DHR3, and DHR39 bind to a common element in the EDG84A promoter. The expression level of the EDG84A promoter-lacZ reporter genes in DHR39-induced and mutant animals, respectively, suggests that DHR39 works as a repressor. The activity of a reporter gene carrying a mutation preventing DHR3 binding was reduced in ftz-f1 mutants and rescued by the induced expression of ßFTZ-F1, suggesting that DHR3 and ßFTZ-F1 activate the EDG84A gene in a redundant manner. A reporter gene carrying a mutation that abolishes DHR39 and FTZ-F1 binding was prematurely expressed, and the expression level of the reporter gene carrying a mutation preventing DHR3 binding was reduced. These findings suggest that the temporal expression of this gene is mainly controlled by ßFTZ-F1 but that the binding of DHR3 is also important. Comparison of the binding site sequence among Drosophila species suggests that DHR3 binding ability was gained after the melanogaster subgroup evolved, and this ability may contribute to the robust expression of this gene. These results show the complicated regulatory mechanisms utilized by multiple nuclear receptors to properly regulate the expression of their target gene through a single target site.

Regulatory mechanisms of ecdysone-inducible Blimp-1 encoding a transcriptional repressor that is important for the prepupal development in Drosophila.

Blimp-1 is an ecdysone-inducible transcription factor that is expressed in the early stage of the prepupal period. The timing of its disappearance determines expression timing of the FTZ-F1 gene, whose temporally restricted expression is essential for the prepupal development. To elucidate the termination mechanism of Blimp-1 gene expression, we examined the regulation of the Blimp-1 gene using an organ culture system. The results showed that the Blimp-1 gene is transcribed in cultured organs taken from a low ecdysteroid period even after extended exposure to 20-hydroxyecdysone, while well-known early genes such as E75A are repressed under the same conditions. Similar selective transcription was observed in the cultured organs obtained from a high ecdysteroid period. We further showed that Blimp-1 transcripts quickly disappeared in the presence of actinomycin D. From these results, we concluded that the Blimp-1 gene is transcribed when the ecdysteroid titer is high, but the expressed mRNA degrades rapidly; these unique regulations limit its expression to the high ecdysteroid stage.

Drosophila Blimp-1 is a transient transcriptional repressor that controls timing of the ecdysone-induced developmental pathway.

Regulatory mechanisms controlling the timing of developmental events are crucial for proper development to occur. ftz-f1 is expressed in a temporally regulated manner following pulses of ecdysteroid and this precise expression is necessary for the development of Drosophila melanogaster. To understand how insect hormone ecdysteroids regulate the timing of FTZ-F1 expression, we purified a DNA binding regulator of ftz-f1. Mass spectroscopy analysis revealed this protein to be a fly homolog of mammalian B lymphocyte-induced maturation protein 1 (Blimp-1). Drosophila Blimp-1 (dBlimp-1) is induced directly by 20-hydroxyecdysone, and its product exists during high-ecdysteroid periods and turns over rapidly. Forced expression of dBlimp-1 and RNA interference analysis indicate that dBlimp-1 acts as a repressor and controls the timing of FTZ-F1 expression. Furthermore, its prolonged expression results in delay of pupation timing. These results suggest that the transient transcriptional repressor dBlimp-1 is important for determining developmental timing in the ecdysone-induced pathway.