Diacylglycerol kinase is downregulated in the Drosophila Seizure Mutant during Spaceflight

Organisms experience accelerated aging in space, exemplified by health effects including bone loss, muscle atrophy, and decreased cardiovascular functional capacity, among others (Verkinos and Schneider, 2010; Kandarpa, 2019; Tavassoli, 1986). As NASA shifts its focus towards manned missions beyond Low Earth Orbit (LEO), studies concerning the longer-term health effects of spaceflight exposure outside of the Earth's magnetic field are necessary for preserving human health during long-duration spaceflight. Research conducted in the space environment provides a unique perspective that is critical for understanding human health conditions, including aging. As aging is characterized by the deterioration of physiological and biochemical functions in different tissues of organisms (López-Otín et al., 2013), the analysis of omics data collected from model organisms subjected to spaceflight conditions may provide important insights on potential targets for promoting longevity.

In this study, a computational/bioinformatics analysis of omics data from GeneLab Data System's GLDS-207 project (Correlated Gene and Protein Expression in Heads from Drosophila Reared in Microgravity) was conducted with the goal of identifying critical targets for promoting longevity. Drosophila is an ideal organism for spaceflight research because of its cost-effectiveness and relative simplicity when compared to the base costs for sustaining more complex organisms beyond LEO experiments (Tolwinski et al., 2017). As the average lifespan of Drosophila is 50–70 days, they are also a good model for studying aging. The data from the head tissue was made available through the Biospecimen Sharing Program, as the original study was focused on cardiac tissue and cardiac disease implications in spaceflight (Walls et al., 2020). Analyses of this data set identified a potential target for promoting longevity, which could be critical for long-term space missions.

First, the data was analyzed to assess the statistical significance. Analysis of DESeq2 outputs comparing the spaceflight and ground control samples for the sei mutants revealed that most differential expressed genes exhibited a p-value < 0.05. The bar graph (Figure 1) shows the p-value distribution for the sei mutant flight versus ground control groups. As the observed changes for most of the differentially expressed genes are significant, the histogram suggests that these changes could be attributed to the effect of space on the sei mutants.

Figure 1.

Statistical significance analysis. This histogram represents the statistical significance of the data based on p-value, specifically for the sei mutant flight and ground control comparison. If the p-value is less than 0.05, the data is significant. As the peak is closest to the origin, this graph indicates that this is an excellent data set for further analyses.

The principal component analysis (PCA) plot was generated as a two-dimensional visualization of the relative correlation in gene expression within specific test groups. The variance between the flight and ground control samples was found to be greater in the sei mutant fly line than in the wildtype. This indicates the increased differential expression of genes in the mutants. Moreover, the variance (72%) in the PCA reveals that the principal impact on the samples is spaceflight (Figure 2).

Figure 2.

The principal component analysis. PCA plot signifies the variance between the spaceflight and ground control groups. More variance in PC1 indicates a higher difference in the pattern of expression between ground controls and flight samples. This pattern is observed in the sei mutant flight and ground control sample comparisons.

A volcano plot was created to characterize this differential gene expression and visualize the most differentially expressed genes (Figure 3). The volcano plot indicated that rdgA, retinal degeneration A, was significantly downregulated. Moreover, this gene has been reported to influence the aging process in other studies (Lin et al., 2014). Down-regulation of this gene in a Drosophila mutant strain during spaceflight may imply its potential role in influencing longevity and altering lifespan during long-term space missions.

Figure 3.

Identification of gene of interest. The volcano plot generated after DESeq 2 analysis for the sei mutant ground control and flight sample comparison is illustrated. The top twenty most significantly up or down-regulated genes are labeled on the plot. Note that rdgA, our gene of interest, is significantly downregulated.

GoSeq pathway analysis was used to identify biological processes and molecular functions that were impacted by differential gene expression. Proteolysis and serine-type endopeptidase activity were found to be highly enriched (Figure 4).

Figure 4.

Gene Ontology Analysis. GoSeq pathway analysis indicated that proteolysis was a highly enriched biological process and serine-type endopeptidase activity was a highly enriched molecular function.

The results of this study indicate that in spaceflight conditions rdgA expression in the sei mutant is decreased. The rdgA gene encodes for DGK, an activator and regulator of the mechanistic target of the rapamycin (mTOR) pathway (Torres-Ayuso et al., 2015). The mTOR pathway is a serine/threonine pathway and has been associated with processes of aging through multiple mechanisms (Papadopoli et al., 2019). Studies have shown that a knockdown of the rdgA gene led to a significant increase in lifespan of about 44% (Lin et al. 2014). Knockdown of DGK extends lifespan and enhances response to oxidative stress.

Spaceflight induces oxidative stress due to an imbalance of reactive oxygen species (ROS) in cells and tissues (Stein, 2002). Oxidative stress has been shown to affect the voltage gated potassium channels in sei mutants (Liu et al., 2002). Although sei mutants exhibited higher resistance to the toxic effects of hydrogen peroxide relative to wild type controls, the underlying mechanism is not known (Hill et al., 2019). Since both spaceflight environment and hydrogen peroxide exposure induce oxidative stress and spaceflight conditions decreased the rdgA expression in sei mutants that survived the spaceflight, it is likely that the decrease in rdgA contributed to its resilience. Moreover, DGK, the enzyme coded by rdgA, has been shown to regulate oxidative stress and aging by inversely modulating TOR signaling. So, the negative effects of oxidative stress, which contributes to the aging of organisms, are potentially reduced by the changes in the expression of the rdgA gene in the sei mutant.

Activation of the mTOR pathway causes excessive protein synthesis, inhibits autophagy, dysregulates mitochondria, and results in accelerated senescence (Chen et al., 2021). As DGK-derived phosphatidic acid acts as a mediator of mTOR signaling (Avila-Flores et al., 2005), the expression level of DGK determines whether the pathway is activated or not. Moreover, DGK inhibition also enhances an organism's protection against oxidative stress (Lin et al., 2014), which is also a major contributor to accelerated aging. Therefore, DGK appears to be a promising target as it directly impacts two critical pathways that lead to accelerated aging. Follow-up studies in higher organisms may lead to new insights and clarify the mechanism by which DGK downregulation could combat accelerated aging in space and on Earth.