multiple miRNAs
4.1 MiRNAs are required for muscle maintenance
In this study, we have shown that the absence of miRNAs that are predicted to target multiple components of the DGC (Dg plus any three other components) affects muscle maintenance in flies. Dg was chosen as a baseline targeting component while grouping these miRNAs as Dg is associated with multiple pathways. Besides Dg-Dys-Syn1 pathway , it has been reported that Dg interacts with Hippo-signaling pathway component Yap to prevent cardiomyocyte proliferation (Morikawa et al., 2017). Inhibition of ubiquitinates ligase Trim32 has been shown to enhance plakoglobin binding to affect muscle atrophy (Cohen et al., 2014) and we further postulated that it can interact with Dg similar to plakoglobin to promote muscle maintenance. Therefore, miRNAs targeting the DGC importantly, Dg can influence more likely muscle maintenance phenotype associated with MDs. Further, Dg is required for correct localization of many other proteins such as Nrx-IV, Cora and GluRs mutations of which are also associated with MD-like phenotypes.
miRNAs are known to mediate stress response in animals to maintain cellular homeostasis.
The expression of many of the miRNAs predicted to target the DGC components have been reported to be deregulated under hyperthermia stress in wild-type animals and/or in absence of Dg or Dys (Marrone et al., 2012). A prolong disease state can display immense stress for organismal health. Therefore, it was interesting to investigate miRNAs regulating a common pathway between stress and MD to decipher MD-related pathogenesis. This study, in particular, determines that in absence of miR-137, miR-927, and miR-966 the muscle integrity in animals was severely compromised. This study further validates various stresses and aging can affect muscle degeneration even in control flies. Further, we were able to show that besides muscle degeneration, muscle atrophy is also increased in control animals during different stress conditions such as temperature stress, sugar starvation, and aging. All miRNA mutants were responsive to stress similar to control animals, indicating long term stress i.e. aging is more detrimental than a short term stresses such as hyperthermia stress and nutrition amelioration. Muscle atrophy was affected more than muscle degeneration in all genotypes in both stress and aging. This further highlights the importance of muscle maintenance in aging or in a prolonged disease state such as cancer that leads to cachexia.
Not only miRNA loss was found to affect muscle integrity, but downregulation of Dg was also detrimental for healthy musculature. Previous studies have shown loss of Dg and Dys cause muscle degeneration and the phenotype is accelerated upon stress (Kucherenko et al., 2011). We further showed that upon downregulation of Dg specifically in adult muscle,
cause muscle degeneration and atrophy in flies. Muscle atrophy was more detrimental as the frequency of muscle atrophy was higher than the frequency of muscle degeneration compared to control indicating, a proper level of Dg is required to maintain a healthy musculature in young and aged animals. Apart from its role in muscle maintenance, number of studies have reported that precise level of Dg is required to maintain energy homeostasis, establishment of NMJ, photoreceptor differentiation, and cellular polarity (Bogdanik et al., 2008; Marrone et al., 2011a; Shcherbata et al., 2007; Yatsenko et al., 2009). In flies, miR-9a and miR-310s had been shown previously to serve as a regulatory molecule to maintain the proper level of Dg to canalize myotendinous junction formation and to buffer MD related type II lissencephaly phenotype. miR-137, miR-927, and miR-966 can further play a similar role in maintaining Dg levels in muscle to protect animals from stress- or age-related muscular dysfunction.
qRT-PCR on miR-137and miR-966 mutants showed upregulated mRNA levels of either, Dys and/or Syn1 even though they were not the predicted to target these genes while miR-927 mutants had either no change in mRNA levels or showed downregulation of Dg, Dys, and Syn1 even though it is predicted to target all three components. There can be a number of reasons following this discrepancy: 1) many miRNAs can have a tissue or sex-specific expression; since qRT-PCR was performed on the whole body of the organism, tissue-specific miRNA expression can be easily masked when normalized to whole body of control genotype. In the future, tissue-specific qRT-PCR could resolve this issue to validate the prediction based miRNA targeting in vivo. 2) The DGC signaling had been shown to have compensatory effects (Cote et al., 2002; Gao and McNally, 2015; Hughes et al., 2018), the fluctuation of mRNA levels between its predicted and non predicted targets could simply be the mimic of compensatory mechanisms of the DGC components.
MiR-137 and miR-966 mutants showed muscle degeneration as early as young age indicating, these miRNAs are required in muscle during development. The severity of muscle degeneration upon aging was more prominent for miR-966 mutants compared to temperature stress and sugar starvation. This further indicated that miR-966 is required to maintain healthy muscle in aging. MiR-966 is also reported to be enriched in hemolymph in old flies (Dhahbi et al., 2016). We have also shown from lifespan analysis that miR-966 mutants, in general, had a higher survival rate compared to control (Supplementary Figure 3). Altogether, these findings further suggest that loss of miR-966 can be more responsive to physiological changes related to aging. Muscle atrophy was also observed in all miRNA
mutants at a young age. This further indicates that all three miRNAs are required during developmental stage to maintain muscle integrity.
Muscle degeneration phenotype can be caused by muscles as well as neuronal defects. GO component term analyzed on conserved predicted mRNA targets of all three candidate miRNAs implied that these miRNA can be involved in the cell membrane and cell periphery related processes. MiR-137 having the most predicted targets, in addition, can be associated with neuronal processes like synapse and NMJs. GO terms for predicted miRNAs targets are in agreement with Dg functions. Luciferase reporter assay further confirmed that all three miRNAs miR-137, miR-927, and miR-966 can downregulate Dg in vitro. Therefore, an in vivo screen on miRNAs gain-of-function was performed to further confirm all three selected miRNAs can downregulate Dg and/or any other muscle-specific target genes. As expected, over-expression of miRNAs gave stronger phenotypes as compared to their loss-of-function mutants. Over-expression of miR-137 was the most severe in flies as its over-expression during developmental stage cause embryonic lethality, while over-expression in adult muscle resulted in strong muscle degeneration or complete absence of individual muscle.
The experiment further confirmed that miR-927 is required more during developing muscle as over-expression of miR-927 with how-Gal4 was lethal during the embryonic stage.
Though the severity of phenotype can also depend on the strength of the UAS-miR-lines used, we observed that over-expression of miR-966 in adult muscle resulted in more severe phenotype compared to developing muscle. Over-expression in developing muscle also affected the muscle maintenance at young age but the effect of aging was prominent in flies.
This further indicates, miR-966 can regulate age-dependent gene expression to maintain healthy muscles in animals. Many muscle-specific miRNAs (myomiRs) have been proposed as biomarkers for pathological and physiological muscle processes. myomiRs such as miR-1, miR-133, miR-206, miR-208, and miR-499 have been proposed as a diagnostic marker for DMD (Cacchiarelli et al., 2011b; Jeanson-Leh et al., 2014; Li et al., 2014). This study further suggests, miR-137, miR-927, and miR-966 can act as a regulatory molecule to regulate Dg level in respons to stress to maintain healthy muscle in flies.