miR-181a Regulates p62/SQSTM1, Parkin and Protein DJ-1 Promoting Mitochondrial Dynamics in Skeletal Muscle Ageing

One of the key mechanisms underlying skeletal muscle functional deterioration during ageing is disrupted mitochondrial dynamics. Regulation of mitochondrial dynamics is essential to maintain a healthy mitochondrial population and prevent the accumulation of damaged mitochondria, however the regulatory mechanisms are poorly understood. We demonstrated loss of mitochondrial content and disrupted mitochondrial dynamics in muscle during ageing concomitant with dysregulation of miR-181a target interactions. Using functional approaches and mitoQc assay, we have established that miR-181a is an endogenous regulator of mitochondrial dynamics through concerted regulation of Park2, p62/SQSTM1 and DJ-1 in vitro. Downregulation of miR-181a with age was associated with an accumulation of autophagy-related proteins and abnormal mitochondria. Restoring miR-181a levels in old mice prevented accumulation of p62, DJ-1 and PARK2, improved mitochondrial quality and muscle function. These results provide physiological evidence for the potential of microRNA-based interventions for age-related muscle atrophy and of wider significance for diseases with disrupted mitochondrial dynamics.


Introduction
Disrupted mitochondrial dynamics is one of the hallmarks of ageing (Lopez-Otin et al., 2013). Altered mitochondrial morphology and content in skeletal muscle of humans and rodents is one of the pathways consistently associated with age-related loss of muscle mass and function (Bratic and Larsson, 2013;Short et al., 2005), with up to a 30% loss of 5 mitochondrial content reported in fast twitch muscles and an accumulation of dysfunctional mitochondria (Chabi et al., 2008). Moreover, accumulation of damaged or dysfunctional mitochondria increases the oxidation of contractile proteins in muscle; associated with disrupted balance between anabolic and catabolic processes and ultimately sarcopenia (Carnio et al., 2014;O'Leary et al., 2013). Mitochondria are dynamic organelles that exist in a highly interconnected network and are continually undergoing fusion and fission (for review see (Archer, 2013)). These processes are tightly regulated and necessary to maintain a healthy mitochondrial population by removing and preventing the accumulation of damaged mitochondria. The regulation of 1 5 mitochondrial remodelling and associated bioenergetic changes, particularly in skeletal muscle, is key for the correct adaptation and response to exercise that results in an increased mitochondrial content with improved fatty acid oxidation and glucose homeostasis (Mansueto et al., 2017). Moreover, the adaptive cellular response of skeletal muscle to exercise requires the autophagic degradation of cellular components, allowing the muscle fibre to rebuild and 2 0 respond to repetitive bouts of exercise (Vainshtein et al., 2014). Interruption of mitochondrial dynamics, particularly in ageing, can result in mitochondrial swelling, loss of cristae, destruction of the inner membrane and impaired respiration (Bratic and Larsson, 2013). The integration of mitochondrial biogenesis and selective degradation via mitophagy is essential for the preservation of healthy muscle, disruption of this balance can result in alterations in 2 5 muscle bioenergetics and loss of muscle mass and function (Hood et al., 2019). Mitophagy is regulated at numerous levels and a number of distinct mitophagic pathways have been elucidated such as ubiquitin-mediated mitophagy including the Pink/Parkin pathway and ubiquitin independent pathways via mitophagy receptors on the outer mitochondrial membrane (e.g. BNIP3), however the exact regulatory mechanisms remain to be fully 3 0 understood (for reviews see (Montava-Garriga and Ganley, 2019;Palikaras et al., 2018). microRNAs (miRs) are small 19-25nt long noncoding RNAs that regulate gene expression post-transcriptionally through binding to complementary target sites within mRNAs, usually 3'UTRs, leading to mRNA degradation and/or inhibition of mRNA translation (Bethune et al., 2012). miRs target multiple genes and are considered a robust mechanism of controlling cellular and tissue homeostasis. The role of miRs in the regulation of key cellular mechanisms has become increasingly recognised, including skeletal muscle homeostasis, development, regeneration and atrophy (Cheung et al., 2012;Goljanek-Whysall et al., 2011;5 Soares et al., 2014) . The expression of a number of specific miRs change in skeletal muscle during exercise and ageing (Brown and Goljanek-Whysall, 2015;Hu et al., 2014;Kim et al., 2014;Nielsen et al., 2010). Although limited, functional studies have demonstrated that miRs play a key role in regulating the expression of genes and pathways altered during exercise and/or ageing, contributing to alterations in skeletal muscle mass (Li et al., 2017;Silva et al., 1 0 2017;Soares et al., 2014). A number of miRs have been found to be both associated and present within mitochondria, indicating a potential regulatory role (Shen et al., 2016).
In this study, we have demonstrated that age-related disruption of mitochondrial dynamics in skeletal muscle can be improved by restoring the expression of miR-181a-5p (miR-181a).

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Quantitative proteomics data revealed a reduced mitochondrial protein content with age, concomitant with the upregulation of mitophagy-associated proteins. Ultrastructural analysis of mitochondria revealed abnormal, large mitochondria in muscle during ageing despite increased expression of autophagy, and in particular mitophagy-associated proteins. Parallel analyses of upstream regulators of mitochondrial dynamics identified miR-181a as targeting 2 0 key autophagy-and mitochondrial dynamics-associated genes. In vitro experiments confirmed miR-181a targets p62 and Park2, and demonstrated that miR-181a can regulate mitophagic flux. Restoration of miR-181a content in muscle of old mice in vivo prevented accumulation of p62, PARK2 and DJ-1 and preserved mitochondrial content, ultimately resulting in increased myofibre size and muscle force. Together, our data indicate that miR-2 5 181a is a potent regulator of muscle mitochondrial dynamics in vitro and in vivo, providing potential therapeutic avenues for age-related muscle atrophy.

Quantitative proteomics reveals decrease in mitochondrial content with age.
To characterise changes in the intracellular muscle environment during ageing and associated adaptive response of muscle to contractions, global label-free analysis was used to quantify the overall changes in the proteome of skeletal muscle from quiescent tibialis anterior (TA) 5 or TA subjected to 15 min of isometric contractions (mimicking acute exercise) from adult and old mice. Significantly changed proteins (fold change > 2 and -10logP > 20) between quiescent or contracted muscle of adult or old mice demonstrate clear differences in the proteomic content of TA muscle (Fig.1A). Despite detected changes in the abundance of some proteins between contracted muscle of adult and old mice, the major significant 1 0 proteomic changes detected were as a result of ageing. The most significantly changed pathways in muscle during ageing were: downregulation of mitochondrial proteins and upregulation of contractile apparatus proteins with age ( Fig 1B & C). This is consistent with previously reported data showing mitochondrial content decrease in muscle during ageing (Chabi et al., 2008;Hepple, 2014;Smith et al., 2018). To further investigate the mechanisms leading to decreased mitochondrial content during ageing, we analysed the expression of known regulators of autophagy and mitochondrial 2 0 dynamics by western blotting and qPCR. General regulators of the autophagic machinery: Sirtuin 1 (SIRT-1) and Forkhead box protein O3 (FOXO3), as well as effector mitophagy proteins, such as PTEN-induced putative kinase 1 (PINK1), Parkin (PARK2), Protein-DJ-1 (PARK7) and the autophagic adaptor protein p62 (Sequestosome 1, Sqstm1), were upregulated in muscle during ageing ( Fig. 2A, B). We also observed downregulation of the 2 5 expression of Pgc1α with age (Fig. S1). Despite upregulation of autophagy-associated proteins, swollen and abnormal mitochondria were detected by EM in muscle of old mice, suggesting defective mitochondrial dynamics (Fig. 2C). This suggests impaired or dysfunctional mitophagy in skeletal muscle from old compared to adult mice, an increase in p62 levels can be associated with an inhibition of autophagy as it is degraded in cells with 3 0 normal autophagic flux. (Fig. 2B). Analysis of an autophagy marker LC3 revealed more pronounced LC3 punctae in muscle from old mice (Fig. 2D). The expression of Lc3b was upregulated in the muscle of old mice and the levels of CoxIV and Nd-1, indicators of mitochondrial content, were downregulated in the muscle of old mice, further suggesting dysfunctional autophagic response in ageing muscle and in the adaptation to exercise (Fig.   2E,F). Together, our data demonstrate an accumulation of key regulators of the mitophagic machinery in muscle during ageing and defective mitochondrial dynamics resulting in accumulation of abnormal mitochondria and loss of mitochondrial content. miR-181a, and 5 not miR-181b, miR-181c or miR-181d, was downregulated in TA of mice during ageing and exercise of adult mice only (Fig. 2G, 2H). This suggests that miR-181a is the key miR-181 family member with a role in muscle ageing. miR-181a as putative regulator of mitochondrial dynamics.

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To determine potential upstream regulators of mitochondrial dynamics we investigated predicted interactions between mitophagy-associated genes and miRs. miR target prediction databases, TargetScan, miRnet and miRWalk, identified miR-181a-5p (miR-181a) as a putative regulator of multiple genes associated with mitochondrial dynamics: previously validated targets (highlighted in bold in Fig. 3A): Park2, Sirt-1, PTEN and Atg-5, and novel 1 5 putative targets: p62, DJ-1, Mfn1, Mfn2 and Tfam (Fig. 3A). The elevated expression of mitophagy-associated proteins observed in TA from old mice coupled with a decreased expression of miR-181a suggested that miR-181a may act as an important regulator of autophagy and mitochondrial dynamics during muscle ageing. To investigate whether miR-181a regulates mitochondrial dynamics we used the mitochondrial uncoupler, carbonyl cyanide m-chlorophenyl hydrazone (CCCP) and autophagic flux inhibitor Bafilomycin A1 (BAF) in a C2C12 myoblast model. Treatment of cells with CCCP decreased expression levels of miR-181a, while miR-181a mimic and 2 5 antagomiR-181a (AM181a) increased and decreased respectively miR-181a levels in C2C12 cells treated with DMSO (control) or CCCP (Fig.S2A). CCCP treatment resulted in formation of LC3 punctae, while BAF treatment resulted in the accumulation of LC3 punctae colocalised with TOM20 (Fig. 3D). miR-181 overexpression resulted in the presence of LC3 positive punctae in DMSO, CCCP and BAF treated cells, whereas inhibition of miR-181a 3 0 resulted in the accumulation of LC3 and TOM20 colocalised punctae in DMSO, CCCP and BAF-treated cells (Fig. 3D). Moreover, inhibition of miR-181a resulted in decreased LC3 II/ I in myoblasts treated with CCCP ( Fig. 3B, C). This suggest inhibition of miR-181a may result in stalled autophagy.
In order to investigate miR-181a-mediated regulation of mitochondrial turnover via mitophagy, we used the Mito-QC reporter construct that contains tandem mCherry-GFP tag fused to the mitochondrial targeting sequence of the outer mitochondrial membrane protein, FIS1 (Allen et al., 2013). The mitochondrial network fluoresces red and green under normal 5 conditions but during mitophagy, with the delivery of mitochondria to the acidic environment of lysosomes, GFP fluorescence is quenched while mCherry remains stable (Allen et al., 2013;McWilliams et al., 2016). CCCP treatment increased the number of mitochondria associated with lysosomes in Scr controls which returned to basal levels after BAF treatment ( Fig. 3E, F). AM181a samples showed reduced levels of mitophagic events after CCCP 1 0 treatment ( Fig. 3E, F), whereas miR-181a overexpression led to increased mitophagy events in DMSO and BAF-treated cells (Fig. 3E, F). miR-181a or AM181a treatment had no effect on cell viability (cytotoxicity assay) and ATP production was mildly increased in myoblasts treated with miR-181a in the presence of CCCP (Fig. S2B).

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To investigate the consequences of mitophagy regulation by miR-181a on mitochondrial content, we analysed the expression of mitochondrial proteins encoded by the mitochondrial genome: Cox I, Nd-1 and encoded by the nuclear genome: Tomm20. The expression of all the mitochondrial genes was increased following miR-181a overexpression and decreased in response to AM181a (Fig. 3G).
We next validated the regulation of miR-181a predicted autophagy-associated targets in an in vitro model of mitochondrial uncoupling. No changes in the expression of these proteins were detected following miR-181a overexpression or inhibition in C2C12 myoblasts (Fig. S2 C, 2 5 D), possibly due to their high turnover. However, in DMSO treated cells, inhibition of miR-181a resulted in increased levels of p62 mRNA and accumulation of p62 protein which did not colocalise with COXIV mitochondrial marker (Fig. 4A, B). miR-181a overexpression resulted in the presence of p62, PARK2 and DJ-1 positive punctae colocalised with the mitochondrial marker COXIV. In CCCP-treated cells, inhibition of miR-181a resulted in 3 0 accumulation of p62, DJ-1 and Park-2 mRNAs and protein with reduced colocalisation with COXIV. miR-181a overexpression resulted in downregulation of the expression of p62, Park2 and DJ-1 to levels comparable to control cells (Fig. 4A, B). We next analysed changes in the levels of Tfam in myoblasts treated with miR-181a mimic or antagomiR, miR-181a overexpression in C2C12 myoblasts treated with CCCP resulted in upregulation of Tfam expression (Fig. 4B). These results indicate that miR-181a increased mitochondrial turnover via mitophagy and potentially concomitant increase in mitochondrial biogenesis as indicated by increase of Tfam expression in C2C12 cells treated with miR-181 and CCCP. This indicates a role for miR-181a in maintaining a population of healthy mitochondria within the 5 cells.
We also analysed changes in localisation of BNIP3 and LAMP1 with TOM20 in C2C12 cells treated with miR-181a or AM181a in control, CCCP or BAF-treated cells. Both experiments demonstrated that AM181a treatment leads to accumulation of BNIP3 or LAMP1 punctae 1 0 colocalised with TOM20, further confirming that low levels of miR-181a are associated with inhibition of mitophagic flux (Fig .S3). However, no miR-181a binding site could be found in the mRNA sequences of Bnip3 or Lamp1.
To investigate direct regulation of autophagy-associated targets by miR-181a, we used 1 5 3'UTR of p62 and Park2, DJ-1 Exon 6 in GFP sensor constructs. Although miR binding sites within exons are very uncommon, we analysed this specific putative binding site due to strong effects of miR-181a on DJ-1 expression (Fig. 4C). GFP reporter constructs were transfected into C2C12 cells in the presence of scrambled or miR-181a mimic. miR-181a treatment led to significant decrease in GFP fluorescence from GFP-p62 and GFP-Park2, but 2 0 not GFP-DJ-1 constructs, as compared to scrambled-treated controls (Fig. 4D). The mutations of miR-181a binding sites rendered all constructs non-responsive to miR-181a overexpression as compared to scrambled controls. This confirms p62 and Park2 as direct, targets of miR-181a, whereas DJ-1 expression may be regulated by miR-181a in an indirect manner (Fig. 4C, D). The expression and localisation of miR-181a targets were analysed in the muscle of adult and old mice treated with saline, miR-181a mimic or AM181a (Figs. 5, 6 and S6). The muscle of control old mice was characterised by the presence of increased p62, DJ-1 and PARK2-3 0 positive myofibres as compared to the muscle of control adult mice confirming disrupted or inhibited autophagy during ageing (Fig. 5A). AM181a treatment of adult mice led to increased number of p62, DJ-1-and PARK2-positive myofibres, whereas fewer p62, DJ-1 and PARK2 positive myofibres were detected in miR-181a-treated old mice compared to their respective controls (Fig. 5A). Inhibition of miR-181a in muscle of adult mice led to accumulation of p62 and DJ-1 mRNA, whereas overexpression of miR-181a in muscle of old mice led to reduced levels of p62, Park2 and DJ-1 mRNA as compared to muscle of control old mice to levels observed in muscle of adult mice (Fig. 5B). This suggests that lower levels of miR-181a in muscle are associated with accumulation of its target genes and dysfunctional 5 mitophagy, potentially leading to accumulation of dysfunctional mitochondria (Fig. 6G). To further investigate the role of miR-181a in regulating mitochondria we analysed the expression of mitochondrial genes (CoxI, and Nd-1) in TA muscle of adult and old mice.
Consistently, in muscle of old mice, miR-181a treatment led to increased expression of the mitochondrial genes (Fig. 5C). Furthermore, we investigated changes in expression of Consistently in miR-181a-treated muscle, the expression of these mitochondrial associated genes was upregulated (Fig. 5C, S5). We also observed changes in the expression of Mfn1 in mice treated with AM181a or miR-181a (Fig. S5). Together these results indicate that miR-1 5 181a promotes mitochondrial dynamics through concomitant activation of mitophagy and mitochondrial biogenesis in skeletal muscle from adult and old mice.

miR-181a regulates mitochondrial quality, myofibre size and muscle force in vivo.
We next investigated the physiological effects of miR-181a on muscle during ageing.

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Changes in miR-181a expression in adult and old mice had no significant effect on body or muscle weight (Fig. S6). However, miR-181a inhibition led to a decrease, whereas miR-181a overexpression led to an increase in myofibre size from adult and old mice respectively ( Fig.   6A, B). The relative proportion of fibre sizes within the population of fibres revealed that miR-181a-treated mice had a higher proportion of larger fibres, whereas AM181a treatment 2 5 resulted in a higher population of smaller fibres in muscle from adult mice (Fig. 6C).
Moreover, a higher proportion of LC3-positive myofibres was detected in muscle of control old mice and AM181a-treated adult and old mice, with almost no LC3-positive fibres detected in adult saline and miR-181a-treated mice ( Fig 6E). These results indicate miR-181a-mediated regulation of mitochondrial content is associated with fibre size.

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The relative oxidative potential of muscle as an indication of mitochondrial function was decreased by miR-181a inhibition in the TA of adult mice, demonstrated by reduced intensity 1 0 of succinate dehydrogenase (SDH) staining (Fig. 6F, G). miR-181a treatment of old mice resulted in a non-significant trend for increased SDH intensity (Fig. 6G). EM analysis of mitochondrial populations revealed the presence of swollen and mitochondria with disordered cristae, with some mitochondria expanding longitudinally between myofibrils, in muscle of control old and AM181a-treated adult mice. miR-181a treatment of old mice led to a lower 5 proportion of structurally abnormal mitochondria and decreased the abundance of abnormally large mitochondria (Fig. 6D).
The physiological relevance of manipulating miR-181a expression was investigated by examining the effects of miR-181a on muscle force. Maximum and specific force of the EDL 1 0 muscle was decreased during ageing ( Fig. 6H) consistent with previous reports (Brooks and Faulkner, 1988). As miR-181a expression is decreased in muscle during ageing, we treated adult mice with AM181a to reduce miR-181a expression. Conversely, old animals were treated with miR-181a mimic in an effort to restore miR-181a expression. Maximum and specific force was decreased in muscle of adult mice treated with AM181a, whereas miR-1 5 181a overexpression in muscle of old mice restored maximum muscle force (Fig. 6H).
Together, these data suggest that a decrease in miR-181a expression results in chronic dysregulated mitophagy in muscle during ageing associated with presence of abnormal mitochondria, loss of mitochondrial content and decline of muscle function.

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In summary, a decrease in miR-181a expression resulted in increased expression of autophagy-associated genes, a decrease in fibre diameter and specific muscle force in adult mice mimicking ageing. Conversely, upregulating miR-181a levels in old mice improved mitochondrial morphology, increased fibre diameter and specific force. This data demonstrates microRNA-mediated fine-tuning of one of the key mechanisms associated with 2 5 ageing, mitochondrial turnover, and provides proof-of-principle for the potential use of microRNA-based therapeutic approaches for ageing-associated disorders such as age-related muscle atrophy.

Discussion
In the present study, we have demonstrated decreased mitochondrial content and quality associated with a dysregulated expression of autophagy and mitochondrial dynamicsassociated proteins in skeletal muscle during ageing. Using bioinformatics and sensor constructs, we have identified miR-181a as a key regulator of autophagy-associated genes, 5 controlling the expression of p62 and Park2, as well as indirect regulation of Park7/Protein DJ-1 expression and localisation. Changes in miR-181a levels and the concomitant changes in the expression of its direct and indirect targets result in altered myofibre size, mitochondrial content and quality and specific muscle force, suggesting miR-181a may be one of the key regulatory mechanisms underlying age-related muscle atrophy. This is the first 1 0 report, to our knowledge, demonstrating a physiologically-relevant mechanism of miR-181amediated fine-tuning of mitochondrial dynamics on multiple levels through concerted regulation of expression of several autophagy-associated genes during ageing.
Mitochondrial quality has an important impact on the health and bioenergetics of healthy following miR-181a overexpression (Fig. 6). Although these changes could be associated with miR-181a predicted target genes: Mfn1 and Mfn2, we did not observe consistent regulation of these genes by miR-181a at the mRNA level, suggesting an indirect regulation by miR-181a (Figs. S1, S5). Finally, we have shown that miR-181a regulates myofibre size and muscle force: adult and old mice treated with AM181a or miR-181a had significant 2 5 changes in the expression of genes associated with mitochondrial dynamics, mitochondrial quality and muscle fibre diameter and specific force of muscle (Fig. 6).
In summary, we propose that miR-181a regulates skeletal muscle homeostasis and muscle metabolism, size and force, by regulating mitochondrial dynamics. miR-181a is 3 0 downregulated during ageing and we propose that concomitant upregulation and accumulation of its mitophagy-associated targets paradoxically leads to a stalling or inhibition of mitophagy, resulting in the accumulation of damaged mitochondria. We have demonstrated that fine-tuning the levels of autophagy regulatory (p62) and mitophagy-associated proteins (DJ-1, Park2) and concomitant changes in the expression of Tfam, by overexpressing miR-181a, leads to improved mitochondrial dynamics resulting in increased myofibre size and muscle function. In this study we focused on the predicted and confirmed targets of miR-181a many of which are associated with the Pink/Parkin mitophagy pathway.
It was therefore surprising to find that inhibition of Parkin, p62 and Protein DJ-1 by miR-5 181a overexpression resulted in increased mitophagy and mitochondrial biogenesis. Low levels of miR-181a in ageing muscle were associated with accumulation of its target proteins and stalled autophagy. Our data suggests that miR-181a regulates mitochondrial dynamics by fine-tuning the Pink/Park pathway and mitochondrial biogenesis, potentially in response to stress factors. Moreover, mitophagy is coordinated by a number of conserved cellular consequences on myofibre size and strength indicating the potential of microRNA-based therapies for age-related muscle atrophy.

Mice
The study was performed using male wild type C57Bl/6 mice (adult: 6 months old; old -22-24months old at the beginning of the treatment). Mice were obtained from Charles River 5 (Margate). All mice were maintained under specific-pathogen free conditions and fed ad libitum a standard chow and maintained under barrier on a 12-h light-dark cycle. For miR-181a-5p expression manipulation, mice were injected with 2mg/kg body weight three times during 4-week period with miR-181a mimic (GE Healthcare, C-310435-05 conjugated to cholesterol) or custom antagomiR-181a (5'-FITC- Olympus Fluoview3000 Laser Confocal microscope and analysed using Image J as described previously (Soriano-Arroquia et al., 2016b). Cytotoxicity and ATP generation were measured using mitochondrial ToxGlo assay (G8000) from Promega as per the manufacturer's instructions.

In situ muscle function analysis
Force measurements of the EDL muscles were performed as described before via peroneal nerve stimulation in adult and old male mice (Sakellariou et al., 2016). Briefly, mice were anaesthetised using ketaset and dormitor. The distal tendon was secured to the lever arm of a 1 5 servomotor (Aurora Scientific). Next, the knee was placed in a fixed position and the peroneal nerve exposed. Bipolar platinum wire electrodes were next placed on both sides of the peroneal nerve. Muscle optimal length (L o ) was determined with during a series of 1 Hz stimulation and set at the length that generated the maximal force. EDL muscles were electrically stimulated to contract at L o and optimal stimulation voltage (10 V) at 2 min calculated by dividing P o by total fibre CSA as described previously.

Muscle staining
TA muscles were cryosectioned at −20 °C through the mid-belly. 10 μ m sections were rinsed with Phosphate Buffered Saline (PBS) and fixed in ice-cold methanol for 5 min. Feret's diameter. Image J software was used to analyse individual muscle fibres as described previously (Sakellariou et al., 2016).

Proteomics and Pathway analysis
TA muscles were immediately dissected and a portion homogenised directly in 50 mM semi-dry blotter, after transfer membranes were stained with Ponceau S to ensure equivalent loading. Membranes were blocked in 3% milk in TBS-T, following washing in TBS-T, membranes were incubated with primary antibodies (as above) at a dilution of 1 in 1000 in blocking buffer. Goat anti-rabbit HRP secondary antibody (Cell Signalling) was diluted 1 in 3000 in TBS-T. Thermo super signal west dura was used for chemiluminescence detection 1 0 using a Chemidoc (BioRad), images were acquired and analysed using Image Lab 5.0 software (BioRad) and normalised using Ponceau S stain.

In vitro miRNA target prediction and validation
miR-181 targets were predicted using TargetScan v.6.2 (http://www.targetscan.org/, release p-value < 0.05 was considered as statistically significant, n = 3-6 as indicated in figure   5 legends. qPCR Data: Expression relative to β 2-microblobulin or Rnu-6 and/or Snord-61 (microRNA) was calculated using delta delta Ct method. p-value was calculated using unpaired Student's t-test. Western blotting: Band intensities were normalized using overall total protein intensity from Ponceau S staining. Relative expression was calculated using 1 0 unpaired Student's t-test with p-value < 0.05 considered significant. Label free proteomics: Proteins were quantified using top3 method using PEAKS7 label free software, proteins were considered significantly changes with p-value < 0.01 and > 2 fold change.

Conflict of Interest:
The authors declare they have no conflict of interest 2 0

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B. miR-181a gain-and loss-of-function in TA of adult and old mice leads to changes in the expression of p62, DJ-1 and Park2 mRNA, relative to β 2-microglobulin.
C. miR-181a overexpression increases the expression of mRNA of mitochondrial genes (Cox I, Nd-1) and regulator of mitochondrial biogenesis (Tfam) in TA of adult and old mice, relative to β 2-microglobulin.

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Error bars show SEM * -p<0.05 Student T-test. Adult -6 months old; old -24 months old male C57BL6/J mice; Ctrl -saline. A. qPCR of miR-181a expression in C2C12 myoblasts following 10 µM CCCP treatment and transfections with scrambled antagomiR (control), miR-181a mimic or antagomiR-181a, respectively, relative to Rnu-6 expression.     intravenous injections, however do not affect body weight or muscle mass. Changes in miR-181a expression in TA muscle of adult and old mice following intravenous injections of miR-181a mimic or antagomiR181a as compared to saline injected mice were detected by qPCR.