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LETTER TO THE EDITOR

Egg-derived tyrosine phosphatase as a Potential Biomarker forMuscle Ageing and Degeneration in Drosophila melanogaster

Ageing is associated with declined activity of behaviors,physiology and metabolic processes (Arking, 2006). In-vestigations in model organisms have indicated the exis-tence of “functional senescence”, the progressive decline ofbiological functions with age and the decline in the activitymay vary from tissue to tissue. Consequently, studies per-taining to the key organs/tissues whose functions deterio-rate/fail with age have led to the development of tissuespecific ageing biomarkers (Grotewiel et al., 2005;Demontis et al., 2013).

Muscle constitutes the bulk of body mass in majority ofeukaryotes. Besides its contractile function, studies frommodel organisms have indicated muscles to play a promi-nent role in ageing. Major age-related changes that areapparent in muscle include alteration in gene expressionpattern, accumulation of poly-ubiquitin protein aggregates,a decline in protein synthesis, increased mitochondrial andnuclear DNA damage and eventually progressive increasein apoptosis. Genetic interventions through signaling eventsnot only showed a delay in age-related muscle deteriora-tion but also increased stress resistance and metabolichomeostasis, which led to extended lifespan (Demontis andPerrimon, 2010). Dietary restriction also plays a role inmodulating lifespan by fine tuning the muscle activities.So, there are overwhelming evidences to suggest thatmuscle does play significant roles during ageing (Demontisand Perrimon, 2010; Demontis et al., 2013). Unraveling theinterconnections between muscle and ageing is imperativeconsidering the massive increase of age-related problemsincluding sarcopenia (Sayer et al., 2013). The identificationof biomarkers based on different disease conditions wouldgreatly help in clinically assessing the etiology for accurateprognosis and treatment. Skeletal muscle specific molecularsignatures that could be used as biomarkers for identifi-cation of sarcopenia and muscle ageing have been reported(Holland and Ohlendieck, 2013). However, the detailedmechanisms involved are not well understood. For suchstudies, accessibility of a suitable model system isindispensable.

- see front matter Copyright � 2014, Institute of Genetics and Develop

Elsevier Limited and Science Press. All rights reserved.

org/10.1016/j.jgg.2014.01.008

The indirect flight muscles (IFMs) of Drosophila mela-nogaster offer an excellent in vivo model system to decipherthe complexities underlying muscle functions and systemicageing. They share tremendous similarities with vertebrateskeletal muscles, in terms of assembly and function and withcardiac muscles in terms of physiology (Moore, 2006; Rai andNongthomba, 2013). In addition, the dispensability of IFMsunder laboratory conditions, availability of various musclemutants and tractable markers and advanced genetic tools(Vigoreaux, 2001) make the IFMs a highly favored modelsystem for ageing studies.

The Gal4-UAS system is an inevitable tool for reversegenetics study in Drosophila (Brand and Perrimon, 1993).Enhancer trap analysis (O’Kane and Gehring, 1987) providesan effective way of selecting strains based on the increased/decreased spatio-temporal activity with age. The identifica-tion of the linked genes from the isolated strains will aid indiscovering tissue specific ageing biomarkers and studyingtheir functions. If a trap gene is involved in the progressionof muscle ageing, we presume that it should show increased/decreased reporter activity with age. Therefore, with the aimto isolate genes which show increased activity with age, wescreened numerous P{GawB} strains reported to beexpressed in fly muscles from Bloomington stock centre,Indiana, USA. Each fly line was crossed with a reporter UAS-GFP line and the expression of GFP was followed in theprogenies. Most of the Gal4 strains that we have studiedshowed a decrease in GFP accumulation with increase in age.But we have been interested in identifying line(s) whichshowed increased expression of GFP with age, which shouldreflect the active function of the trapped gene(s) during theageing process. From the analyses of 38 strains, P{GawB}DJ694 was found to show an age dependent increase in GFPexpression (Fig. 1A and B). In the first few weeks of eclo-sion, GFP expression was weak in the IFMs, which thensurged to its peak at around 30 days. This line was shown tohave increased b-galactosidase reporter activity with age(Seroude et al., 2002). So, we chose to characterize thisstrain.

mental Biology, Chinese Academy of Sciences, and Genetics Society of China.

Fig. 1. Establishment of EDTP as a potential ageing marker for Drosophila IFMs.

A and B: Fluorescence images of whole fly thoraces showing differential GFP expression driven by P{GawB}DJ694 on the 3rd day (3 d) and 30th day (30 d). C:

Genomic representation of the EDTP gene structure, annotated transcripts and P{GawB}DJ694 insertion site with orientation (gene region is snapshot taken from

flybase.org). D: Quantification of EDTP transcript in 3 days and 30 days old fly IFMs by real time PCR. Mann-Whitney test indicated significant increase

(*P < 0.05). E: Agarose gel showing no transcript expression of CG18467 in young and aged IFMs but expressed moderately in adult brain. F: Comparison of

EDTP expression in young IFM mutants e Canton S (CS), up1, up101, wupAhdp-2 and tnMB10738. One-way ANOVA with Dunnett’s multiple comparison post test

revealed significant increase of transcript levels (*P < 0.05, **P < 0.01, ***P < 0.001). G and H: Confocal images of IFM myofibrils in wild type and mutant

tnMB10738. Red is Phalloidin TRITC and green anti-Mlp60 A marks the Z-discs (Scale ¼ 5 mm).

222 Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 221e224

Analysis of the P{GawB}DJ694 flanking genomic DNAsequences, recovered by inverse PCR, confirmed that theelement is inserted at nucleotide position 13,330,712 of thelocus AE013599 at 54B cytological region on the secondchromosome of D. melanogaster genome. This physicallocation corresponds to the first intron of the Egg-derivedtyrosine phosphatase (EDTP) or CG6542 gene. This genehas been annotated to produce two transcripts differing by21 nucleotides in one of the exon and encodes a putativeprotein-tyrosine phosphatase enzyme (flybase.org). Furtheranalysis of the flanking sequences recovered from the 50-end of the P-element indicated that P{GawB}DJ694 isoriented along the plus strand while EDTP is encoded bythe DNA on minus strand (Fig. 1C). In other words, thedirection of P{GawB}DJ694 and EDTP gene are oppositeto each other. Therefore, it was hard to presume that theexpressed Gal4 in IFMs, visualized through reporter GFP,indeed corresponds to the inserted gene. This situationcould occur only when the weak P-element promoter pre-ceding Gal4 and EDTP gene regulators comes under thesame enhancer simultaneously. To test this hypothesis, wechecked the expression levels of EDTP transcripts, isolatedfrom dissected IFMs of 3 days and 30 days old flies, usingreal time PCR. In agreement with the GFP expressionpattern, EDTP transcript level was found to be significantlyelevated in the IFMs of 30 days old flies as compared to

3 days (Fig. 1D) (Mann-Whitney test, P < 0.05). Anotherannotated gene CG18467 was found to be located upstreamof the insertion site in the same orientation. Since therewas a possibility that P{GawB}DJ694 was inserted in aputative downstream enhancer of CG18467, we checked itsexpression pattern in aged and young IFMs. However, wecould not detect any expression of the gene in the IFMs,though there was moderate expression in adult brain(Fig. 1E), corroborating with Fly Atlas Anatomicalexpression data in Flybase. Therefore, despite its reverseorientation, P{GawB}DJ694 was found to follow theexpression pattern of the EDTP in IFMs.

Normal functioning of muscle requires strict regulation ofthe component proteins and their maintenance. Aged IFMs areknown to show defective muscle ultrastructure with increasedstiffness and reduced force production (Miller et al., 2008). Asthe vital role of muscle in ageing is evident, we quantified theEDTP transcript level in the IFMs of young flies whose musclefunctions are disrupted. We chose three different mutantswhich showed muscle degeneration in the IFMs, i.e., up1 andup101, alleles of Troponin T (upheld) (Nongthomba et al.,2007), and wupAhdp-2, an allele of Troponin I (heldup) (Bealland Fyrberg, 1991). In these mutants, the normal myofi-brillar structures are seen completely broken down within fewdays after eclosion because of the unregulated actin andmyosin interactions. EDTP transcripts in the 3e5 days old

223Letter to the Editor / Journal of Genetics and Genomics 41 (2014) 221e224

IFMs of these mutants were significantly increased, i.e., up1

(P < 0.001), up101 and hdp2 (P < 0.01) (Fig. 1F). Markedincrease in the expression levels of EDTP in the mutant IFMsindicates that EDTP could be involved in muscle tear-wearpathways and may serve as biomarker for muscledegeneration.

Cellular homeostasis is critically regulated by thecontinuous synthesis and degradation of proteins and thewhole metabolic processes involved undergo ageing-relatedfunctional decline. It has been speculated that senescencein protein turnover might lead to accumulation of defectiveproteins which in turn might influence ageing (Stadtman,2004). To test this hypothesis, we chose a mutant alleleof abba (thin), a homolog of vertebrate TRIM32 (LaBeau-DiMenna et al., 2012), which encodes an E3 ubiquitinligase enzyme essential for the targeted protein degradationpathway. abba is expressed highly in the IFMs (unpub-lished microarray data from our lab) and mutations inTRIM32 have been found to cause limb girdle musculardystrophy type 2H in humans (Frosk et al., 2002). We useda mutant allele of abba, tnMB10738, a minos mediatedinsertion allele, which fails to produce the full lengthmRNA in IFMs (unpublished data from our lab). Themutant allele causes defective myofibrillar phenotype(Fig. 1H) compared to wild type (Fig. 1G). Upon quanti-fication, EDTP transcript level was found to be signifi-cantly high in the IFMs of mutant flies which were only 2-days old as indicated in Fig. 1E (P < 0.05). Probably, dueto the dysfunction of the proteasome degradation pathway,IFMs of young flies could mimic the old age situationwhere poly-ubiquitin aggregates are accumulated. So, theincreased levels of EDTP in the young IFMs of alleletnMB10738 highlight that EDTP may play an important rolein ubiquitin dependent targeted proteosomal degradationpathways and muscle ageing.

EDTP is homolog of myotubularin-related protein 14(MTMR14), a member of the highly conserved myotubularinfamily. It has been implicated in centronuclear myopathyand reported to play a role in autophagy and calcium ho-meostasis (Amoasii et al., 2012). EDTP could be critical fordephosphorylation of many muscle proteins through itsputative phosphatase function to maintain the complexphysiological functions and cellular homeostasis (Salviet al., 2012). Considering all these facts, EDTP in muscleappears to be critical for regulating systemic ageing.An intensive study could shed lights on the role of EDTPin muscle function and ageing, thereby establishing its po-tential as an ageing and degeneration biomarker of themuscle.

ACKNOWLEDGEMENTS

We thank the laboratory members for their critical com-ments. This work was supported by financial assistance fromDepartment of Science and Technology and Department ofBiotechnology, Government of India and Indian Institute ofScience to Upendra Nongthomba.

SUPPLEMENTARY DATA

Supplementary data related to this article can be found athttp://dx.doi.org/10.1016/j.jgg.2014.01.008.

Salam Herojeet Singha,b, Nallur B. Ramachandrab,*,Upendra Nongthombaa,*

aMolecular Reproduction, Development and Genetics,

Indian Institute of Science, Bangalore 560 012, India

bDepartment of Studies in Zoology, University of Mysore,

Manasagangotri, Mysore 570 006, India

*Corresponding authors. Tel: þ91 80 2293 3258,

fax: þ91 80 2360 0999 (U. Nongthomba); Tel: þ91 821 241 9781/9888,

fax: þ91 821 251 6056 (N. B. Ramachandra).

E-mail addresses: nbruom@gmail.com (N. B. Ramachandra);

upendra.nongthomba@gmail.com,

upendra@mrdg.iisc.ernet.in (U. Nongthomba)

Received 9 September 2013

Revised 11 January 2014

Accepted 13 January 2014

Available online 3 February 2014

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