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The microRNA miR-34 modulates aging and neurodegeneration in Drosophila

Human neurodegenerative diseases possess the temporal hallmark of afflicting the elderly population. Hence, aging is among the most significant factors to impinge on disease onset and progression1, yet little is known of molecular pathways that connect these processes. Central to understanding this connection is to unmask the nature of pathways that functionally integrate aging, chronic maintenance of the brain and modulation of neurodegenerative disease. microRNAs (miRNA) are emerging as critical players in gene regulation during development, yet their role in adult-onset, age-associated processes are only beginning to be revealed. Here we report that the conserved miRNA miR-34 regulates age-associated events and long-term brain integrity in Drosophila, presenting such a molecular link between aging and neurodegeneration. Fly miR-34 expression is adult-onset, brain-enriched and age-modulated. Whereas miR-34 loss triggers a gene profile of accelerated brain aging, late-onset brain degeneration and a catastrophic decline in survival, miR-34 upregulation extends median lifespan and mitigates neurodegeneration induced by human pathogenic polyglutamine (polyQ) disease protein. Some of the age-associated effects of miR-34 require adult-onset translational repression of Eip74EF, an essential ETS domain transcription factor involved in steroid hormone pathways. These studies indicate that miRNA-dependent pathways may impact adult-onset, age-associated events by silencing developmental genes that later have a deleterious influence on adult life cycle and disease, and highlight fly miR-34 as a key miRNA with a role in this process

Human neurodegenerative diseases have the temporal hallmark of afflicting the elderly population. Ageing is one of the most prominent factors to influence disease onset and progression 1 , yet little is known about the molecular pathways that connect these processes. To understand this connection it is necessary to identify the pathways that functionally integrate ageing, chronic maintenance of the brain and modulation of neurodegenerative disease. MicroRNAs (miRNA) are emerging as critical factors in gene regulation during development; however, their role in adult-onset, age-associated processes is only beginning to be revealed. Here we report that the conserved miRNA miR-34 regulates age-associated events and long-term brain integrity in Drosophila, providing a molecular link between ageing and neurodegeneration. Fly mir-34 expression exhibits adult-onset, brain-enriched and age-modulated characteristics. Whereas mir-34 loss triggers a gene profile of accelerated brain ageing, late-onset brain degeneration and a catastrophic decline in survival, mir-34 upregulation extends median lifespan and mitigates neurodegeneration induced by human pathogenic polyglutamine disease protein. Some of the age-associated effects of miR-34 require adult-onset translational repression of Eip74EF, an essential ETS domain transcription factor involved in steroid hormone pathways. Our studies indicate that miRNA-dependent pathways may have an impact on adult-onset, age-associated events by silencing developmental genes that later have a deleterious influence on adult life cycle and disease, and highlight fly miR-34 as a key miRNA with a role in this process.
Recent evidence reveals that miRNA pathways are important in the adult nervous system, notably in the maintenance of neurons and in the regulation of genes and pathways associated with neurodegenerative disease 2,3 . Given these findings, we considered that there may be a fundamental role for select miRNAs in ageing. We examined flies carrying a hypomorphic mutation in loquacious (loqs), a key gene in fly miRNA processing 4 (Supplementary Fig. 1a). Flies bearing the loqs f00791 mutation were viable, but detailed examination indicated a significantly shortened lifespan ( Supplementary Fig. 1b). Further analysis indicated that loqs f00791 flies showed late-onset brain morphological deterioration: although normal as young adults, by 25 days loqs f00791 flies developed large vacuoles in the retina and lamina of the brain ( Supplementary Fig. 1c). Although developmental processes may contribute to shortened lifespan, the adult-onset brain degeneration of loqs f00791 mutants indicated that one or more specific miRNAs may be critically involved in age-associated events impacting on long-term brain integrity.
To explore this question, we determined whether specific miRNAs displayed age-modulated expression in the brain. RNA was isolated from dissected brains of adult flies of young (3 days), mid (30 days) and old time points (60 days). Using an array for Drosophila miRNAs, 29 were expressed in the adult brain (Fig. 1a). Whereas most miRNAs maintained a steady level or decreased with age, one miRNA, mir-34, increased (Fig. 1a). Small RNA northern blot analysis confirmed that mir-34 expression was barely detectable during development, but became high in the adult and was further upregulated with age (Supplementary Fig. 2a, b). Expression of mir-34 was affected in loqs f00791 flies ( Supplementary Fig. 1d). miR-34 falls into a category of Drosophila miRNAs whose processing requires the exoribonuclease nibbler (nbr) 5,6 . In the adult, mature miR-34 displayed three major differentially sized forms (24 nucleotides, 22 nucleotides and 21 nucleotides) with a uniform 59 end, descending by single nucleotides at the 39 end which result from nbr-mediated trimming; only isoform c became upregulated with age ( Supplementary Fig. 2c and Fig. 1b, c; see also refs 5-7).
miR-34 is a markedly conserved miRNA, with orthologues in fly, Caenorhabditis elegans, mouse and human showing identical seed sequence ( Supplementary Fig. 2d). To define miR-34 function, flies deleted for the gene were generated ( Supplementary Fig. 3a) Table 4). Analysis of age-associated functions revealed that young mutants (3 days) had normal locomotion and stress resistance, but by 20 days the mutants had dramatic climbing deficits and were markedly stress-sensitive compared to age-matched controls (Fig. 2b). Because mir-34 expression was brain-enriched, we also examined the brain. Typically, older flies show sporadic, agecorrelated vacuoles in the brain-a morphological hallmark of neural deterioration 8 . mir-34 mutants were born with normal brain morphology, but showed dramatic vacuolization with age, indicative of loss of brain integrity (Fig. 2c). Rescue with a 9-kb genomic DNA fragment containing mir-34 and its endogenous cis-regulatory elements (Supplementary Fig. 3a, b) partially restored the age-associated expression of mir-34 to mir-34 null flies in the same homogeneous genetic background ( Supplementary Fig. 3d). Although rescue was not complete, indicative of a complexity in genomic elements that regulate mir-34, rescue was sufficient to mitigate the mutant effects, indicating that miR-34 function normally underlies these age-associated aspects (Supplementary Table 1). These data indicated that mir-34 mutants were normal as young adults, but with age developed deficits reflective of much older animals, including loss of locomotion, stress sensitivity and brain deterioration,

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coupled with shortened lifespan. We therefore hypothesized that loss of mir-34 accelerated brain ageing. To address this, we transcriptionally profiled the fly brain (3 days, 30 days and 60 days) from wild-type animals. On the basis of a linear regression model 9 , we extracted 173 probe sets from this profile the expression of which was tightly correlated with the progression of normal ageing (Fig. 2d and Supplementary Tables 2 and 3). We next made another set of brain transcriptional profiles for mir-34 mutants and controls of matched chronological age (3 days and 20 days). We measured relative changes of these probe sets between 3 days and 20 days within each genotype, and compared the extent of such changes between mir-34 mutants and controls. This indicated that the overall pattern of these probe sets was significantly different between the two genotypes (P 5 0.006, two-sample, paired Wilcoxon test; Fig. 2d). In particular, most positively correlated probe sets displayed a faster pace of increase in mir-34 mutants compared to controls-thus showing accelerated age-associated expression changes in mir-34 mutants (Fig. 2d). This result, combined with the physiological and histological evidence of more rapid loss of ageassociated functions, suggested that mir-34 mutants were undergoing accelerated brain ageing. miRNAs function by binding to the 39 UTRs of target mRNAs and often result in downregulation of protein translation. We therefore reasoned that age-associated activities of miR-34 might be mediated through silencing of critical targets that have a negative impact on the adult animal. miRNA-target prediction algorithms indicated miR-34 binding sites within the 39 UTR of the Eip74EF gene; notably, these binding sites were conserved in the orthologous Eip74EF genes from different Drosophila species ( Supplementary Fig. 4a). We confirmed the miR-34 interaction through mutations in the seed sequences of the predicted miR-34 binding sites in the 39 UTR of the Eip74EF mRNA ( Supplementary Fig. 4b). The Eip74EF gene is a component of steroid hormone signalling pathways. Although such pathways have generally been studied for effects during development, data have implicated these pathways in lifespan regulation 10 .
The Eip74EF gene encodes two major protein isoforms, E74A and E74B (referred to as the E74A and E74B genes, respectively 11 ); the isoforms share the same 39 UTR ( Supplementary Fig. 4a). Northern blots indicated that transcription of E74A, but not E74B, persisted in adults, overlapping the time period when mir-34 is expressed ( Supplementary Fig. 4c). Given this, we focused on E74A as a regulated target of miR-34 in the adult. Despite robust expression of the mRNA transcript, the E74A protein was expressed at low levels in adult heads throughout lifespan (Fig. 3a, b and Supplementary Fig. 4d). In flies lacking miR-34, E74A protein was markedly increased (Fig. 3b); E74A was also de-regulated in the loqs f00791 mutant flies ( Supplementary  Fig. 1e). Genomic rescue of mir-34 mitigated this de-regulation of the E74A protein (Fig. 3c). Fine temporal analysis indicated that the E74A protein was highly expressed in young flies, but underwent a marked decrease within a 24-h time window (Supplementary Fig. 5). This temporal pattern seemed to be mutually exclusive to that of miR-34 (see Supplementary Fig. 2a). Moreover, in flies lacking miR-34, the downregulation of E74A protein during this critical period was dampened ( Supplementary Fig. 5). This evidence indicates that adult-onset expression of mir-34 functions, at least in part, to attenuate E74A protein expression in the young adult, and maintain that repression through adulthood ( Supplementary Fig. 4d).
We next determined whether deregulated expression of E74A protein contributed to the age-associated defects in mir-34 mutants. Because E74A function is essential during development, with strong mutations leading to pre-adult lethality 11 , we used the mild, but viable, E74A BG01805 hypomorphic mutation (Supplementary Fig. 4a). When the E74A BG01805 mutation was combined with mir-34 mutant flies in the same homogenous genetic background, proper regulation of E74A protein was partially restored (Fig. 3d), and age-associated defects due to loss of mir-34, including shortened lifespan and brain vacuolization, were mitigated (Fig. 3e, f; E74A BG01805 mutants alone have a normal lifespan ( Supplementary Fig. 6a)). To assess further the adult activity of E74A, we upregulated E74A in the adult with an E74A transgene that lacks miR-34 binding sites driven by a temperature-sensitive promoter 12 . At 29 uC, these flies demonstrated increased levels of E74A expression in the adult (Supplementary Fig. 6b). Notably, these animals also showed late-onset brain degeneration ( Supplementary Fig. 6c) and a significantly shortened lifespan ( Supplementary Fig. 6d). These data indicate that deregulated expression of E74A has a negative impact on normal ageing, and that one function of miR-34 is to silence E74A in the adult to prevent the adult-stage deleterious activity of E74A on brain integrity and viability.
Notably, during the course of these studies, we noted that mir-34 mutants also displayed a defect in protein misfolding-a molecular process implicated in ageing and common to many human neurodegenerative diseases 13 . Whereas normally with age, the fly brain accumulates a low level of inclusions that immunostain for stress chaperones like Hsp70/Hsc70, mir-34 mutants showed a marked increase compared to control flies of matched age (30 days) ( Supplementary Fig. 7). Given that mir-34 expression increases with age, and mir-34 loss shows altered chaperone accumulation, we tested whether mir-34 expression itself is upregulated by stresses like heat shock or oxidative toxins, but found no evidence to support this (data not shown). However, given that loss of mir-34 caused an increase in protein misfolding, this raised the possibility that upregulation of mir-34 expression might mitigate disease-associated protein misfolding. In Drosophila, expression of a pathogenic ataxin-3 polyglutamine (polyQ) disease protein (SCA3trQ78) leads to inclusion formation, a decrease in polyQ protein solubility and progressive neural loss 14 (Supplementary Fig. 8a). Upregulation of mir-34 markedly mitigated polyQ degeneration, such that inclusion formation was slowed, the protein retained greater solubility, and neural degeneration was suppressed (Fig. 3g, h and Supplementary Fig. 8b-d). Lowering E74A expression by heterozygous reduction in flies expressing pathogenic polyQ protein revealed a minimal effect (data not shown), indicating that E74A may not be a target of miR-34 activity in this process. However, our studies with E74A were of necessity limited to hypomorphic alleles that may not uncover the full extent of E74A function mediated by miR-34. Furthermore, additional targets of miR-34 may be involved in different aspects of miR-34-directed pathways, including disease.
Given this effect to mitigate disease-associated neural toxicity with upregulation of mir-34, and that mir-34 expression naturally increases with age, we investigated whether enhanced expression of mir-34 in wild-type flies could modulate the ageing process. We increased miR-34 dosage in wild-type flies with genomic rescue transgenes, which express mir-34 under its endogenous regulatory elements (see Supplementary Fig. 2a). Analysis of multiple independent transgenics in the same genetic background with that of control indicated that upregulation of miR-34 levels with genomic constructs (,20%, Supplementary Fig. 3d) promoted median survival rate by ,10% compared to wild type ( Fig. 3i; other traits, such as the occurrence of brain vacuolization, despite being an age-associated phenomenon, are sporadic and low in normal flies, thus were difficult to assess). Thus, upregulation of mir-34 expression can protect from neurodegenerative disease and extend median lifespan.
Our findings indicate that miR-34 in Drosophila presents a key miRNA that couples long-term maintenance of the brain with healthy ageing of the organism. miR-34 activity, enhanced by its agemodulated expression and processing, is critically involved in silencing of the E74A transcript through adulthood and in modulation of protein homeostasis with age, as well as in polyQ disease. Select neural cell types may be especially vulnerable in ageing and disease 15 ; miR-34 function may have an impact on the integrity or activity of these systems. Intriguingly, E74A seems to confer sharply opposing function on animal fitness at different life stages, being essential during preadult development 11 , but harmful to the adult during ageing (this study). This biological property-of a gene being beneficial at one life stage, but damaging at another-is referred to as antagonistic pleiotropy 16 . Genes associated with antagonistic pleiotropy are likely to be evolutionarily retained due to their earlier beneficial function 17 . Their adult-onset activities, however, antagonize the ageing process if they are not properly regulated. miRNA pathways provide a tantalizing mechanism by which to suppress potentially deleterious age-related activities of such genes; a number of miRNAs have been noted to show age-modulated expression and activity 18,19 . Roles of   (4,8,9) were analysed. Genotypes: control, 5905; mir-34 (1), mir-34 genomic rescue in 5905 homogenous genetic background.

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select miRNAs normally expressed in the adult may be of evolutionary advantage to tune-down events that promote age-associated decline and potentially disease, in order to prolong healthy lifespan and longevity. Upregulation of lin-4, a C. elegans miRNA with a known developmental role, extends nematode lifespan 18 , raising the possibility that this upregulation, like the natural increase of mir-34 expression in Drosophila, functions to silence genes that have a negative impact on ageing and potentially promote disease. Notably, mir-34 expression is elevated with age in C. elegans 19,20 , and mammalian mir-34 orthologues are highly expressed in the adult brain 21 and have also been noted to increase with age and be misregulated in degenerative disease in humans [22][23][24][25][26] . Current data regarding miR-34 function indicate that it is neutral or adverse in C. elegans 19,27 , and can be either protective or contributory to age-associated events in vertebrates [22][23][24][25][26] . Thus, miR-34 seems to be a key miRNA poised to integrate age-associated physiology; the precise function will reflect the diverse spatiotemporal expression and activity of distinct orthologues, the mRNA target spectrum, as well as the complexity of the adult brain and life cycle. The conservation of miR-34, coupled with in-depth comparative analysis of mir-34 expression, 39 end processing, targets and pathways in the ageing process of nematodes, flies and mammals, make it a tempting subject for understanding features of ageing and disease susceptibility.

METHODS SUMMARY
Flies were grown in standard media at 25 uC unless otherwise specified. Stock lines and GAL4 driver lines were obtained from the Drosophila Stock centre at Bloomington, or are described 14 . Deletion of the mir-34 region was made by sitespecific recombination. Fly transgenics were generated by standard procedures. Flies were generated or backcrossed a minimum of five generations into a controlled uniform homogeneous genetic background (line 5905 (FlyBase ID FBst0005905, w 1118 )), to assure that all phenotypes were robust and not associated with variation in genetic background. In this uniform homogeneous genetic background, the lifespan of control flies is highly uniform with repetition when 150 or more individuals are used for lifespan analysis. Negative geotaxis and thermo stress were used to examine fly locomotion and stress resistance, respectively. Adult male heads were processed for paraffin sections as described 14 . To determine lifespan, newly eclosed males were collected and maintained at 15 flies per vial, transferred to fresh vials every 2 days while scored for survival. A total of 150-200 flies were used per genotype per lifespan; all experiments were repeated multiple times (see Supplementary Table 4). Lifespans were analysed in Excel (Microsoft) and by Prism software (GraphPad) for survival curves and statistics. Techniques of molecular biology, western immunoblots and histology were standard. Fly brain mRNA was prepared using Trizol reagent for array and mRNA analysis, miRNA arrays were miRCURY LNA arrays version 8.1 (Exiqon), and mRNA expression was profiled using Affymetrix Drosophila 2.0 chips (Affymetrix). The microarray data can be found in the Gene Expression Omnibus (GEO) of NCBI through accession number GSE25009.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.

METHODS
Genetic background. Fly lines were from the Bloomington Stock centre or are described 14 . To control for background effects, and to assess significance of all effects, flies were generated in the same uniform homogeneous genetic background (line 5905 (FlyBase ID FBst0005905, w 1118 )), or backcrossed a minimum of five generations into this uniform genetic background. This assured that, for all phenotypes, even modest and consistent effects were associated with the gene manipulations and not a variation in background. With these carefully controlled experiments, the lifespan of control flies was highly uniform upon repetition, when 150 or more individuals were used for lifespan analysis (see Supplementary Table 4). mir-34 deletion mutants. Deletion of the mir-34 region was made by site-specific recombination between two piggyBac insertions, using FLP-FRT-mediated sitespecific recombination 28 . The loss of other genes in the region was then fully rescued by genomic transgenes, so that a line selectively lacking only mir-34 was generated. Two FRT-bearing insertions, PBac[XP]d02752 and PBac[RB]Fmr1 e02790 , were used (Exelixis collection), which encompass the mir-34 region. Genetic crosses were made to combine these two transposon elements with heat-inducible FLP recombinase. After 48 h of egg laying, parents were removed, and vials containing progeny were placed in a 37 uC water bath for a 1-h heat shock. Progeny flies were treated with daily 1-h heat shock, for an additional 4 days. Young virgin female progeny flies were collected and crossed to males with 3rd chromosome balancers.
In the subsequent generation, progeny males were used to generate additional progeny for PCR confirmation. Progeny flies bearing the deletion were positive for PCR verification, using primers from neighbouring genomic DNA and ones from transposons ( Fly age-associated phenotypes. Negative geotaxis and thermo stress were used to examine fly locomotion and stress resistance, respectively. To perform negative geotaxis, groups of 15 adult male flies of indicated age were transferred into a 14-ml polystyrene round-bottom tube (Falcon), and placed in the dark for 30-min recovery. The assay was conducted in the dark, with only a red light on. Climbing ability was scored as the percentage of flies failing to climb higher than 1.5 cm from the bottom of the tube, within 15 s after gently being banged to the bottom. Three repeats were performed for each group and the result averaged. For each genotype at a given age, a minimum of 200 flies were tested. For heat sensitivity, groups of 15 adult males of indicated age were transferred into 14-ml polystyrene roundbottom tubes (Falcon) then placed in a 25 uC incubator for 30 min recovery. Heat stress was applied by immersing the vial containing the flies into a 37 uC water bath for 1 h, followed by a 30-min recovery at 25 uC, then another 1-h heat stress at 37 uC. Flies were then transferred into regular food vials and maintained at 25 uC. Dead flies were counted after 24 h. To assess brain morphology, adult male heads were processed for paraffin sections as described 29 , and brain vacuoles were counted through continuous sections generated from each head (n 5 10 heads counted for each genotype). Molecular biology. Fly genomic DNA was prepared from whole flies with the Puregene DNA purification kit (Qiagen). To generate mir-34 pUAST constructs, PCR amplification was conducted using genomic DNA as template, with primer pairs of pUAST mir-34-I (286 bp, PCR primer 59-CCGTTACACACGACT ATTCTCAAT-39/59-CCATCTGATACAGGTCCTACATTTTCTAAAA-39) and pUAST mir-34-II (936 bp, PCR primer 59-ACCTTGAGCGCTTCAACTCT-39/ 59-CACTCTTTCTCGTTTGCATGG-39). PCR products were then ligated into the pUAST vector. mir-277/dfmr1 rescue construct was made in the pCaSpeR4 vector, which contained two parts. Part 1 was a genomic DNA fragment (7,530 bp) harbouring the mir-277 sequence (PCR primers: 59-GGTCGTGCATGACGAG ATTA-39/59-GGATGTTTTGCGACCAACTT-39), and part 2 was a genomic fragment containing dfmr1 genomic sequence, derived from the pBS WTR construct (a gift from T. Jongens 30 ), by BamH1 and Ppum1. The mir-34 genomic rescue construct was also made in the pCaSpeR4 vector, with two parts. One was a genomic DNA fragment (6,855 bp) upstream of mir-277 sequence (PCR primers: 59-GGTCGTGCATGACGAGATTA-39/59-GGATGCATTTTATCGTT AGGC-39), and the other was a genomic DNA fragment (2,111 bp) containing mir-34 sequence (PCR primers: 59-GCAGGAAAATGCGATAAATGA-39/ 59-TCGTTACAACATGGAAATCCTC-39). The resultant construct, therefore, contains mir-34 sequence, including most upstream fragment, with the exclusion of 108 bp of mir-277 sequence. In addition, a modified mir-34 genomic rescue construct was made (pCaSpeR4 vector), which contains same upstream and downstream ends of the original mir-34 genomic rescue construct, with a small deletion of miR-277 mature sequence. The genomic regulation of mir-34 seems complex, as despite these standard manipulations for gene rescue, the genomic rescue expression of mir-34 and extent of phenotypic rescue of mir-34 mutants was only partial. We attempted upregulation of mir-34 with the GAL4-UAS system, including with the conditional gene switch system in adults. Upregulation of mir-34 during development in non-germline tissues (when it normally is not expressed; Supplementary Fig. 2a) was deleterious, and we were unable to upregulate mir-34 expression more robustly than with the genomic constructs. For western immunoblots, 10 adult male heads per sample were homogenized in 50 ml of Laemmli buffer (Bio-Rad) supplemented with 5% 2-mercaptoethanol, heated to 95 uC for 5 min and 10 ml loaded onto 4-12% Bis-Tris gels (NuPage), then transferred to nitrocellulose membrane (Biorad) and blotted by standard protocols. Primary antibodies used were anti-tubulin (1:10,000, E7, Developmental Studies Hybridoma Bank), anti-E74A (a gift of C. Thummel). Secondary antibodies for immunoblots were goat anti-mouse conjugated to HRP (1:2,000, Chemicon) and developed by chemiluminescence (ECL, Amersham). The final image was obtained by Fuji scanner (Fujifilm).
Luciferase assays were performed using standard approaches 5 . Specifically, 8 3 10 4 DL1 cells were plated and bathed in 30 ml of serum-free medium with 60 ng of dsRNA in each well of a 96-well plate. The next day, 1.6 ng of pMT-Firefly, 400 ng of pMT-mir-34 and 400 ng of pMT-renilla E74A wild-type or mutant 39 UTR reporters were transfected by Effectene (Qiagen). Two days after transfection, the expression of the reporters and mir-34 was induced by CuSO 4 . Twentyfour hours after induction, luminescence assays were performed by the Dual-Glo Luciferase Assay System (Promega). The mir-34 seed sequences in the 39 UTR of E74A were mutated as noted in Supplementary Fig. 3, using the Quik change mutagenesis system (Stratagene). Primers to knockdown Ago1 are described 5 .
The miRNA-target prediction algorithms TargetScan (v5.1) 31 and PicTar (fly) 32 were used to determine miR-34 target mRNA candidates. miRNA microarray analysis. For miRNA array analysis, Iso31 flies (isogenized w 1118 ) were used. Flies were killed by brief submersion in ethanol under CO 2 anaesthesia, followed by two PBS washes (Sigma). To control for circadian effects, all flies were processed between 11:00 and 13:00. Brains were removed manually and collected in an Eppendorf microcentrifuge tube stored on ice. For each miRNA microarray replicate, 200-300 brains were collected for each time point,

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with a ,50/50 ratio of males and females. RNA was prepared using the miRvana RNA extraction system (Ambion) yielding ,2.5 mg per 100 brains. RNA was eluted into 80 ml of RNase free water (Fisher Scientific) and stored at 280 uC. miRNA profiling was carried out at the Penn microarray core facility using miRCURY LNA arrays (Exiqon) and protocols. Exiqon's Hy3/H5-labelling kit was used (Exiqon). RNA samples were labelled with Hy3 and hybridized together with a Hy5-labelled common reference standard. The common reference standard consisted of equal amounts of RNA from brains of 3 days, 30 days and 60 days flies. The miRNA microarray data were analysed at the Penn Bioinformatics Core. Raw data was imported into Gene Spring 1.0 (Agilent) and normalized using a global LOESS regression algorithm (locally weighted scatterplot smoothing). Relative expression levels were calculated as the log 2 normalized signal intensity difference between the Hy3 and Hy5 intensity. Present/absent flagging was analysed by Exiqon (Exiqon). Expression levels (fold changes) for the 30 day and 60 day time point were calculated relative to the 3 day time point. The data sets were exported into Spotfire DecisionSite 9.0 (Tibco) for visualization and filtering. mRNA microarray analysis. For ageing microarray analysis, fly stock Iso31 was used. For mir-34 mutant microarray analysis, mir-34 null line-1 in 5905 background was used, with fly 5905 line, as control. To generate an ageing profile, flies were aged to 3 days, 30 days and 60 days, and 30-50 brains dissected per time point, per replicate, as above (50-50 males and females). For each time point, three replicates were conducted. For mir-34 mutant microarray analysis, time points were 3 days and 20 days, and for each time point, 20 brains from male flies of the appropriate genotype were used, with five replicates in total. Microarray hybridization and reading was performed at the Penn Microarray Core Facility. For mRNA microarrays, total RNA was reverse transcribed to ss-cDNA, followed by two PCR cycles using the Ovation RNA amplification system V2 (Ovation). Quality control on both RNA and ss-cDNA was performed using an 2100 Agilent Bioanalyzer (Quantum Analytics). The cDNA was labelled using the FL-Ovation cDNA Biotin Module V2 (Ovation), hybridized to Affymetrix Drosophila 2.0 chips (Affymetrix) and scanned with an Axon Instruments 4000B Scanner using GenePix Pro 6.0 image acquisition software (Molecular Devices). Affymetrix .cel (probe intensity) files were exported from GeneChip Operating Software (Affymetrix). The .cel files were imported to ArrayAssist Lite (Agilent) in which GCRMA probe-set expression levels and Affymetrix absent/present/marginal flags were calculated. Statistical analysis for those genes passing the flag filter was performed using Partek Genomics Suite (Partek). The signal values were log 2 transformed and a 2-way ANOVA was performed.
Transcriptional analysis of ageing status. We first used the wild type to extract age-associated probe sets and then compared the relative changes of these probe sets in a separate set of transcriptional profiles generated for the wild type and mir-34 mutant. For transcriptional profiles of normal aged brains, the GCRMA package RMA (J. Z. Wu, J. MacDonald and J. Gentry, GCRMA: background adjustment using sequence information, R package version 2.14) for R/Bioconductor 33 was used to generate log 2 expression levels for probe-set IDs from the original .cel files. Then, a linear regression model was used to compute the significance of a correlation between age and gene expression 9 . This approach assumes a linear relationship between age and log 2 expression level: In this equation, Y ij is the log 2 gene expression level of probe set i in sample j, A j is the age for individual j. The coefficients b 1i is regression coefficients reflecting the rate of change in gene expression with respect to age. Probe sets with expression significantly correlated with age (P # 0.001 for b 1i ) were determined. Then the same probe sets were used to estimate the relative expression in separate profiles of mir-34 mutants and age-matched controls. The average levels of each individual probe set were calculated for the difference between 20 day and 3 day, within the same genotype (that is, D20 day/D3 day) for each gene in controls and mir-34 mutants, respectively. These differences were then compared between genotypes (that is, mir-34 mutants 2 controls). The significance of the difference between genotypes was analysed using a paired Wilcoxon test. The difference between control and mutant samples in positively correlated genes (Fig. 2d) is not by chance (P 5 0.0001).