Mir-184 affects lifespan via regulating the Activin pathway in Drosophila

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1 1 Mir-184 affects lifespan via regulating the Activin pathway in Drosophila Senior Thesis Presented to The Faculty of the School of Arts and Sciences Brandeis University Undergraduate Program in the Department of Biology Dr. Michael Rosbash, Advisor In partial fulfillment of the requirements for the degree of Bachelor of Arts by Beth He May 11, 2016 Copyright by Beth He

2 2 Abstract: MicroRNAs (mirnas) are highly conserved, small noncoding RNAs that modulate posttranscriptional gene expression through as many as 100 mrna targets. Previously, many mirnas have been shown to play a role in regulating the aging process in multiple model organisms. This study shows that the levels of an abundant mirna, mir-184, decrease with age in fly heads and manipulation of its levels in the whole fly as well as in specific tissues, such as glia and muscle, affect lifespan. Furthermore, we also identified mir-184 as a new member of the activin pathway as it is a downstream target of dfoxo and negatively regulates activin in the regulation of glia and muscle metabolism and aging. Introduction: Aging is an interesting biological phenomenon that is highly conserved and regulated across species via numerous evolutionarily derived mechanisms (López-Otín et al., 2013). The majority of molecular mechanisms involved in aging also converge in nutrient sensors, which regulate intracellular gene expression and metabolism (Dimmeler and Nicotera, 2013). Recent studies suggest that the process of aging is regulated and shaped by a complex network of many partially interconnected genetic pathways, concentrated on insulin signaling and modulation (Inukai and Slack, 2013). This insulin and insulin-like growth factor-1 (IIS) pathway is the most conserved aging-regulating mechanism across organisms (López-Otín et al., 2013 and Bai et al., 2013). Phenotypically, aging is defined as the process of temporal loss of physiological integrity and function resulting in disrupted healthy cellular behavior (López-Otín et al., 2013). MicroRNAs (mirnas) are implicated in a wide range of processes, including metabolism (IIS pathway), senescence, autophagy, proliferation, apoptosis, gametogenesis, development, tissue

3 3 repair, and aging (Huang et al., 2013 and Chen et al., 2014). mirnas are small, endogenous, noncoding RNAs that bind to target mrnas and down-regulate their expression through mrna degradation or translational repression (Garza-Manero et al., 2014). Since their discovery, mirnas have been subject to great interest because of their evolutionary conservation and high involvement in gene expression regulation (Huang et al, 2013 and Chen et al., 2014). It has been estimated that nearly 60% of human protein-coding genes are mediated by mirnas (Huang et al., 2013). They are particularly interesting since mirnas can have multiple targets and thus, the possibility of regulating different molecular pathways and effecting many biological processes (Garg and Cohen, 2014). As such, advanced understanding of mirnas could lead to detailed understanding of complex and diverse relationships and regulators of various molecular pathways (Inukai and Slack, 2013). Such complicated networks of mirnas can be seen through their role in aging. Overall trends of mirnas expression levels have been found to regulate throughout the process of aging. Moreover, many mirnas have been identified as related to aging in different model organisms (Inukai and Slack, 2013). Previous studies suggest that some mirnas may not simply regulate aging process, but might be necessary for vitality and longevity. mirnas lin-4 and mir-14 down-regulation reduced the lifespan of C. elegans and Drosophila, respectively (Ambros, 2003 and Kato and Slack, 2013 and Garg and Cohen, 2014). Both mirnas target and regulate the age-associated IIS metabolic pathway (Sun and Lai, 2013). This study hopes to elucidate more mirnas involved in aging and identify one of their mechanisms of influencing the aging process. To do so, experiments were done using Drosophila as a model organism because of its well understood genetic background and easily manipulated genome. Since the discovery of mirnas

4 4 in Drosophila, the UAS-GAL4 system has been used to extensively gather information about mirna function in animals (Lucas and Raikhel, 2013). In Drosophila, the yeast protein GAL4 and its upstream activation sequence (UAS) are used for transgene expression. Conditional GAL4 proteins are expressed and tissue specific promoters are utilized to allow for the activation of UAS transgenes. As such, the GAL4/UAS system allows the spatial and temporal specific expression of downstream genes. (Osterwalder et al., 2001). Materials and Methods: mirna Deep Sequencing In order to study and compare mirna enrichment levels in young vs. old flies and heads vs. brains, mirna deep-sequencing libraries were made following the protocol(s) described by Abruzzi et al., 2015 (Done by Xiao Chen). qrt-pcr Total RNA was extracted from 20 fly body + heads in Trizol reagent (Invitrogen). The total RNA was treated with DNase and quantified using a NanoDrop. Between ng of total RNA was used for quantification using SuperScript One-Step RT-PCR reagent (Invitrogen, as described by Vodala, 2012) and measured against an ABI prism 7300 sequence Detection System (Applied Biosystems). mrna abundance for all genes was normalized against rpl32, by comparative C T method (Done by Xiao Chen). Northern Blot Total RNA was isolated from male heads per genotype, by cutting off the heads with a sharp razor, and then placing them in into the Trizol reagent. Then, the heads were ground by

5 5 pestle and RNA was isolated per the instructions of the manufacturer s protocol (Trizol reagent, Invitrogen). Each lane contained 3µg of RNA, and the RNA was distributed on a 15% NuPage Tris-UREA gel with 1xTBE buffer. The blot transfer was completed with a 0.5xTBE buffer. Then the RNA blots were pre-hybridized with Oligohyb (Ambion), and then incubated with radioactively labeled RNA probes for about 12hrs overnight. The antisense strand of mirna-184 was used as the probe (Done by Xiao Chen). Genetic Background Whole Fly Mir-184 deletion Mutants: The homozygous mir-184 deletion mutant flies were designed as described in Chen et al., Tissue-Specific Mutants (using mirna Sponge): Females virgins with either tissue-specific drivers or GeneSwitch tissue specific drivers, from Bloomington Stock center, were crossed with UAS- mir184sp (knockdown) or scramble (control) lines. Three days after the progeny enclosed, they were transferred to the aging food as described in the procedure below (Done by Beth He). Lifespan Experiments Knockout and Overexpression Line Diet: Flies were reared and maintained at either 25 C or 29 C and 12-hour light/dark. They were maintained on an agar-based diet of 20g agar, 100g sugar, and 100 g yeast, all in 1100mL of water.

6 6 The mifepristone inducible-gal4 system (GeneSwitch) was utilized to control the expression of the UAS constructs in certain adult tissues. Such tissue-specific promoters were activated only in adulthood by adding the drug RU486 (mifepristone, Sigma, St. Louis, MO, USA) to the diet. 1g of mifepristone was diluted in 200 ml of 95% EtOH and kept as stock solutions. 40 ul of stock is used for 1 ml of aging food (Done by Beth He). Lifespan Flies for each tissue-specific driver UAS/GAL4 line were collected based on control and mutant and sex, four categories of eighty flies for each line. Number of dead flies were counted three times/week and flipped into fresh vials until all had died. The rate of survival was calculated by dividing number of flies alive at each transfer session by the total number of flies that started the experiment (Done by Beth He). 3 UTR Activin Mutation The 3 UTR from Drosophila gdna was cloned into the psicheck2 vector. The mir-184 binding site was mutated using a mutagenesis kit from Agilent (Done by Xiao Chen). mrna Expression Levels Total RNA was extracted from heads in Trizol reagent (Invitrogen). The total RNA was treated with DNase and quantified using a NanoDrop. Between ng of total RNA was used for quantification using SuperScript One-Step RT-PCR reagent (Invitrogen) and measured against an ABI prism 7300 sequence Detection System (Applied Biosystems). mrna abundance for all genes was normalized against mir-184 expression, by comparative C T method (Done by Xiao Chen).

7 7 Luciferase Luciferase assay was performed with standard protocol as described by Liu et al., Specifically, two psicheck2 vectors containing either wild-type 3 UTR or mutated 3 UTR were co-transfected with 2 additional plasmids (tub-gal4 and UAS-mir-1840) into S2 cells. After three days, the Renilla/Luciferase ratio was measured using Tecan (Done by Xiao Chen). Results: Previous studies have shown that as humans get older, mirna expression decreases (Dimmeler and Nicotera, 2013). To see if a similar mechanism exists in Drosophila, we performed experiments to explore the relationship between mirnas and aging. The results suggest that mir-184 likely plays an important role in aging via the IIS pathways. Mir-184 is highly expressed in Drosophila heads and brains, decreasing with age To see which mirnas are most prevalent in Drosophila, mirna deep sequencing was done in heads and dissected brains. While the levels of mir-184 are abundant in both tissues, mir-184 is heavily enriched in neurons (dissected brains) (Figure 1).

8 8 Figure 1: An mirna deep sequencing assay demonstrates that mir-184 (selected above) is abundant in both heads and brains, and more enriched in brains. Both axes represent RPKM (log 2). Mir-184 was used in following experiments because of its high expression, and thus, greater molecular functionality. qrt-pcr was used to compare mir-184 expression levels in heads and bodies. Mir-184 levels were normalized rpl32, a commonly used house-keeping gene. The results show that mir-184 expression levels in heads are over three times that found in the Drosophila body (Figure 2).

9 9 Figure 2: Mir-184 expression levels are nearly three fold higher in Drosophila heads as compared to the body. Comparison of mir-184 levels in Drosophila heads and bodies using a qrt-pcr assay. Since mir-184 is more abundant in heads, a second qrt-pcr assay was performed using head RNA to test if mir-184 expression levels alter with age. It was found that relative mir-184 levels are lower in older Drosophila (0.64) as compared to young flies (1) (Figure 3).

10 10 Figure 3: mir-184 expression levels are dependent on age. Older flies have lower (0.64) expression levels than young ones (1). Mir-184 levels are normalized to rpl32. Levels in young vs. old dme-mir-92a dme-mir-285 dme-mir-1 dme-mir-9c dme-mir-184 dme-mir-9b dme-mir-133 dme-mir- dme-mir-276a dme-mir-998 dme-mir-305 dme-mir-275 dme-mir-277 dme-bantam dme-mir- dme-let-7 dme-mir-252 dme-mir-13a dme-mir- dme-mir-315 dme-mir-308 dme-mir-2a-1 dme-mir- dme-mir-210 dme-mir-307a dme-mir-316 dme-mir-996 dme-mir-137 dme-mir-981 dme-mir-279 dme-mir- dme-mir-304 dme-mir-284 dme-mir-9a dme-mir-970 dme-mir-986 dme-mir- dme-mir- dme-mir-927 dme-mir-2b-1 dme-mir-33 dme-mir-11 dme-mir-306 dme-mir-263a dme-mir- dme-mir-2c Figure 4: Many mirna expression levels decrease when age. mirna deep sequences assay, quantitatively comparing ratio of mirna expression levels in old and young flies, demonstrates that mir- 184 is one the top five of candidates whose levels decrease with age. The qrt-pcr result of Figure 3 and mirna deep sequencing result of Figure 4 were corroborated by a northern blot of mir-184 levels in old and young flies (Figure 5). When using mir-184 samples from young (7 day) and old (21 day) flies, the levels of mir-184 in the aged flies were noticeably lower than before. Levels of 2s rrna are shown as a control and are unchanged with increasing age.

11 11 Figure 5: mir-184 expression levels are lower in older flies than in young flies. Northern blot analysis of RNA extracted from young (7D) and old (21D) flies. 2S rrna serves as a control. These results illustrate that mir-184 expression levels decrease with age, as observed previously in humans by Dimmeler and Nicotera, To study the importance of mir-184 on vitality and its role in longevity, lifespan experiments were done to see if and how knockout, tissue specific knockdowns, and overexpression of mir-184 affects aging. Decreases in mir-184 levels lead to a shortened lifespan In vivo lifespan experiments were conducted to see the impact of variable mir-184 expression on aging. To test this, the average lifespan of wild-type and mir-184 knockout (KO) flies were measured. The mutant mir-184 flies solely lacked mir-184, but maintained normal wild-type expression of neighboring genes. Lifespan experiments were done with various drivers and manipulation of mir-184 expression levels for observable effects on aging. For all the following lifespan experiments, homozygous mir-184 knockdown lines were used utilizing the Drosophila UAS/GAL4 system.

12 12 When compared to yellow-white (yw) control flies, mir-184 knockout (KO) flies exhibit shorter lifespans in both sexes, with greater shortening of lifespan in males, with the maximum lifespan for males decreasing by 21 days and for females by 10 days. 100% 80% Mir-184 Knockout in YW Wild-Type Rate of Survival 60% 40% 20% 0% -20% Age (Days) yw males yw females 184KO males 184KO females Figure 6: mir-184 KO flies show decreased survival rates when compared to yw controls. KO males had the lowest survival rate. The maximum lifespan for yw wild-type for both sexes is 76 days, and 55 and 66 days, for mir-184 male and female knockout, respectively. X-axis: Age (Days), Y-Axis: Rate of Survival, calculated as described in Materials and Methods. Mir-184 KO flies had decreased survival rates when compared to yw controls, and introduced a sex-based discrepancy unseen in yw wild-type flies (Figure 6). To test if these effects of mir-184 knockout are background specific, the experiment was repeated in w1118 wild-type background. While the results are less dramatic, the mir-184 KO still has a shortened lifespan with males having the lowest survival rates.

13 13 Mir-184 Knockout in w1118 Wild-Type 100% 80% Rate of Survival 60% 40% 20% 0% -20% Age (Days) w1118 males w1118 females w1118; 184KO males w1118; 184KO females Figure 7: mir-184 KO flies showed decreased survival rates when compared to w1118 wild-types with KO males being the least likely to survive. X-Axis: Age (Days), Y-Axis: Rate of Survival, calculated as described in Materials and Methods. The initial results presented by Figures 2, 3, 4 and 5 indicate that mir-184 is abundant in the brain with its expression decreasing as the flies age. Additionally, KO mir-184 in flies facilitates reduction of lifespan, suggesting that mir-184 is important for the vitality of Drosophila. To test which specific tissue(s) mir-184 is having an effect, the mirna sponge tool was used to knock down (KD) mir-184 in certain regions of Drosophila, using certain tissue specific drivers. MiRNA sponges are incredibly useful tools because they are able to selectively bind to particular target mirnas and suppress their function. As demonstrated by the image below, high sponge expression (and therefore function) results in the relief of repression of the target mrna of that mirna (Figure 8).

14 14 Figure 8. mirna sequester mirnas and allow their target mrna(s) to function normally. Figure adapted from Ebert and Sharp, The following lifespan results from various different GAL4 lines with mir-184 sponge lines (184sp) were compared with controls of scramble sponge lines (scr). These tissue specific experiments began with a tubulin (tub) driver which expresses in the whole body of Drosophila. Knocking down mir-184 in tub-gal4 flies shortens lifespan, demonstrating a similar phenotype as previous experiments with KO mir-184 in the entire fly (Figure 9). This provided support that the sponge technique produced similar results as complete KO flies, which allowed for further tissue specific experiments using the mirna sponge. However, the sponge KD mir-184 flies only shortened the average lifespan of females. In previous aging experiments with yw and w1118 wild-type flies, both genders had shortened lifespans when mir-184 was down-regulated (Figures 6 and 7). This discrepancy is presumably due to the variable expression patterns of different drivers.

15 15 Rate of Survival 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Whole Body mir-184 Knockdown Age (Days) tub-scr males tub-184sp males tub-scr females tub-184sp females Figure 9: A whole body driver (tub) was used to down-regulate mir-184 and showed that the sponge technique was able to reproduce previous aging results (Figures 6 and 7). KD mir-184 resulted in shorter lifespans than the control (tub-scr), however, only females had shorter lifespans. X-Axis: Age (Days), Y- Axis: Rate of Survival, calculated as described in Materials and Methods. A fat body specific gene-switch driver was used to see if knocking down mir-184 resulted in any observable aging differences. The fat body GAL4 system with driver #8151 was used and resulted in no effect.

16 16 Fat Body mir-184 Knockdown 100% 80% Rate of Survival 60% 40% 20% 0% -20% Age (Days) 8151-scr males 8151-scr females sp maless sp femaless Figure 10: mir-184 KD in the fat body had no effect on aging. X-Axis: Age (Days) Y-Axis: Rate of Survival, calculated as described in Materials and Methods. To test the effects of mir-184 knockdown in brains on aging, the Elav-GAL4 driver (a commonly used pan-neuronal specific driver) was used and no measurable effects were observed on aging. 100% 80% Pan-neuronal mir-184 Knockdown Rate of Survival 60% 40% 20% 0% -20% Age (Days) elav-scr males elav-184sp males elav-scr females elav-184sp females Figure 11: Using a neuronal-specific driver, elav, the elav-mir184sp had similar aging rates as the control elav-scr, across sexes, with a lifespan of days. X-Axis: Age (Days) Y-Axis: Rate of Survival, calculated as described in Materials and Methods.

17 17 As observed with the Elav-GAL4 driver, knocking down mir-184 with a differentiated muscle driver, #55133, resulted in no significant change in lifespan. Differentiated Muscle mir-184 Knockdown 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% scr males scr females SP males SP females Figure 12: With differentiated muscle driver, #55133, the knockdown of mir-184 had no lifespan effect. All four fly categories had a maximum lifespan of 51 days. X-Axis: Age (Days), Y-Axis: Rate of Survival, calculated as described in Materials and Methods. However, the knockdown of mir-184 using a muscle driver (#38464) presented a shortened lifespan when compared to scr controls. The mir-184 knockdowns died at a much more rapid rate than the controls.

18 18 Muscle mir-184 Knockdown Rate of Survival 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Age (Days) scr males scr females SP males SP females Figure 13: With muscle driver, #38464, the knockdown mir-184 flies had a shortened lifespan, demonstrating the driver dependent aging phenotype in muscles. X-axis: Age (Days) and Y-Axis: Rate of Survival, calculated as described in Materials and Methods. Lastly, a glia tissue specific driver, repo, was used to see if knocking down mir-184 affected aging. In both sexes, the knockdown of mir-184 using the repo-gal4 driver decreased the rate of survival, by about 10 days.

19 19 Glial mir-184 Knockdown Rate of Survival 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Age (Days) repo-scr males repo-184sp maless repo-scr females repo-184sp femaless Figure 14: Knockdown of mir-184 in glia tissue, using repo driver, resulted in shortened lifespan phenotype for both males and females. X-Axis: Age (Days), Y-Axis: Rate of Survival, calculated as described in Materials and Methods. In summary, the results demonstrated that knocking down mir-184 had variable effects on aging when solely activated in specific tissues. The KD mir-184 had no effect on fat body and neuronal specific drivers while shortening the lifespans when activated in specific drivers of muscle and glia. The table below summarizes the results from lifespan experiments lacking mir-184 from development (with except of the fat body driver, Figure 10):

20 20 Background or Driver Tissue Specificity Mir-184 KO/KD Phenotypic Change Reference Figure Yellow-white No Knockout Shortened Lifespan, 6 (yw) both sexes w1118 No Knockout Shortened Lifespan, 7 both sexes Tubulin Whole body Knockdown Shortened Lifespan, 9 both sexes 8151 Fat Body Knockdown None 10 Elav Pan-neuronal Knockdown None Differentiated Knockdown None 12 Muscle Muscle Knockdown Shortened Lifespan, 13 both sexes Repo Glia Knockdown shortened Lifespan, both sexes 14 Figure 15: Summary of KO/KD results from development. Knocking down mir-184 in glia (CNS +PNS) and some muscle tissue shortens lifespan for both sexes. Aside from the fat body specific driver (#8151, which is a gene-switch driver), the tissue specific drivers used above down-regulated mir-184 from development. Recent studies have identified mir-184 as a modulator for germ cell development in Drosophila (Asgari, 2013 and Lucas and Raikhel, 2013). To see if mir-184 is necessary for normal germ cell formation and if the observed lifespan phenotypes are due to development disruption, gene-switch drivers were used to knockdown mir-184 only in adulthood. To test whether the effect in glia was due to a requirement of mir-184 in development, the mir- 184 sponge was expressed by a steroid inducible driver that expresses in the PNS glia (#41001) There was no significant effect of the mir-184 knock-down in adult PNS glia.

21 21 PNS Glia mir-184 Knockdown Rate of Survival 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Age (Days) scr males SP males scr females SP females Figure 16: Knockdown of mir-184 in PNS glia tissue resulted in wild-type lifespan phenotype of 63 days. X-Axis: Age (Days), Y-Axis: Rate of Survival, calculated as described in Materials and Methods. However, in contrast, another inducible driver that expresses in both CNS and PNS glia (#40320), produced consistent results with the glial-driver, repo, (Figure 14) indicating that knockdown of mir-184 in adult glia shortens lifespan and this effect is unlikely to be due to developmental effects. These results were found in both males and females.

22 22 CNS and PNS Glia mir-184 Knockdown Rate of Survival 100% 80% 60% 40% 20% 0% Age (Days) scr males scr females sp males sp females Figure 17: Knockdown of mir-184 in glia tissue, using CNS and PNS activated driver, resulted in shortened lifespan phenotype, especially in females. X-Axis: Age (Days) Y-Axis: Rate of Survival, calculated as described in Materials and Methods. Additionally, another inducible driver that expresses in both CNS and PNS glia (#40275) only showed shortened lifespan in females: 100% CNS and PNS Glia mir-184 Knockdown Rate of Survival 80% 60% 40% 20% 0% Age (Days) scr males SP males scr females SP females Figure 18: Knockdown of mir-184 in glia tissue, using another CNS and PNS activated driver, resulted in shortened lifespan phenotype only in females. X-Axis: Age (Days), Y-Axis: Rate of Survival, calculated as described in Materials and Methods.

23 23 In addition, various muscle tissue specific drivers were used to see if the shortened lifespan phenotype was universally consistent. The following data demonstrates that the effect of mir-184 knockout on muscles is dependent on the driver. The inducible GAL4 muscle driver, #41013, demonstrated extended lifespan phenotype with mir-184 tissue specific knockdown. Muscle mir-184 Knockdown 100% 90% 80% Rate of Survival 70% 60% 50% 40% 30% 20% 10% 0% Age (Days) scr males SP males scr females SP females Figure 19: With muscle driver, #41013, the knockdown of mir-184 extended lifespan. The scr wild-type males lifespan was 58 days, while for females it was 65 days. The mir-184 KD extended average lifespan for both sexes to 61 days and 72 days, for males and female, respectively. X-axis: Age (Days) and Y-Axis: Rate of Survival, calculated as described in Materials and Methods. However, #40308 gene-switch driver, which expresses both in muscles and CNS glia, demonstrated shortened lifespan phenotype, with the most significant results in females.

24 24 CNS Glia and Muscle mir-184 Knockdown Rate of Survival 100% 80% 60% 40% 20% 0% -20% Age (Days) scr males scr females SP males SP females Figure 20: Knockdown of mir-184 in glia tissue, using muscle and CNS activated driver, resulted in shortened lifespan phenotype significantly in females. X-axis: Age (Days) and Y-Axis: Rate of Survival, calculated as described in Materials and Methods. The muscle and glia tissue specific inducible driver was found to have the strongest effect on aging. The females of the sp line expressed the most significant shortened lifespan phenotype (Figure 20). The following table summarizes the results found from the gene-switch inducible, tissue specific lifespan experiment. These mir-184 knockdown lifespan phenotype screens suggest that manipulating mir-184 only during adulthood is sufficient to change lifespan with certain tissue specific drivers. Gene-Switch Driver Tissue Specificity Phenotypic Change Reference Figure PNS Glia None CNS + PNS Glia Shortened Lifespan, both sexes CNS + PNS Glia Shortened Lifespan, only females Muscle Extends Lifespan, both sexes Muscle + CNS Glia Shortened Lifespan, both sexes 20 Figure 21: Knocking down mir-184 of adults in some glia and muscle tissues shortens lifespan.

25 25 In conclusion, the GAL4 screens with various drivers located in three specific tissues: fat body, neuron, glia, and differentiated muscle, demonstrated that knockdown of mir-184 in particular regions of Drosophila affects aging. Suppressing expression mir-184 in neurons and fat body has no effect on aging. Inversely mir-184 knockdown had noticeably strong effects on aging and shortened lifespan when localized in both glia and certain muscles. However, it is important to note that the variable results with different tissues could reflect the strength of the driver rather than significant differences between tissues. Overexpression of mir-184 extends the lifespan of Drosophila Since knocking down mir-184 in certain tissues of Drosophila shortened lifespan, preliminary overexpression experiments were conducted to provide greater evidence on the functional importance of mir-184 in aging. Because down-regulation of mir-184 in glia cells shortens lifespan, the same glial driver, #40275, was used to overexpress mir-184 to see if it is sufficient to extend lifespan. Figure 22 demonstrates that mir-184 overexpression only extended average lifespans in females.

26 26 Glial mir-184 Overexpression 100% 80% Rate of Survival 60% 40% 20% 0% -20% Age (Days) w1118 males OE males w1118 females OE females Figure 22: Overexpression of mir-184 with the glial driver, 40275, extends lifespan only in females. X- axis: Age (Days) and Y-Axis: Rate of Survival, calculated as described in Materials and Methods. Similarly, when using the same muscle tissue specific driver that resulted in shortened lifespan phenotype when mir-184 was knocked down (#38464), the lifespan of females extended suggesting greater functional importance on aging regulation of mir-184 (Figure 23).

27 27 Rate of Survival 100% 90% 80% 70% 60% 50% 40% 30% 20% 10% 0% Muscle mir-184 Overexpression Age (Days) 184KO 184KO;38464-mir-184 Figure 23: Muscle driver, 38464, overexpression in the mir-184 KO background extends lifespan in females. X-axis: Age (Days), Y-Axis: Rate of Survival, calculated as described in Materials and Methods. In conclusion, these aging experiments using a UAS-GAL4 system and mirna sponge, found that mir-184 affects lifespan primarily in glia and muscle. In demonstrated tissues, knocking down mir-184 only during adulthood is sufficient to shorten lifespan, while increasing mir-184 in glia and muscles during adulthood extends lifespan, particularly in females. These experiments support that mir-184 is an important component of the aging molecular mechanism. Mir-184 Regulation of Age-Dependent IIS Pathway Many findings imply and give evidence that the inhibition of the IIS pathway extends the lifespan of numerous model organisms (Dimmeler and Nicotera, 2013 and Bai et al., 2013). Activin is not only involved in the IIS pathway, but also plays a large role in regulating the process of aging in Drosophila (Bai et al., 2013). To see if mir-184 modulates aging through

28 28 interaction with the IIS pathway, the following experiments were done to elucidate the relationship between mir-184 and activin. To see if activin is a direct target of mir-184, an mrna sequencing experiment of fly heads was done. The activin (actbeta) expression was measured in flies with mir-184 KO and overexpression (Drosophila Tim-GAL4;Tubulin-GAL80 ; UAS-mir-184), demonstrating an ts inverse relationship between mir-184 and Activin expression (Figure 24). 1.8 Activin (mrna) Activin Expression mir-184 KO/yw tim-tub C/18C Figure 24: Expression of Activin (actbeta) is inversely correlated with mir-184 expression. Left column of mir-184 knockout sample has high Activin expression, while overexpression (right column) of mir-184 using the tubulin-gal4 driver results in down-regulation of Activin. Since many mirnas regulate the IIS pathway via 3 UTR binding to age-associated targets, mir- 184 could be active in the aging process via direct binding to activin (Kato and Slack, 2013). To test this hypothesis, an in vitro S2 cell luciferase system was employed. Renilla was fused to the activin 3 UTR to act as an indicator of Activin protein levels in the system, with Luciferase as a loading control. If activin expression level is suppressed by the mirna (and therefore a target of mir-184), the Renilla levels will decrease, resulting in a lower renilla/luciferase ratio. In this

29 29 experiment, activin wild-type 3 UTR was used as a control and the mir-184 binding site in the activin 3 UTR was mutated. Since mir-184 is endogenously expressed in S2 cells at a high level, no additional mir-184 is transfected. Through these experiments, it was found that a mutated mir- 184 binding site in activin resulted in higher luciferase expression, suggesting a de-suppression effect. Ratio of Renilla/Luciferase Mir-184 Activin Binding Site Activin activin WT 3 UTR Activin activin-short mut 3 UTR Figure 25: mir-184 site mutated Activin had 1.5-fold higher Luciferase expression. The resulting data explains that disruption of the mir-184 binding site of Activin significantly affects Luciferase expression levels by nearly 2.7 fold (Figure 25). This indicates that mir-184 directly suppresses activin in-vitro, further suggesting that activin is a direct target of mir-184.

30 30 Discussion: Many mirnas across model organisms have been implicated in the aging process through interaction with the IIS pathway. The findings of this study indicate mir-184 in Drosophila as a key mirna, not only involved in germ line development (Asgari, 2013), but also can be necessary in the stability of the healthy aging of the organism. Mir-184 activity presents age modulated expression and coupling with the processes of the IIS pathway. The results obtained suggest that via direct interaction with activin, mir-184 has key roles in the heavily ageinfluencing insulin pathway. Previous studies by Asgari, and Lucas and Raikhel, identified mir-184 as important in the development of Drosophila through germ line proliferation and maintenance (Asgari, 2013 and Lucas and Raikhel, 2013). Mir-184 levels globally decrease with the normal aging of flies, suggesting additional biological involvement in aging (Figures 3 and 5). Furthermore, our lifespan experiment results show that overall knockout of mir-184 shortens the lifespan of both sexes (Figures 6 and 7). Such global mir-184 knockout experiments were done with two different wild-type backgrounds, yw and w1118, indicating that the shortened lifespan phenotype is related to mir-184 inactivation rather than differences in background. Interestingly, tissue specific mir-184 expression lifespan experiments imply that mir-184 knockdown influences aging when mir-184 is inactivated in certain tissues. GAL4 mir-184 knockdown lifespan experiments from development (aside from fat body (Figure 10), which is gene-switch) signify that lack of mir-184 had no effect when inactivated in fat body and neural tissue, but demonstrated shortened lifespan phenotype when knocked down in glial and certain muscle tissues (Figures 10-14).

31 31 Gene-switch lifespan experiments were done with glial and muscle tissue specific drivers to see if mir-184 knockdown is sufficient to shorten lifespan in adulthood. As previously mentioned, mir-184 has been found to play an important development role in germ cells. Therefore, the results from the GAL4 lifespan experiments stated above could have been the consequences of mir-184 inactivation in glia/muscles during development, disrupting essential developmental pathways. Glial tissue specific drivers of the PNS and CNS/PNS were used to see how inactivation of mir-184 in adulthood influences aging. A strong shortened lifespan phenotype is observed when mir-184 is inactivated in both CNS and PNS glia (Figures 16-18). However, mir-184 knockdown in adults affected lifespan in different ways depending on the driver. The muscle driver #41013 (Figure 19) extended the lifespan of knockdown flies for both sexes, while when mir-184 is inactivated in muscles coupled with CNS glia, using driver #40308 (Figure 20), the most noticeable and strong shortened lifespan phenotype is observed. Considering the relationship between mir-184 knockdown in muscle and faster aging is not universal across muscle tissue types, the data suggests that glia mir-184 inactivation has the strongest connection with shortened lifespan phenotype. Furthermore, overexpression of mir-184 in glia and certain muscle tissue of adult Drosophila is sufficient to extend lifespan. When mir-184 is overexpressed in the same tissues as those that demonstrated shortened phenotypes with mir-184 knockdown, strong extended lifespan phenotype is observed (Figures 22-23). Thus, traditional expression of mir-184 in certain tissues of adults is sufficient for healthy Drosophila aging process. Metabolism is one of the many processes involved in the healthy aging of an organism. Specifically, the insulin/insulin-like growth factor-1 signalling (IIS) pathway has been strongly implicated in the aging process network, with its inhibition increasing the lifespan of numerous

32 32 model organisms (Dimmeler and Nicotera, 2013). Other mirnas associated with the aging network have been found to play roles in the regulation of the IIS pathway (Inukai and Slack, 2013). This study found that mir-184 can be involved in the aging process by being necessary and sufficient for the suppression of activin. The expression levels of mir-184 and activin are inversely reciprocal (Figure 24), and the mutation of the mir-184 binding site in activin results in much greater Activin protein expression (Figure 25) indicating that mir-184 directly binds to activin, relating the age-dependent IIS pathway to mir-184 through activin expression levels. The results of this study suggest that one of the ways mir-184 could be involved in the aging process is through participation in the IIS pathway. Generally, the reduction of insulin/igf-1 signaling increases the lifespan of Drosophila (Bai et al., 2013 and Grönke et al., 2010). The transcription factor FOXO is an important contributor to the regulation of the aging effects of the IIS pathway. The Drosophila homolog of FOXO, dfoxo transcriptionally regulates genes that could monitor numerous insulin pathway phenotypes, including cell proliferation, metabolism, and aging (Bai et al., 2013). Moreover, the overexpression of dfoxo seems to extend the lifespan of the organism (Garg and Cohen, 2014). Thus, the IIS pathway sufficiently mediates Drosophila lifespan partly through dfoxo regulation of muscle activin signaling and its downstream targets (Bai et al., 2013). The literature presents a complex IIS pathway in the muscles of Drosophila. Bai et al., 2013, elucidated this pathway by finding that dfoxo binds and suppresses dawdle (daw) and that activin is able to inhibit the transcription of autophagyspecific gene 8a (Atg8a) by signaling through Smad binding element smox. The Drosophila IIS pathway story is further complicated with the addition of babo, and the fact that down-regulation of daw and its downstream signaling partners babo and smox extend lifespan. As such, previous

33 33 literature results suggest a pathway for the relationship between insulin/insulin-like growth factor-1 signaling and aging. Such a pathway in Drosophila muscle is depicted below: Figure 26: The IIS pathway in Drosophila muscles, describing the relationship between dfoxo/activin and the aging process. Figure adapted from Bai et al., Results from this study suggest that mir-184 participates in the aging process by directly regulating activin (Figures 24-25). Following traditional mirna molecular function, the expression of mir-184 down-regulates or suppresses activin expression, which inhibits increased lifespan phenotype. This proposed mir-184 involvement in the IIS pathway of muscles, is further supported by the lifespan results of this study suggesting that in certain muscles mir-184 expression levels directly impact aging (Figures 13 and 23). Moreover, data from the Marr and Lau lab suggest that Drosophila dfoxo activates mir-184 in S2 cells (personal communication), further supporting mir-184 involvement in aging, while introducing another regulating component to the IIS aging pathway. This possible pathway is presented below:

34 34 Figure 27: This proposed pathway coupling data from this study and data from the Lau/Marr Labs suggest how mir-184 interacts with the age-dependent IIS pathway. Throughout the tissue specific lifespan experiments, mir-184 knockdown in the general panneuronal driver, elav (Figure 11), had no effect on aging, while mir-184 knockdown in CNS/PNS glia resulted in shortened lifespans (Figures 14, 17-18). As mir-184 knockdown in both certain muscle tissues and CNS/PNS glia presented shortened lifespan phenotypes, the agedependent IIS pathway of muscles could also be applicable to certain glial tissues (Figures 26-27). Secondly, the coupling of mir-184 knockdown in muscles and CNS glia resulted in the most significant shortened lifespan phenotype (Figure 20). Although the exact IIS pathway of Drosophila brains is not as well understood as in muscles, the expression of Atg8a in the CNS of Drosophila extends lifespan, following the model in muscles (Bai et al., 2013). As such, it is possible that down-regulating mir-184 in CNS/PNS glia over-expressed activin, which therefore, increased babo inhibition of Atg8a, allowing for shortened lifespan. As neural insulin secretion is controlled remotely by muscle activin, the observed lifespan phenotype of mir-184 knockdown

35 35 in muscles and CNS glia might have resulted from both the muscle pathway depicted in Figures 26-27, and the subsequent decrease in Atg8a levels in both muscle and glia (Bai et al., 2013). The results of the lifespan experiments of this study suggest that only manipulation of mir-184 expression in certain tissues is sufficient to affect lifespan. Although some of our results could be attributed to unseen differences between tissue specific drivers, current literature suggests that it can also be related tissue dependent IIS aging pathways and regulators. For example, the down-regulation of modulators (daw, babo, smox) downstream of activin have been found to extend lifespan only in muscle and not fat body (Bai et al., 2013). Likewise, other key components in the IIS aging pathway have been implicated in tissue specific activation and mechanisms. Tissue specific activation of dfoxo regulates lifespan in adults of the organism (Hwangbo et al., 2004). The data presented in this study identifies mir-184 role in aging through its relationships with dfoxo and Activin. While dfoxo regulates aging in muscle via Activin ligands (daw, smox, babo), it maintains other aging pathways through insulin-like peptides (DILPs) (Bai et al., 2013). Much like mirnas, DILPs are proteins that are evolutionarily conserved, involved in the modulation of various biological processes including metabolism, longevity and aging (Bai et al., 2012). dfoxo in fat body tissue modulates lifespan by inducing fat body tissue specific dilp6 transcription, which when over-expressed systemically reduces insulin signalling and in doing so, increases lifespan (Bai et al., 2012 and 2013). As such, in our study the lack of consistent aging impact of mir-184 expression level manipulation does not necessarily negate its involvement in Drosophila aging, but rather, emphasizes its likely involvement in tissue specific, age-dependent IIS regulation pathways.

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