RiboTag translatomic profiling of Drosophila oenocytes under aging and oxidative stress

Background Aging is accompanied with loss of tissue homeostasis and accumulation of cellular damages. As one of the important metabolic centers, aged liver shows altered lipid metabolism, impaired detoxification pathway, increased inflammation and oxidative stress response. However, the mechanisms for these age-related changes still remain unclear. In fruit flies, Drosophila melanogaster, liver-like functions are controlled by two distinct tissues, fat body and oenocytes. Although the role of fat body in aging regulation has been well studied, little is known about how oenocytes age and what are their roles in aging regulation. To address these questions, we used cell-type-specific ribosome profiling (RiboTag) to study the impacts of aging and oxidative stress on oenocyte translatome in Drosophila. Results We show that aging and oxidant paraquat significantly increased the levels of reactive oxygen species (ROS) in adult oenocytes of Drosophila, and aged oenocytes exhibited reduced sensitivity to paraquat treatment. Through RiboTag sequencing, we identified 3324 and 949 differentially expressed genes in oenocytes under aging and paraquat treatment, respectively. Aging and paraquat exhibit both shared and distinct regulations on oenocyte translatome. Among all age-regulated genes, mitochondrial, proteasome, peroxisome, fatty acid metabolism, and cytochrome P450 pathways were down-regulated, whereas DNA replication and glutathione metabolic pathways were up-regulated. Interestingly, most of the peroxisomal genes were down-regulated in aged oenocytes, including peroxisomal biogenesis factors and beta-oxidation genes. Further analysis of the oenocyte translatome showed that oenocytes highly expressed genes involving in liver-like processes (e.g., ketogenesis). Many age-related transcriptional changes in oenocytes are similar to aging liver, including up-regulation of Ras/MAPK signaling pathway and down-regulation of peroxisome and fatty acid metabolism. Conclusions Our oenocyte-specific translatome analysis identified many genes and pathways that are shared between Drosophila oenocytes and mammalian liver, highlighting the molecular and functional similarities between the two tissues. Many of these genes are altered in both aged oenocytes and aged liver, suggesting a conserved molecular mechanism underlying oenocyte and liver aging. Thus, our translatome analysis will contribute significantly to the understanding of oenocyte biology, and its role in lipid metabolism, stress response and aging regulation.


Conservation in age-related transcriptional changes between oenocytes and liver 373
Since our analyses suggest that Drosophila oenocytes may perform liver-like functions, 374 we wonder if oenocyte and liver exhibit similar transcriptional changes during aging. To test this, 375 we compared age-related transcriptomic profiles between Drosophila oenocytes and mouse liver 376 [10]. We first searched for fly orthologues of mouse liver genes using Drosophila Integrative 377 Ortholog Prediction Tool (DIOPT) [43]. Out of 1052 protein-coding genes that are differentially 378 expressed in aging mouse liver, 735 of them have putative orthologues in Drosophila genome, 379 corresponding to 881 Drosophila genes (Fig. 7A). About 30% of these Drosophila orthologues 380 (252 out of 881) also showed differential expression during oenocyte aging, suggesting a large 381 conservation between liver and oenocyte aging (Table S1: List 17-18). 382 Gene ontology analysis showed that several key biological processes were altered in aged 383 liver, including immune response, apoptosis, peroxisome, bile acid biosynthesis, and fatty acid 384 metabolism (Table S2: List 17-18). Among these biological processes, peroxisome and fatty acid 385 metabolism are shared between liver and oenocyte aging (Fig. 7A). Next, we took a close look at 386 the pathways that contain same orthologues between fly and mouse. Genes up-regulated in both 387 aged oenocytes and liver were enriched in pathways like Mitogen-activated protein kinase 388 (MAPK), Ras signaling, NF-κB, and JAK-STAT (Fig. 7B), while down-regulated genes were 389 found in peroxisome, fatty acid metabolism, and oxidative phosphorylation pathways ( Fig. 7C) 390 (Table S2: List 17-18). Using STRING protein network analysis, we found that large number of 391 Ras/MAPK signaling components were up-regulated under both oenocyte and liver aging (Figs. 392 7D&7E), suggesting that age-dependent dysregulation of these pathways are conserved between 393 fly and mammal. 394 Lastly, we examined age-related transcriptomic changes between oenocytes and several 395 other fly tissues, such as fat body, midgut, and heart. The age-related transcriptional profiles in 396 these fly tissues were obtained from recent genomic studies [44][45][46] (Table S1: List 19-20). 397 Pathway analysis (using STRING) on these tissue transcriptomes revealed a tissue-specific 398 transcriptional profiles during fly aging (Fig. 7F). Each tissue has its own and unique age-399 regulated biological processes and pathways ( Fig. 7G) (Table S2: List 20-21). For example, 400 genes that were differentially expressed in aged oenocytes are enriched for proteasome and 401 ribosome-related functions, while aged fat body showed transcriptional changes in aminoglycan 402 metabolism, chitin metabolism, and detoxification. In aging heart, immune response, glycolysis 403 and gluconeogenesis were enriched. And ion transport, DNA replication, and fatty acid 404 degradation were altered in aging midgut (Fig. 7G). Taken together, aged oenocytes share similar 405 transcriptional profiles with aging liver, while they also exhibit unique features compared to 406 other fly tissues. 407 408

Discussion 409
Oenocytes are one of the poorly studied yet important cells in insects [21,22]. Although 410 previous studies show that oenocytes play a crucial role in lipid metabolism (e.g., synthesis of 411 cuticular hydrocarbon and pheromone), many other oenocyte-regulated physiological functions 412 remain to be determined. Among the uncharacterized functions, we know very little about 413 oenocyte aging and the role of oenocytes in aging regulation. To address these issues, we 414 performed RiboTag sequencing to characterize Drosophila oenocyte translatome under aging 415 and oxidative stress. We show that both aging and paraquat up-regulated DNA repair pathway, 416 while down-regulating immune response and fatty acid elongation. In addition, aged oenocytes 417 were associated with impaired peroxisome, mitochondrial, proteasome, and cytochrome P450 418 pathways. Our RiboTag sequencing also revealed many shared tissue-specific pathways and age-419 related transcriptional changes between fly oenocytes and mammalian liver, highlighting 420 evolutionarily conserved mechanisms underlying oenocyte and liver aging and potential 421 functional homologies between the two tissues. 422 423 1. Oenocyte-specific expressed genes are involved in insect-specific and conserved liver-like 424

functions. 425
Previous functional and histological analyses showed that oenocytes contain large 426 amounts of smooth ER and acidophilic cytoplasm (high protein and lipid contents) [47,48], 427 which is consistent with their roles in lipid synthesis and processing, especially the production of 428 VLCFA and hydrocarbon [20,24,49,50]. Interestingly, Drosophila oenocytes uptake and 429 process fatty acids that are released from the storage tissue fat body during food deprivation [14]. 430 The coordination between fat body and oenocytes in mobilizing lipid storage during fasting is 431 quite similar to the adipose-liver axis in mammals. Besides lipid metabolism, many other oenocyte-associated functions (e.g., detoxification and ecdysteroid biosynthesis) have not yet 433 been thoroughly examined at the molecular level. It is unclear whether some of these functions 434 are also conserved liver-like functions, or they are merely insect-specific roles. 435 To better understand oenocyte function, we conducted oenocyte-specific translatome 436 profiling in adult Drosophila and identified 423 genes that were highly expressed in oenocytes 437 (at least 5-fold higher than whole body expression). These genes were enriched in pathways like 438 fatty acid elongation, proteasome-mediated protein catabolism, xenobiotic metabolism, 439 ketogenesis, and peroxisome pathways. There was only a small overlap between oenocyte-440 enriched and fat body-enriched genes, suggesting that the two tissues regulate distinct functions 441 in Drosophila. Comparing to the genes and pathways enriched in human liver, we found that 442 oenocytes shared several biological processes with liver, such as ketogenesis, peroxisomal beta-443 oxidation, ROS metabolism, long-chain fatty acid metabolism, and xenobiotic metabolism. This 444 is consistent with a previous study showing that Drosophila oenocytes expressed high levels of 445 lipid metabolic genes similar to those of mammalian liver [14]. One enriched pathway in 446 Drosophila oenocytes that was not observed in the previous study is the ketogenesis pathway. It 447 is well-known that ketone bodies (acetoacetate, β-hydroxybutyrate, and acetone) are primarily 448 produced by liver when glucose is not available as fuel source [51]. Ketogenesis in insects, 449 however, is not well studied. Ketone bodies have been detected in hemolymph, fat body, and 450 thoracic muscle of adult desert locust and cockroach [52][53][54]. It is speculated that ketone bodies 451 are produced in fat body according to the ex vivo tissue culture assay in locust [53]. However, fat 452 body (along with many other tissues) can also oxidize ketone bodies, which is quite different 453 from mammals where the ketogenesis tissue liver cannot oxidize ketone [53]. It might be 454 possible that in previous ex vivo tissue culture studies, the ketone production came from a 455 contaminated tissue (like oenocytes), rather than fat body. Based on our oenocyte translatome 456 analysis, most of the ketogenesis genes are highly expressed in oenocytes, but not in fat body. 457 Our data suggest that oenocytes are likely the major ketogenesis tissue. A careful function and 458 genetic analysis, such as cell ablation or tissue-specific gene silencing, will need to be performed 459 to examine whether oenocytes are responsible for ketogenesis in Drosophila and in other insect 460

species. 461
Insect hydrocarbons serve as important waterproofing components, and species-and sex-462 specific recognition signals. The biosynthesis of hydrocarbons are involved in fatty acid 463 elongation, desaturation, reduction, and oxidative decarbonylation [55]. Our oenocyte 464 translatome analysis revealed an enrichment of genes in microsomal fatty acid elongation 465 system, such as CG18609, spidey, CG6746, and Sc2. This is consistent with oenocyte's role in 466 hydrocarbon production and its abundant smooth ER content. In microsomal fatty acid 467 elongation system, spidey (also known as Kar) encodes for the only very-long-chain 3-ketoacyl-468 CoA reductase in Drosophila genome, and it has been implicated in oenocyte VLCFA synthesis 469 and waterproof of the trachea system [50], as well as the production of cuticular hydrocarbon,  In adult insects (especially in females), ovary is the major tissue for ecdysteroid 479 biosynthesis [57,58]. It remains to be determined whether other adult tissues are also capable to 480 synthesize ecdysteroids. Interestingly, we found two Halloween genes (phantom and shadow) 481 Our data revealed that aging and oxidative stress decreased the expression of most of the 509 peroxisome biogenesis and protein import genes, which may lead to reduced peroxisome 510 function, including hydrogen peroxide metabolism. Decreased expression of receptor protein 511 Pex5 and reduced peroxisomal enzyme import were previously observed in aged C. elegans [38] 512 and during human fibroblast senescence [65]. Among many key peroxisomal enzymes, the 513 importing of antioxidant catalase was significantly affected during fibroblast senescence, which 514 led to accumulation of hydrogen peroxide and further disruption of peroxisome import [65]. 515 Similar to early studies in aging rat liver [66-68], we found that the expression of many 516 peroxisomal antioxidant enzymes (e.g., Cat, SOD1, Prx5) decreased in aged oenocytes. The 517 combined dysregulation of peroxisomal gene expression and protein import may attribute to 518 elevated toxic reactive oxygen species, and impaired oenocyte functions. Furthermore, 519 generation of excess peroxisomal ROS could disrupt mitochondria redox balance, leading to 520 mitochondrial dysfunction and tissue aging [69].

Conservation between oenocytes and liver aging. 545
The comparison of aging transcriptomes between fly oenocytes and mouse liver revealed 546 many shared pathways between the two tissues. Among these conserved pathways, MAPK and 547 Ras signaling pathways were significantly up-regulated in both aged oenocytes and liver. MAPK 548 signaling is one of the major regulatory pathways involved in stress responses (e.g., oxidative 549 stress). The typical MAPK pathway includes three branches: c-Jun N-terminal kinase (JNK), 550 p38/MAPK, and extracellular signal-regulated kinase (ERK). Previous studies show that all three 551 MAPK cascades are elevated under aging, probably due to increased oxidative stress [79,80]. Using RiboTag sequencing, we characterized the first oenocyte translatome profiles in 573 Drosophila. Our analysis uncovered many previously unexplored oenocyte-specific molecular 574 pathways, especially those associated with oxidative stress and aging. Some of these pathways 575 were found enriched in both fly oenocytes and mammalian liver, suggesting a functional 576 homolog between the two tissues. We believe that the analysis of oenocyte translatome will 577 contribute significantly to our understanding of oenocyte biology, as well as the molecular 578 mechanisms for its role in stress response and aging regulation. 579 580

Oenocyte-enriched genes and tissue-specific aging transcriptome analysis
Young group) to the whole body transcriptome profiles from previous studies (two wild-type 650 backgrounds: w 1118 : GSM2647344, GSM2647345, GSM2647345. yw: GSM694258, 651 GSM694259).The sequencing reads with FPKM ≥ 0.01 were normalized by quantile 652 normalization function using preprocessCore package. 653 (https://www.bioconductor.org/packages/release/bioc/html/preprocessCore.html). Oenocyte-654 enriched genes were defined as those with 5-fold higher FPKM in oenocytes comparing to whole to gene symbols was set to false. Permutation type was set to gene set; enrichment statistic used 668 as weighted analysis; metric for ranking genes was set to Signal to Noise. 669

Gene ontology and pathway analysis 670
Functional annotation analysis of differentially expressed genes was performed using 671 Ras/MAPK protein network in STRING, "kmeans clustering" option was used and number of 674 clusters was set to 2 or 3. 675

Quantitative real-time polymerase chain reaction (qRT-PCR) 676
qRT-PCR was performed using Quantstudio 3 Real-Time PCR system and SYBR green 677 master mixture (Thermo Fisher Scientific, Waltham, MA, USA Catalog number: A25778). To 678 determine the most stable housekeeping gene, the Ct values for four housekeeping genes were examined in all twelve cDNA samples obtained from different treatments. Using an Excel-based 680 tool, Bestkeeper [97], we confirmed that Gapdh1 is the least-variable housekeeping gene across 681 samples (Table S3). All gene expression levels were normalized to Gapdh1 by the method of 682 comparative Ct [98]. Mean and standard errors for each gene were obtained from the averages of 683 three biological replicates, with one or two technical repeats. Primer sequences are available in 684 Table S4. showing the overlap of differentially expressed genes in aged oenocytes, fat body, heart, and 770 midgut. (G) GO terms enriched in aged oenocytes, fat body, heart, and midgut. 771      mRpS9  mRpS7  mRpS6  mRpS21  mRpS2  mRpS18C  mRpS18A  mRpS17  mRpS16  mRpS14  mRpS11  mRpS10  mRpL9  mRpL4  mRpL36  mRpL35  mRpL34  mRpL33  mRpL32  mRpL30  mRpL3  mRpL28  mRpL27  mRpL24  mRpL23  mRpL22  mRpL21  mRpL20  mRpL2  mRpL19  mRpL18  mRpL17  mRpL16  mRpL15  mRpL14  mRpL13  mRpL12  mRpL11  mRpL10  Log2(FC-aged/young) P e x 3 P e x 1 6 P e x 1 9 P e x 5 P e x 7 P e x 1 3 P e x 1 4 P e x 2 P e x 1 0 P e x 1 2 P e x 1 P e x 6 P e