Unique age-related transcriptional signature in the nervous system of the long-lived red sea urchin Mesocentrotus franciscanus

The red sea urchin, Mesocentrotus franciscanus, is one the earth’s longest-lived animals, reported to live more than 100 years with indeterminate growth, life-long reproduction and no increase in mortality rate with age. To gain insight into mechanisms associated with longevity and negligible senescence, age-related transcriptional profiles were examined in tissues of the red sea urchin. Genome-wide transcriptional profiling using RNA-Seq revealed few age-related changes in gene expression in muscle and esophagus tissue. In contrast, radial nerve showed an unexpected level of complexity with the expression of 3,370 genes significantly altered more than two-fold with age, including genes involved in nerve function, signaling, metabolism, transcriptional regulation and chromatin modification. There was an age-related upregulation in expression of genes involved in synaptogenesis, axonogenesis and neuroprotection suggesting preservation of neuronal processes with age. There was also an upregulation in expression of positive regulators and key components of the AMPK pathway, autophagy, proteasome function, and the unfolded protein response. This unique age-related gene expression profile in the red sea urchin nervous system may play a role in mitigating the detrimental effects of aging in this long-lived animal.

RNA Sequencing and age-related differential gene expression in M. franciscanus tissues. RNA from Aristotle's lantern muscle (ALM), esophagus (ES), and radial nerve cord (RN) from sea urchins of each size category (Small and Large) for the collections conducted over three different summers were used for RNA-Seq analysis (Table 1). RNA was pooled from individuals for each tissue type and size category (ALM-S, ALM-L, ES-S, ES-L, RN-S and RN-L) and samples from each of the three sampling years were treated as independent replicates Sample Test Diameter (mm) Est. Age a (years)

Number of animals (n)
Mf -Small (2010) 42 25 and Ebert, 1998 26 . The values are expressed as mean ± standard error.  Supplementary Fig. S1. BUSCO analysis suggested a near-complete transcriptome with 96.4% of the 978 BUSCO genes found complete as single copies (24.8%) or duplicated (71.6%), and 3% present but fragmented. Functional annotation of the assembled transcript contigs was performed by comparison against sequences of S. purpuratus (SPU genes, peptides, and transcriptome from www.EchinoBase.org). This resulted in 44,088 of the 127,198 transcripts matching to 17,292 SPU proteins with top hits averaging 77.7% identity. Gene names used to refer to M. franciscanus genes in the text correspond to the gene names of S. purpuratus homologs from EchinoBase, omitting the "Sp-" prefix. Count-based differential gene expression between Small (young) and Large (old) samples was analyzed within each tissue type and averaged over all three years to yield 144, 123 and 3,370 genes that were significantly different in ALM, ES and RN, respectively [q-value (FDR) < 0.05, log2 fold-change < −1 or>1].
Twelve genes were selected for verification by quantitative RT-PCR (9 for RN and 3 for ALM) using RNA from individual sea urchins collected in 2010. In all cases the qRT-PCR expression levels were significantly different between the Small (young) and Large (old) sea urchin groups and all genes showed the same trend of increased or decreased expression when compared to the RNA-Seq results, although the magnitude of the change was different in some cases ( Fig. 2; Supplementary Table S1).

Functional analysis of differentially expressed genes in Aristotle's lantern muscle (ALM).
The expression of 144 genes was significantly altered with age in ALM (65 upregulated and 79 downregulated) [log2 fold-change < −1 or >1, q-value (FDR) < 0.05]. Thirty-nine genes encoded uncharacterized proteins and the remaining 105 genes were assigned to functional categories based on a modified version of the custom sea urchin gene ontology of Tu et al. 27 Table S2). The functional categories most affected with age were Adhesion/ECM (extra cellular matrix), Signaling, Biomineralization, Metabolism, Immunity, and Cytoskeleton/Cilia, each with 10 to 20 genes significantly altered. In the Signaling category, there was an upregulation with age in expression of the gene encoding myostatin (GDF8, Growth differentiation Factor 8) a member of the Transforming Growth Factor beta superfamily that inhibits muscle growth 28 . This is consistent with the slowed growth rate observed with increasing size of sea urchins 19 . There was a decrease in expression of 13 genes whose products play a role in biomineralization (Table S2). The presence of biomineralization gene transcripts may be the result of sclerocytes that co-isolated with muscle tissue when it was scraped from the pyramids of Aristotle's lantern. It is interesting that no genes associated with mitochondrial function or protein homeostasis were significantly altered with age in ALM.

Functional analysis of differentially expressed genes in esophagus (ES). The expression of 123
genes was significantly altered with age in ES (53 upregulated and 70 downregulated) [log2 fold-change < −1 or >1 level, q-value (FDR) < 0.05]. Of the 123 differentially expressed genes, 49 encoded uncharacterized proteins and 74 were assigned to functional categories using the modified custom sea urchin gene ontology of Tu et al. 27 (Fig. 3b; Supplementary Table S3). The category of "Metabolism" was most affected with 3 upregulated and 12 downregulated genes. Nine genes in the Cytoskeleton/Cilia category were altered with age including upregulated expression of 4 genes associated with cilia formation and function [dynein regulatory complex protein 9 (Iqcg), Cilia and Flagella Associated Protein 126 (CFAP126), Cilia and Flagella Associated Protein 69 (CFAP69), sperm-associated antigen 8 (Spag8)]. In sea urchin larvae, the proximal zone of the esophagus is densely ciliated to assist in the transport of food 29 , a function that may be preserved with age in adults. Only 1 gene was significantly altered with age in each of the Mitochondria, Apoptosis, Autophagy, and Calcium Toolkit categories, and only 2 genes were altered with age in the Protein Homeostasis and Signaling categories ( Fig. 3b; Supplementary Table S3). The black bars represent RN and the red bars represent ALM. Gene names: Tnks1 -Tankyrase-1, Capncalpain, Wnt1 -Wingless-type MMTV integration site-1, Mab21 -Mab-21-like 2, Lkb1 -Liver Kinase B1/ Serine Threonine Kinase 11, Hyou1 -Hypoxia up-regulated 1, Neurx -Neurexin, Glur6 -Glutamate receptor ionotropic kainite-like 2, MAP3K9 -mitogen activated protein kinase kinase kinase 9, EBP -echinenonebinding protein, SM30E -spicule matrix protein 30E, MstnB -myostatin B. expression of 3,370 genes in RN was significantly altered with age at the log2 fold-change < −1 or >1 level [q-value (FDR) < 0.05] (2,291 upregulated and 1,079 downregulated) (Supplementary Table S4). The genes whose expression was most altered with age were selected for more detailed analysis including all genes that exhibited a log2 fold change of <−2 or >2. Using this criterion, the expression of 1,292 genes was significantly changed with age (907 upregulated and 385 downregulated). The differentially expressed RN gene sequences were assigned to functional categories based on a modified version of the custom sea urchin gene ontology of Tu et al. 27 (Fig. 4; Supplementary Table S5). This resulted in 1,006 out of 1,292 differentially expressed RN genes being assigned to functional groups (726 upregulated and 280 downregulated), with the remaining 286 genes (181 upregulated and 105 downregulated) being uncharacterized. The most dramatic expression differences were in the categories of DNA/RNA Metabolism, Signaling, Metabolism, and Nerve function (Fig. 4).
A large number of age-related differentially expressed genes were associated with the "Nerve Function" category (115 upregulated and 12 downregulated) ( Fig. 4; Supplementary Table S5). Many genes that play a role in maintenance of nerve function and neuroprotection were altered with age (38 upregulated and 4 downregulated) including an upregulation of the nerve growth factor Neurotrophin (Nt) and Neurotrophic Tyrosine Receptor Kinase (Trk). Twelve genes associated with synaptogenesis and 18 genes associated with axonogenesis, neurogenesis, and neurite outgrowth were up-regulated with age (Table 2). Many genes associated with neurotransmitter production, transport and function were altered with age (45 upregulated and 8 downregulated). The neurotransmitter receptors affected by age are shown in Table 2 and include metabotropic glutamate receptors (5 upregulated) and ionotrophic glutamate receptors (3 kainate type upregulated, 3 AMPA type downregulated). Three GABA receptors and regulators of GABA production (Clptm1 and Gad) as well as one glycine www.nature.com/scientificreports www.nature.com/scientificreports/ receptor and a transporter that removes glycine from the synaptic cleft (Glyt) were also upregulated. Ten genes associated with acetylcholine (ACh) neurotransmission were altered with age (2 muscarinic ACh receptors and 7 nicotinic ACh receptors were upregulated while 1 nicotinic ACh receptor was downregulated) ( Table 2), as well as genes involved in choline transport and metabolism [High Affinity Choline Transporter 1 (Slc5a7_1) and Acetylcholinesterase (AchE), both upregulated]. Biogenic amine neurotransmitters were also affected with age including an upregulation of an adrenergic receptor subunit and modulators of adrenergic receptor activity [beta-arrestin 1 (Arrb1) and alpha-arrestin 3 (Arrdc3)] as well as dopamine receptor and dopamine metabolism [Dopamine beta-hydroxylase (Dbh) and tyrosine hydroxylase (Th)]. Serotonin receptor 1a was upregulated while receptor 1b was downregulated with age, and a melatonin receptor was downregulated ( Table 2). Several neuropeptide receptors were upregulated with age including neuropeptide S receptor, somatostatin receptors, galanin receptor, tachykinin receptor and octopamine receptor ( Table 2). A number of the genes assigned to the "Calcium Toolkit" category are involved in the regulation of neuronal signaling pathways, including the downregulated genes: Calmodulin Like 3 (CalmL3), Calpain (Capn), Regucalcin (Rgn), and upregulated genes: Transient Receptor Proteins (Trpc5_4, Trpc5_6, Trpv6L, Trpc7) and Doublecortin Like Kinase 1 (Dclk1L). Additional upregulated genes in this category are involved in calcium homeostasis, neuronal excitability, and excitation-contraction coupling or light sensing (Supplementary Table S5).
The expression of 66 genes encoding products assigned to the "Protein Trafficking" category was upregulated while 5 were downregulated with age ( Fig. 4; Supplementary Table S5). The upregulated genes include 7 that play a role in post-Golgi transport and secretory or synaptic vesicle function and 7 genes that play a role in ER and Golgi structural organization and stability. There was also an increase in expression of five members of the ER stress response that clears misfolded proteins from the ER [i.e. Membralin (Mbr1), UDP-Glucose Glycoprotein Glucosyltransferase 1 (Ugcgl1), Thioredoxin Domain Containing 5 (Txndc5), Tripartite Motif Containing 13 (Trim13), Eukaryotic Translation Initiation Factor 2 Alpha Kinase 3 (Eif2ak3perk)].
A large number of genes associated with the "Cytoskeleton/Cilia" category was modulated with age (53 upregulated and 43 downregulated) including genes that play a role in axon formation and neuronal migration ( Fig. 4; Supplementary Table S5). Upregulated genes are involved in maintenance of subcellular spatial organization and integrity, cell motility, and organelle positioning including 26 genes associated with cytoskeletal structure and dynamics, and 16 genes encoding molecular motors for intracellular transport of vesicles and organelles (e.g. 10 kinesins, 2 dyneins, 4 myosins). Many genes encoding proteins associated with cilia formation and function were altered with age (5 upregulated and 15 downregulated). Although the role of cilia in the nervous system of adult sea urchins is not well characterized 30 , there is increasing evidence in humans that primary cilia may play a role in neural stem cell activity, neurogenesis, neuronal maturation, and maintenance 31 .
Numerous genes associated with the adhesion and extracellular matrix category "Adhesion/ECM" were modulated with age (30 upregulated and 37 downregulated) indicating active ECM remodeling ( Fig. 4; Supplementary  Table S5). Ten genes encoding structural components of the ECM were upregulated while 21 were downregulated including 7 collagen genes and lysyl oxidase (LoxL2) which catalyzes the cross-linking of collagen molecules. Eight genes that play a role in cell-cell or cell-matrix adhesion were downregulated with age, while 14 genes were upregulated including 3 integrins and 1 cadherin. Five genes encoding matrix metalloproteases were increased in expression with age while five genes encoding tissue inhibitors of metalloproteinase (TIMP) were downregulated.
A large number of genes encoding components of the signaling pathways category "Signaling" were altered with age in RN (111 upregulated and 19 downregulated), representing a wide range of cellular processes   Table S5). G-protein coupled receptors (GPCR) were among the most affected with 29 upregulated and 1 downregulated. Downstream of GPCR, genes associated with cAMP dependent signal transduction (4 upregulated and 1 downregulated) and upregulation of A-Kinase Anchoring Protein 1 (Akap1) which modulates protein kinase A activity were altered with age. In addition, four genes encoding proteins in the phosphoinositide phospholipase C (PLC) and protein kinase C (PKC) pathways were upregulated with age. Other pathways affected with age were the Ras GTPase family members (16 upregulated and 2 downregulated) and components of the mitogen-activated protein kinase (MAPK) pathway (9 upregulated).
Several genes associated with the Wnt signaling pathway (3 upregulated and 5 downregulated), the Notch signaling pathway (2 upregulated), and the TGFβ/BMP signaling pathway (4 upregulated and 2 downregulated) were affected with age (Supplementary Table S5). Only 3 genes that play a role in insulin signaling were altered with age in red sea urchin nerve tissue: an insulin-like growth factor binding protein (IgfalsL) was upregulated while an insulin-like growth factor binding protein (IgfbpL7) and sorbin and SH3 domain containing 1 (Sorbs1) were downregulated (Supplementary Table S5).
Gene expression patterns indicated age-related modulation of several genes associated with the 5' AMP-activated protein kinase (AMPK) and mechanistic target of rapamycin (mTOR) pathways (Supplementary  Table S5). This included upregulation in expression of Phosphatase and Tensin Homolog B (PtenB) and Inositol Polyphosphate-5-Phosphatase F (Inpp5f), which negatively regulate AKT (an upstream positive regulator of mTORC1), and Serine/Threonine Kinase 11 (Lkb1/STK11) which phosphorylates and activates AMPK as well as Prkag2, the regulatory subunit of AMPK. It should be noted that expression of Ulk1 (Unc-51 like autophagy activating kinase 1) which plays a role in facilitating AMPK induced autophagy and inactivation of mTORC1 was increased with age in the red sea urchin RN tissue at log2 fold-change of 1.65 (Supplementary Table S4, SPU_027245). AMPK inhibits mTORC1, but has been shown to activate mTORC2 32 . mTORC2 plays a role in cytoskeletal organization and cell migration and Mip1 (Sin1), a component of mTORC2, was upregulated in sea urchin RN with age.
In the "Protein Homeostasis" category, 14 ribosomal protein genes were downregulated, while there was an upregulation of translation initiation factor Eif4e3, a negative regulator of translation of target mRNAs 33 and Eif2ak3perk which phosphorylates and inactivates EIF2 leading to reduced global protein synthesis (Supplementary Table S5). In addition, 15 genes involved in ubiquitin-proteasome function increased in expression suggesting enhanced protein degradation in RN with age (Supplementary Table S5).
Sixteen genes encoding lysosomal proteins were upregulated with age including many known to play a role in autophagy ( Fig. 4; Supplementary Table S5). These include upregulation of Atg14 (Autophagy-related 14 or Becn1-associated) a key regulator of autophagy, Ubiquitin Specific Protease 36 (Usp36) that plays a role in activation of autophagy, phosphatidylinositol 3-phosphate-binding protein (Wdfy3) that functions as a master conductor for aggregate clearance by autophagy, as well as RB1-inducible coiled-coil 1 (Rb1cc), Tectonin Beta-Propeller Repeat Containing 1 (Tecpr1), Lysosomal Associated Membrane Protein 1 (Lamp1), and Estrogen-Induced Gene 121-Like Protein (Kiaa1324L) which regulate events of autophagosome formation and function, and autophagin-1 (Atg4hb) which encodes a cysteine protease required for autophagy.
Expression of 44 genes associated with the chemical "Defensome" was altered with age, including 20 upregulated and 24 downregulated ( Fig. 4; Supplementary Table S5). Among the upregulated genes were 10 members of the ATP-binding cassette transporters (2 AbcA, 3 AbcB, 4 AbcC, 1 AbcG family members) that transport various molecules across extra-and intra-cellular membranes and play an important role in efflux of xenobiotic compounds. Seven chaperones or co-chaperones were upregulated with age, including hypoxia upregulated 1 (Hyou1), which belongs to the heat shock protein 70 family and has a role in cytoprotective cellular mechanisms triggered by oxygen deprivation. Among the downregulated genes were several that play a role in xenobiotic metabolism, conjugation, and elimination including 6 members of the cytochrome P450 family, 2 UDP-glucuronosyltransferases, 2 sulfotransferases, 6 glutathione S-transferases, and an organic cationic transporter.
In the "Metabolism" category there were 84 upregulated and 47 downregulated genes including genes involved in amino acid, carbohydrate, and lipid metabolism, as well as a variety of other specialized metabolic functions ( Fig. 4; Supplementary Table S5). Genes involved in lipid metabolism including fatty acid, cholesterol, and sphingolipid metabolism were among the most affected with 28 upregulated and 9 downregulated. Six genes encoding amino acid transporters were upregulated with age, while 5 genes encoding monocarboxylic acid transporters were downregulated. Hypoxia Inducible Factor Prolyl 4-Hydroxlase (PH-4) was among the downregulated genes and Iodothyronine deiodinase type I (Dio1) showed the largest reduction in expression with age (−5.2 log2 fold-change). Iodothyronine deiodinase is important for regulation of thyroid hormones essential for growth, development and basal metabolism and is known to play a role in development and metamorphosis in sea urchins 34 . Reduced expression of iodothyronine deiodinase is consistent with the slowed growth and decreased metabolic rate that occurs in sea urchins with increase in size 19,[35][36][37] .
The expression of 52 genes associated with "Immunity" was altered with age (34 upregulated and 18 downregulated) ( Fig. 4; Supplementary Table S5). Among the upregulated genes there were 11 Scavenger Receptor Cysteine-Rich (SRCR), 7 NACHT and LRR containing proteins (NLR), 3 Toll-like receptors (TLR), 2 lectins, 2 macrophage MHC receptor 2-like genes, and 7 additional genes involved in innate immune cell signaling and antiviral response. Among the downregulated genes there were 6 carbohydrate binding lectins that play a role in recognition of pathogens, apoptotic and necrotic cells. Also downregulated were 5 genes encoding SRCR proteins, 1 TLR, 2 antimicrobial peptides, and amassin, a protein that facilitates clotting of coelomocytes.
Only 5 genes whose products play a role in "Apoptosis" were significantly altered with age ( Fig. 4; Supplementary Table S5). There was an upregulation of two isoforms of Apoptotic Protease-Activating Factor (Apaf), caspase-3 substrate Gas2 that alters the actin cytoskeleton during apoptosis, and an inhibitor of apoptosis (Kiaa0317L). Downregulated was the pro-apoptotic protein Interferon Alpha Inducible Protein 27 (Ifi27) that promotes INF-induced apoptosis.
Eleven genes in the "Biomineralization" category were downregulated with age in RN tissue ( Fig. 4; Supplementary Table S5). Whether these genes are expressed in nerve tissue or due to sclerocytes that co-isolated with the RN tissue was not investigated.

Ingenuity Pathway Analysis (IPA) of differentially expressed M. franciscanus Rn genes. IPA
was used as an additional approach to identify canonical pathways, networks, and functions associated with the age-related differentially expressed genes in sea urchin RN tissue [log2 fold-change < −2 or >2 level, q-value (FDR) < 0.05]. Human gene identifiers corresponding to the differentially expressed sea urchin RN genes were used as input for IPA pathway analysis (Supplementary Table S6). A total of 1,038 out of 1,042 input gene identifiers mapped in IPA and identified 22 significantly enriched pathways with age (BH FDR adj P-value < 0.05) ( Fig. 5; Supplementary Table S7). The pathways most affected included signaling pathways associated with nervous system function such as PKA, cAMP, GPCR, calcium, and synaptogenesis signaling. In addition, AMPK signaling, D-myo-inositol-tetrakisphosphate signaling, xenobiotic metabolism signaling, and EIF2 signaling were amongst the top 22 affected pathways.
Of the 22 pathways significantly affected by age, 10 had a z-score ≥ 2.0 or ≤ −2.0 suggesting overall activation or inhibition of the pathway, respectively (Supplementary Table S7). These included the following activated pathways: cAMP, synaptogenesis, Gαs, α-adrenergic signaling, and D-myo-inositol-tetrakisphosphate biosynthesis. The inhibited pathways include: EIF2 signaling and melatonin degradation pathways. Overall, the IPA results were consistent with those from curation of the differentially expressed genes into the custom sea urchin ontology groups, providing an additional validation of the results.

Comparison of age-related gene expression between sea urchin tissues and species. At the log2
fold-change level of < −1 and >1, 68 of the 144 differentially expressed genes in ALM, and 42 of the 123 genes in ES were also differentially expressed in RN (Supplementary Table S2; Supplementary Table S3). Only 3 genes were shared exclusively between ES and ALM and only 3 genes were shared between all 3 tissue types (Supplementary Table S3). Two of the common genes encode uncharacterized proteins (SPU_001941 and SPU_014904) and the third gene encodes Solute Carrier Family 16, Member 12-like-2 (Slc16a12L_2), a monocarboxylic acid transporter that mediates creatine transport across the plasma membrane, which showed decreased expression with age in all three tissue types.
The age-related pattern of gene expression in RN of the red sea urchin was compared to that previously reported for the purple sea urchin S. purpuratus 23  www.nature.com/scientificreports www.nature.com/scientificreports/ the opposite expression in M. franciscanus versus S. purpuratus RN. The common downregulated genes include Homeobox transcription factor (Hlx), HIF-Prolyl 4-Hydroxylase (Ph4), Parkin Coregulated Chaperone-Binding Protein (Pacrg), Collagen-Fibrillar V/XI (6Afcol), and Wnt1. The common upregulated genes were the transcriptional repressor Scratch, Prokineticin Receptor-2 (Prokr2), KIAA0391-protein-like that functions in mitochondrial tRNA maturation, and Syntaxin 1 A (Stx1) that plays a role in the docking of synaptic vesicle with the presynaptic plasma membrane. The limited number of overlapping genes that behave similarly with age in RN of these two sea urchin species may, at least in part, be the result of technical differences in the two approaches used to evaluate gene expression (microarray versus RNA-Seq) and the elimination of low expressors from the microarray analysis.

Discussion
It has been proposed that lifelong transcriptional stability is a key determinant of longevity and negligible senescence 24 . Consistent with this idea, muscle and esophagus tissue from the long-lived red sea urchin M. franciscanus showed a high level of transcriptional stability with a limited number of significant age-related changes. In contrast, radial nerve cord (RN) showed more than 3,000 age-related differentially expressed genes at a two-fold level, and 1,292 at a four-fold level, representing a broad range of cellular functions and pathways including nerve function, signaling, metabolism, and DNA maintenance. Altered expression of many genes involved in transcriptional regulation, epigenetic modification, chromatin structure and remodeling indicated a strong transcriptional response to aging in sea urchin RN tissue. Transcriptional studies on six species (entire organisms for C. elegans and Drosophila and brains of mice, rats, chimpanzees and humans) found an age-associated decrease in expression of genes involved in synaptic transmission, vesicular transport, calcium homeostasis, mitochondrial function and an induction of expression of genes associated with stress-response such as DNA repair, antioxidant defense and immune function 38,39 . The observation that many of the transcriptional changes observed in the red sea urchin nerve tissue differ from those observed in mammals and invertebrate model organisms suggests a novel age-related transcriptional program in these long-lived animals; however, whether the changes represent an orchestrated transcriptional program or are in response to stochastic events remains to be determined.
A loss of synaptic integrity and decline in synaptic gene expression is characteristic of the aging nervous system across phyla [39][40][41] . In the aging human frontal cortex, genes involved in synaptic plasticity that underlie learning and memory are among the most significantly affected 38 including the reduced expression of genes encoding subunits of ionotropic and metabotropic glutamate receptors as well as the GABA receptor 38 . Similar to other animals, the sea urchin RN showed an age-related decrease in expression of three AMPA glutamate receptor subunits. In contrast, there was an upregulation in the expression of several subunits of metabotropic glutamate receptors, ionotropic (kainate) glutamate receptors, and one AMPA glutamate receptor. In addition, there was an increase in expression of receptor subunits and regulators of GABA, glycine and acetylcholine neurotransmission as well as biogenic amine and neuropeptide neurotransmitters. Therefore, aging sea urchin neurons appear to be upregulating expression of numerous genes involved in neurotransmission, while downregulating specific subunits of AMPA, serotonin, and melatonin receptors.
In addition to neurotransmitters, there was increased age-related expression of numerous genes with a role in synaptogenesis, axonogenesis, and neuroprotection, including an upregulation of the nerve growth factor Neurotrophin and Neurotrophic Tyrosine Receptor Kinase associated with neuronal maintenance and survival. There was also modulation in expression of many genes involved in cytoskeletal dynamics and vesicular transport www.nature.com/scientificreports www.nature.com/scientificreports/ in aging sea urchin RN tissue. Many of the upregulated genes were involved in Golgi function, post-Golgi transport, secretory vesicle function, and synaptic vesicle cycling and localization. The cytoskeleton genes upregulated with age in sea urchin RN tissue are involved in maintenance of subcellular spatial organization and integrity, cell motility, and organelle positioning (e.g. ankyrin, spectrin) and also genes encoding molecular motors for intracellular transport of vesicles and organelles (e.g. kinesin, dynein). The neuronal cytoskeleton is required for axon formation and axonal transport but also provides the structural basis for several specialized axonal structures at the synapses essential for localizing the synaptic vesicles to the plasma membrane 42 . A number of neurodegenerative diseases have been linked to defective axonal cytoskeletal regulation 42 and the upregulation of key cytoskeletal genes in sea urchin nerve tissue suggests preservation of this aspect of nerve function with age.
Synaptic function and plasticity are dependent upon the regulation of neuronal calcium fluxes and calcium-mediated signaling pathways 39 . Calcium homeostasis and the synaptic calcium signaling system in the mammalian brain is affected by age with reduced expression of calmodulin 1 and 3, CAM kinase II and IV, calcineurin B, multiple protein kinase C isoforms, Na + /Ca 2+ exchangers, voltage gated calcium channels (VGCC), transient receptor potential TRP receptors, and plasma membrane calcium ATPase 39,43 . In the red sea urchin RN tissue, many genes associated with calcium signaling showed the opposite trend with upregulation of a plasma membrane ATPase, two VGCC, three Na/Ca exchangers, four TRP receptors, and two protein kinase C isoforms, suggesting that the red sea urchin is able to effectively modulate calcium homeostasis and calcium signaling pathways to avoid the age-related changes that are typically seen in humans and other animals.
Gene expression patterns indicated modulation of several genes associated with the AMPK and mTOR pathways which play an important role as master regulators of cellular metabolic homeostasis. These pathways respond to signals such as amino acid levels, bioenergetic status, hormone and growth factor signaling as well as oxidative stress, hypoxia, or DNA damage 44 . AMPK is a serine-tyrosine kinase that acts as the master metabolic regulator responding to cellular energy status (AMP/ADP:ATP ratio) to inhibit anabolic processes and stimulate catabolism 45 . mTOR forms two complexes, mTORC1 and mTORC2, which differ in their composition and downstream targets 46 . mTORC1 activity promotes protein, lipid, and nucleotide synthesis as well as mitochondrial energy production while inhibiting autophagy to support cell growth and proliferation 46 . mTORC2 plays a role in cytoskeletal organization and cell survival 32 . AMPK activation leads to inhibition of mTORC1 through direct and indirect mechanisms, and can lead to stimulation of mTORC2 32 . The predicted upregulation of AMPK and downregulation of mTORC1 in sea urchin RN tissue was supported by the observed downregulation in expression of a number of genes involved in protein synthesis as well as an upregulation in expression of genes involved in autophagy. Activation of AMPK or inhibition of mTORC1 by genetic, nutrient, or pharmacological manipulations increases longevity and confers protection against age-related pathologies to model organisms 45,[47][48][49] . The improved healthspan and increased lifespan are thought to be largely due to increased levels of autophagy which serves to recycle cellular components to meet the biosynthetic and energy demands of the cell. Autophagy is known to play a protective role against onset of neurodegeneration in animal models and its downregulation in normal aging is thought to contribute to development of chronic neurodegenerative diseases 40,44,50 . Modulation of AMPK and mTOR pathways resulting in decreased cell growth and proliferation, and increased autophagy as illustrated in the model shown in Fig. 6, may serve to mitigate the detrimental effects of aging in the sea urchin nervous system.
Protein folding in the ER is a critical step in the biogenesis of most secretory and transmembrane proteins and the ER stress response or unfolded protein response (UPR) plays a fundamental role in the maintenance of cellular protein homeostasis 51 . UPR is characterized by the induction of chaperones to assist in the refolding of proteins, degradation of misfolded proteins by the proteasome, and attenuation of protein translation which is mediated by the serine-threonine kinase PERK 51 . PERK phosphorylates the initiation factor eIF2α thereby reducing global translation while at the same time activating translation of selected messages. Aging is typically associated with a decline in expression and activity of key UPR components leading to the accumulation of misfolded proteins and protein aggregates 51 . It is therefore interesting that sea urchin RN tissue showed an upregulation in expression of several genes whose products play a role in the ER stress response (i.e. Eif2ak3perk, Mbr1, Ugcgl1, Txndc5, Trim13). This was accompanied by an increase in expression of genes whose products play a role in the ubiquitin-proteasome pathway and autophagy and a decrease in protein synthesis. There was also increased expression of many chaperones in the "Defensome" category. The upregulation of UPR is expected to act as a protective process alleviating ER stress, however; prolonged ER stress has been shown to result in inflammatory signaling while unmitigated and excessive stress leads to apoptosis 51 . In red sea urchin RN tissue, the immune gene profile does not support a pro-inflammatory response and very few genes associated with apoptosis were altered with age.
The predicted increase in AMPK signaling and concomitant decrease in mTORC1 signaling and decreased global protein synthesis in aging sea urchin nerve tissue raises an important question. Synaptic function and plasticity rely on the expression of new proteins, and mTOR function has been reported to be critical to synaptic plasticity 52 . The solution to this problem may lie in the fact that there is localized protein translation in neurons. Localized protein synthesis is now well documented at synapses in both vertebrates and invertebrates (i.e. Aplysia) leading to the intriguing possibility that while overall protein synthesis is reduced via suppression of mTOR (resulting in the general benefits that promote longevity), local protein synthesis at the synapses may confer plasticity and preservation of neuronal function. The localized translation at synapses is accomplished by transporting mRNAs out of the nucleus where they bind to RNA-binding proteins. These RNP complexes are then translocated along microtubules via motor proteins to synapses where the mRNA is made available to the local translation machinery 53 . Further, it has been shown that Netrin1 plays a key role in promoting translation at sites of synaptic contact or synaptic stimulation 54 . Interestingly, one of the first players identified in this local protein translation was Microtubule-Associated Protein 2 (MAP2), the corresponding transcript for which was upregulated with age in sea urchin RN tissue. In addition, there was increased expression of molecular motors www.nature.com/scientificreports www.nature.com/scientificreports/ (kinesin, dynein), genes that encode proteins that play a role in export of mRNA from the nucleus (Tpr, Ranbp3, Nup210), and Netrin-1 in sea urchin RN tissue. This mechanism may provide a means to locally establish, maintain, and modify the synaptic proteome with age in the red sea urchin nerve tissue.
It has been proposed that the nervous system may act as a central regulator of aging by coordinating the physiology of extraneural tissues 40,55 . In support of this, manipulating longevity genes in the central nervous system of model organisms such as worms, flies, and mice is sufficient to counteract the effects of aging at a systemic level 55 . Promoting autophagy in the nervous system of adult Drosophila not only prevents the age-dependent accumulation of damage in neurons but also leads to increased lifespan 56,57 . It is therefore tempting to speculate that beneficial changes in the nervous system of the red sea urchin may have more widespread benefits for other issues and the organism as a whole.
There are several shortcomings of this study that should be considered in evaluating the outcome. This study only compares two time points during the life span of the sea urchins and therefore may be missing potentially important trends that occur over the life of these animals. In addition, the maximum life span potential of this animal is not known and therefore we cannot definitely say that the oldest animals are approaching their maximum limit. For each tissue, RNA was pooled from the young or old sea urchins for each of the respective collections prior to RNA-Seq analysis and interindividual variation was only assessed in a small number of genes used for qRT-PCR. It has been reported that pooling of samples can be beneficial when differential expression is the sole objective of the study and/or when biological variation is high relative to measurement error 58 , as is often the case with wild caught animals. Using the whole radial nerve cord, does not provide information about different cell types in the nervous system; a shortcoming that could be addressed with single-cell transcriptomics. Finally, one must consider that gene expression does not always correlate with protein production and function and so inferences about function based on gene expression should be viewed with caution.
The age-related pattern of gene expression in the nervous system of the red sea urchin tissues was unique compared to humans and short-lived model animals; however, there was some overlap in gene expression between different tissues and species of long-lived sea urchins. The underlying factors that drive the observed changes in gene expression in the red sea urchin remain to be identified, and it is unclear whether the changes represent a predetermined sequence of programmed transcriptional regulation or a transcriptional response to stochastic changes that occur over time. The unique age-related gene expression profile, including altered expression of a number of transcriptional regulators and chromatin modifiers, hints to a negligible senescence promoting program. However, this assertion requires verification, and functional verification is required to determine if the observed changes in gene expression act to preserve tissue function and mitigate aging in these animals. . Each year, 6 small animals (<5 cm test diameter) and 4-6 large animals (>15 cm test diameter) were collected. Following collection, sea urchins were left to acclimate in an outdoor, flow-through aquarium at the Pacific Biological Station in Nanaimo, BC for about one week to ensure all animals were exposed to a uniform environment prior to dissection. Muscle tissue from Aristotle's lantern (ALM), esophagus (ES) and radial nerve cord (RN) were dissected from the sea urchins and immediately immersed in RNA later solution (Qiagen, Valencia, CA) and frozen at −80 °C.
RNA was extracted from the tissues using Trizol reagent (Invitrogen/Life Technologies, Carlsbad, CA) followed by the RNA clean-up protocol of the RNeasy mini kit (Qiagen) with a 15 minute DNase I digestion step. Total RNA concentrations were determined by measuring absorbance at 260 nm using an Agilent 8453 Spectrophotometer (Agilent, Santa Clara, CA) and RNA integrity was confirmed by electrophoresis on 1% formaldehyde agarose gels. Figure 6. Schematic representation of the predicted activation of Lkb1, AMPK, Ulk1, and mTORC2 and inhibition of mTORC1 in sea urchin radial nerve tissue inferred from the age-related differential gene expression. Predicted increased activity is depicted in blue while predicted inhibition of activity is depicted in red with the predicted outcomes being increased autophagy, cytoskeletal rearrangement, and cell survival and decreased protein synthesis and mitochondrial energy metabolism.
Function and canonical pathway analysis. The age-related differentially expressed genes were manually curated into functional groups using the custom-built sea urchin gene ontology major functional classes 27 with the following modifications: DNA replication, histone, transcription factor, cell cycle and ZNF were combined into one category called "DNA/RNA metabolism", the categories of adhesion and metalloprotease were combined into one category called "Adhesion/ECM", and GPCR Rhodopsin, GTPase, kinase, phosphatase, and signaling were combined into one category called "Signaling". Three new categories relevant to aging biology "Mitochondria", "Autophagy", "Protein homeostasis" were added to this classification for a total of 15 major functional classes. Any gene predictions that were not assigned an identity in EchinoBase were subjected to BLAST searches of the NCBI Reference Sequences (RefSeq) collection in an attempt to assign an identity for further functional characterization, and those for which there was no significant match or have no characterized function, were classified as "uncharacterized".
Ingenuity Pathway analysis (IPA; QIAGEN Inc., Redwood City. CA) was used to identify significant networks, functions, and canonical pathways altered with age in sea urchin radial nerve tissue 59 . For input into IPA analysis, the human ENSEMBL identifiers with highest homology to the sea urchin protein sequences were selected using BLASTP (Supplementary Table S6). P-values provided by IPA were adjusted to account for multiple testing using the Benjamini-Hochberg FDR method.
Verification of differentially expressed genes using quantitative RT-PCR. Differential expression of selected genes was verified using quantitative reverse-transcription polymerase chain reaction (qRT-PCR) on the ABI 7300 Real Time-PCR machine using the SYBR Green detection system (Applied Biosystems, Foster City, CA, USA). RNA from individual sea urchin tissues was reverse transcribed using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). Primers for qRT-PCR were designed using Primer Express software (version 3.0) (Applied Biosystems) based on sequences for M. franciscanus selected using the annotated S. purpuratus genes blasted against M. franciscanus short sequence reads in NCBI (accession SRX000089) and synthesized by Bio-Synthesis (TX, USA) (Supplementary Table S9). The expression level of genes was determined using the delta-delta-CT method and normalized using a normalization factor derived from the geometric mean of expression of three stably expressed control genes 60 . Up to 10 control genes (mAct, GAPDH, cyclophilin7, profilin, HPRT, ATub5, PARP1, RPL8, PCNA, B-tubulin2) were tested and the gene stability measure (M) was determined to select the three most stable control genes for radial nerve cord (ATub5, HPRT, PARP1) and muscle (PCNA, RPL8 and B-tubulin2), with M values <0.4 60,61 . No-template-controls were included on each reaction plate to check for contamination and negative controls consisting of untranscribed RNA (No-RT controls) were performed for each RNA extraction to check for genomic DNA contamination. All verifications were performed using RNA from the tissues of small (young) and large (old) individual sea urchins from the 2010 collection, the same RNA that was used for RNA-Seq analysis.

Data availability
The RNA-Seq data have been deposited in NCBI's Gene Expression Omnibus (GEO) and Sequence Read Archive (SRA). All data are associated with BioProject accession PRJNA562282. The raw sequence FastQ files can be found in SRA project SRP219667 (BioSamples: SAMN12637227-SAMN12637246). DGE count tables and transcript assembly are accessible through GEO Series accession number GSE136352 (https://www.ncbi.nlm. nih.gov/geo/query/acc.cgi?acc=GSE136352). All additional data is available in the Supplementary information.