Proteomic mapping of Drosophila transgenic elav.L-GAL4/+ brain as a tool to illuminate neuropathology mechanisms

Drosophila brain has emerged as a powerful model system for the investigation of genes being related to neurological pathologies. To map the proteomic landscape of fly brain, in a high-resolution scale, we herein employed a nano liquid chromatography-tandem mass spectrometry technology, and high-content catalogues of 7,663 unique peptides and 2,335 single proteins were generated. Protein-data processing, through UniProt, DAVID, KEGG and PANTHER bioinformatics subroutines, led to fly brain-protein classification, according to sub-cellular topology, molecular function, implication in signaling and contribution to neuronal diseases. Given the importance of Ubiquitin Proteasome System (UPS) in neuropathologies and by using the almost completely reassembled UPS, we genetically targeted genes encoding components of the ubiquitination-dependent protein-degradation machinery. This analysis showed that driving RNAi toward proteasome components and regulators, using the GAL4-elav.L driver, resulted in changes to longevity and climbing-activity patterns during aging. Our proteomic map is expected to advance the existing knowledge regarding brain biology in animal species of major translational-research value and economical interest.

High-resolution proteοmics: peptide generation -nLC-MS/MS. Total-protein extracts were prepared from 100 D. melanogaster whole-brains, having been isolated, via manual dissection, from single-transgenic elav.L-GAL4/+ young adult (2-3-day-old) female flies. Protein extraction and processing were carried out as previously described 37 . Generated peptides were analyzed using an LTQ Orbitrap Elite instrument (Thermo Scientific, IL, USA), with the mass spectrometer being coupled to a Dionex Ultimate 3000 HPLC system. The extracted ion chromatogram was further processed using the Proteome Discoverer software (Thermo Scientific, IL, USA) and the Sequest search engine. The database chosen for protein identification searches was the D. melanogaster reference proteome, directly downloaded from UniProt 2.16 resource, without further modifications. Identification criteria included a precursor-mass tolerance of 10 ppm and fragment-mass tolerance of 0.05 Da. Trypsin was selected as the cleavage enzyme, with a maximum of "0" missed-cleavage parameter. A false-discovery rate-threshold of 0.5% ensured the reliability of protein identification procedure.
Longevity measurement. Populations of 20-25 flies (males and females in separate vials) were thoroughly analyzed in terms of their respective longevities. Survival curves were generated by daily counting the number of deceased flies. The results of each viability experiment consisted of at least 100 flies, from three different fly crosses, were statistically analyzed. All viability experiments were performed at the same time for control and RNAi-downregulated strains.
Intriguingly, ubiquitous activation of RNAi machinery, through GeneSwitch system, has been previously reported to result in RNAi sequence-independent side-effects on Drosophila lifespan under aging 38 . However, RNAi expression restricted to certain tissues may not be detrimental to lifespan 38 . In accordance, we have recently shown, through engagement of the elav.L-GAL4/UAS and RNAi genetic platforms, that neuronal cell-specific targeting of CCS (copper chaperone for SOD1) gene affected climbing activity of Drosophila female flies during aging but not their longevity 39 , therefore suggesting that -at least-in this system the activation of RNAi machinery alone cannot affect aging. (A) Sub-cellular-topology classification of 2,335 fly-brain proteins having been obtained and identified via engagement of a high-resolution nLC-MS/MS proteomics technology. (B) Fly-brain proteome categorization in "Biological Processes" and "Molecular Functions". (C) Major "Signaling Pathways" having been assembled in the fly-brain setting. Due to functional overlaps, a number of proteins are categorized in more than one group. (A,B) DAVID v.6.8 -Gene Ontology subroutine. (C) DAVID v.6.7 -PANTHER. a line drawn at the 66 ml (2/3) mark. Next, flies were gently tapped to the bottom of the cylinder, in order to start climbing (against gravity) all together. The number of flies that reached above the 66 ml mark, after a 20 sec time-period, was recorded. The assay was repeated 10 times for each group, allowing for 1 min rest-period in between 2 successive trials. Results were, then, converted to percentages, and the average pass rate per genotype and time point was computed. For each examined genotype, at least 5 different groups, from independent genetic crosses, per time-point were analyzed. Comparisons between control and RNAi-targeted fly-groups were carried out at the same time.
Statistical analysis. Statistical analysis was performed using the Statistical Package for Social Sciences (IBM SPSS v23.0 for Windows IBM Corp., NY, USA). For climbing assays, the results were presented graphically as an average pass rate per genotype/time-point with sample standard deviation (±SSD) value. Differences between compared genotypes were evaluated using the independent t-test analysis. All data from lifespan experiments were analyzed with the Kaplan-Meier survival test, using log rank and Breslow test statistics. Significance was accepted at p < 0.05 (*) and p < 0.01 (**).

Results and Discussion
High-resolution mapping of Drosophila melanogaster brain proteome: organelle compartmentalization and functional dissection of the mapped proteins. The elav.L-GAL4 is a strong neuronal cell-specific driver [51][52][53] , routinely used for gene-function analysis studies in Drosophila nervous system. Protein extracts from 100 manually dissected brains, derived from elav.L-GAL4 heterozygote D. melanogaster female flies (elav.L-GAL4/+) of 2 to 3 days old, were processed through a high-resolution nLC-MS/MS proteomics approach, and 7,663 unique peptides and 2,335 single proteins were identified (Supplementary Table 1). Out of this large collection of fly-brain proteins, 777 were classified to reside in the cell membranes (including external and internal ones), 538 in the nucleus (including nucleolus), 120 in the extracellular region and 1,124 in the cytoplasm (Fig. 1A). Furthermore, regarding cytoplasmic proteins, a number of organelle-related ones were recognized in the mitochondrion (n = 287), vesicle (n = 91), endoplasmic reticulum (n = 83), ribosome (n = 66), Golgi apparatus (n = 63), peroxisome (n = 27) and lysosome (n = 19) (Fig. 1A).
In an effort to reconstruct the fundamental pathways that critically control the physiology of neuronal cells, we analyzed Drosophila brain proteome via the PANTHER-pathway classification system being utilized through DAVID 6.7 bioinformatics resource (Fig. 1C). Proteins that belong to major signaling pathways and processes, such as axon guidance (n = 12) and angiogenesis (n = 25), which are crucial in neuronal-wiring mechanisms during brain development, were recognized. Pathways numerically enriched with brain proteins embrace the Wnt (n = 35), the integrin (n = 31), the FGF (n = 21), the EGF (n = 20) and the heterotrimeric G-protein pathway (n = 29). Interestingly, dopamine pathway, which besides the synthesis of neurotransmitter dopamine is also involved in functions such as learning, reward and motivation, is presented with 16 proteins (Fig. 1C). www.nature.com/scientificreports www.nature.com/scientificreports/ The employment of liquid chromatography coupled with tandem-mass spectrometry has been previously employed to uncover the Drosophila head proteome of wild type flies 54 . In that study, a total number of 4,812 proteins were detected in an enriched membrane fraction of fly heads. More recently, Kuznetsova and collaborators analyzed the Drosophila brain proteome in a mixed population of equal male and female wild type flies of different ages, and identified 4,005 proteins 55 . Since the protein dataset of Kuznetsova and collaborators 55 has been obtained from fly brains, albeit from diverse proteomic backgrounds, we, next, proceeded to brain proteome comparison, using UniProt accession and Flybase ID numbers, revealing approximately 1,727 shared proteins (Fig. S1). Without excluding technical differences of the employed protocols, the 608 unique proteins of our study may indicate an effect of GAL4 activity on the regulation of their expression. Orange-colored boxes: proteins of the present study identified in the fly-brain proteome. Green-colored boxes: proteins that were absent from our study (for either technical or biological reasons). White boxes: proteins that are completely missing from Drosophila melanogaster proteome.
Next, and since over 50% of fly genes show sequence homologies to human genes and approximately 75% of all known human-disease genes are believed to have functional fly homologues 1,3,7-9,56 , the identification in Drosophila brain proteome of proteins related to human pathologies was expected. Specifically, Drosophila brain protein-homologues of human ones associated with Parkinson's disease (n = 26), Huntington's disease (n = 26), Alzheimer's disease (n = 20) and chemokine/cytokine-mediated inflammation (n = 33) were recognized (Fig. 2).  Deregulation of UPS affects the length and number of axons and dendrites, while it also leads to deficient synaptic function that is believed to be an early effect of neurodegenerative diseases, rendering UPS an important pharmacological target 4,20,[57][58][59][60][61][62] . Thereby, we focused on proteins related to UPS components and regulators. A total of 27 proteins belonging to the proteasome complex were identified in our proteomic database, 13 of which were presented in the 20S core and the remaining 14 in the 19S regulatory complex (9 to base and 5 to lid sub-complex) (Fig. 3A). Additionally, 15 proteins being associated with the ubiquitin tagging of substrate-targets destined for proteasomal degradation were also recognized. Among them, 9 proteins were linked to cullin-RING E3 ubiquitin ligases (Fig. 3A), the largest superfamily of E3 ubiquitin ligases. Notably, molecular reconstruction of the 26S Drosophila brain proteasome through KEGG-pathway maps resulted in a rather complete protein model of both 20S and 19S proteasome complexes (Fig. 3B).
Targeted disruption of 26S proteasome components in Drosophila neuronal tissues results in severe lifespan reduction and kinetic pathology. Based on the importance of UPS integrity to neuropathologies, we, next, investigated the effects of proteasome impairment in Drosophila neuronal cells during aging, with the use of GAL4/UAS genetic platform. GAL4/UAS, the most widely used system in Drosophila for achieving ectopic gene expression, allows the selective activation of any cloned gene or RNAi in a wide variety of tissue-and cell-specific patterns. Briefly, in one Drosophila line the GAL4 transcriptional activator of the yeast is introduced into its genome under the control of a cell/tissue-specific endogenous promoter, while in another line the target transgene or RNAi is cloned downstream of a UAS sequence. In the progeny of a cross between these lines, the target gene/RNAi of interest is expressed in the same cell/tissue-specific pattern as the GAL4 activator [63][64][65] . Hence, we genetically targeted critical 26S-proteasome subunits, using the neuronal-specific elav.L-GAL4 driver. Specifically, we examined representative subunits from both 19S-RP and 20S-CP, one of which (β5) carries proteolytic activity.
According to previous reports, Rpn2 serves as scaffolding subunit of the 19S proteasome-base ring and together with Rpn1 comprise the two largest proteasome subunits of RP, which are both necessary for the coordinate functions of substrate shuttles, ubiquitin receptors and deubiquitinating enzymes [66][67][68] . After downregulating the Rpn2 subunit, specifically in neuronal cells, using the RNAi-based technology, a reduction of approximately 20 days in the viability of both male and female flies was observed at their 50% survival rate, as compared to control populations (Fig. 4A,B). Likewise, their climbing activity, especially in male flies, was also negatively affected, following an age-dependent declining pattern (Fig. 5A,B). Altogether, Rpn2 seems to represent an essential www.nature.com/scientificreports www.nature.com/scientificreports/ proteasome subunit controlling neuronal proteostasis. These findings are strongly supported by a previous report from our laboratory, describing the development of distinct, dysmorphic phenotypes in Drosophila eye and wing, in response to RNAi-mediated downregulation of Rpn1 or Rpn2 proteasome subunit 69 .
We, next, proceeded to investigate the role of outer α-rings of 20S-CP in normal nervous system. These are comprised of 7 different α subunits (α1-7), which provide the binding sites for 19S-RP and form the molecular gate responsible for regulating substrate entrance into the inner catalytic cavity of β-rings 28,70 . Flies with reduced levels of α5 proteasome subunit, specifically in neuronal cells, of both sexes, managed to survive only for few days (Fig. 4C,D), while they also presented dramatically diminished climbing activities (Fig. 5C,D), thus underscoring the indispensable contribution of α5 subunit to nervous-system physiological development and function in Drosophila. Similarly to Rpn2, the tissue-specific downregulated expression of α5 subunit in Drosophila has also been previously associated with developmentally malformed eyes and wings 69 . Interestingly, the critical role of α5 subunit in proteasomal activity has been demonstrated in yeast, with α5 mutations (such as the substitution of an α-pocket lysine residue) giving rise to proteasome holoenzymes that carry immature β subunits and reduced peptidase activities 71 . Strikingly, mutations in α5 subunit of Saccharomyces cerevisiae have proved to notably affect its lifespan and revealed that the 20S-CP gate opening is directly controlled by the α5 subunit 72 .
The inner β-rings are composed of the inactive (unable to hydrolyze) β3, β4, β6, and β7 subunits, and the active β1, β2 and β5 subunits, which contain proteolytic sites of different specificities. The β5 subunit with a chymotrypsin-like activity hydrolyzes proteins after hydrophobic amino acid residues and carries the major proteasome-related proteolytic activity 33,73 . We herein show that male and female flies being depleted of either β5 (Fig. 4E,F) or β6 (Fig. 4G,H) proteasome subunit, specifically in neuronal tissues, are presented with a life expectancy of only few days. Furthermore, they are characterized by total climbing (kinetic) deficiency, given their (both sexes) inability to pass the specified cylinder mark during the respective climbing assay (Fig. 5E-H). These observations are in line with previous ones, describing flies that bear a dominant-negative mutation in the β6 gene, which die as undifferentiated pupae with failures in head eversion at a restrictive temperature 74 . This mutation has been also reported to alter cell-fate determination in the adult Drosophila sense-organ lineage 75 . It has been previously proposed that a deficient β6-proteasome subunit may disrupt proteasomal www.nature.com/scientificreports www.nature.com/scientificreports/ activity by altering the β2-β6 interfacing regions 75,76 . Importantly, previous studies have associated β5 and β6 proteasome-subunit deregulation with disrupted Drosophila eye and wing development 69,77,78 . In accordance to our findings, data from transgenic mice have shown that reduction in β5-associated chymotrypsin-like activity leads to multiple early-aging phenotypes and shortened lifespan 79 . On the contrary, overexpression of β5 subunit can improve proteostasis during aging and can increase longevity in Drosophila 80 . Ιt seems that certain thresholds of 26S-proteasome activities are required for Drosophila nervous-system physiological development and function.

RNAi-mediated downregulation of the E2 ubiquitin-conjugating enzyme UbCD1 specifically in
Drosophila neuronal tissues compromises longevity and climbing activity. Ubiquitination of substrates destined for proteasomal degradation depends on the sequential actions of ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s) and ubiquitin-ligases (E3s), which mediate the final transfer of ubiquitin to the selected substrates 81 . E2s contain a highly conserved 150-200 amino acid ubiquitin-conjugating catalytic-core domain and are involved in the transfer of ubiquitin or ubiquitin-like proteins to downstream conjugation targets. E2s are responsible for the selection of correct modifier, the determination of length, type and topology of attached ubiquitin chain, and the processivity of chain-assembly reaction, thereby defining the fate assignment of the modified protein 73,[81][82][83][84] . It has recently emerged that E2s, besides their typical role in protein degradation, are also involved in processes controlling protein function, sorting and localization, while their aberrant regulation has been associated with many disease pathways 81,85 . Among them, UbcD1, or effete, encodes a highly conserved E2 enzyme, with approximately 80% sequence identity to the yeast Ubc4/Ubc5 homologue, which mediates the selective protein-ubiquitination and degradation processes 86 . UbcD1 has been also associated with proper telomere behavior during cell division 87 , Hedgehog signaling by controlling Ci stability 88 , female germline stem-cell maintenance 89 , and regulation of apoptosis through its involvement in the poly-ubiquitination and degradation of DIAP1 90,91 . UbcD1 has been directly associated with Drosophila SCF complex of proteasome-dependent proteolysis [92][93][94] and, as it is illustrated in Fig. 3A, the cullin-RING E3 ligase (SCF) complex can be readily detected in Drosophila nervous system. Therefore, in an in vivo attempt to prove the importance of neuronal-specific UbcD1 targeting to the lifespan and climbing activity of D. melanogaster during aging, we genetically downregulated its expression using the elav.L-GAL4 and UbcD1_RNAi transgenic flies. UbcD1-targeted male (Fig. 4I) and female (Fig. 4J) flies were presented with a maximum life expectancy of only 20 days, while their vast majority were dying within the first 10 days. Notably, both male (Fig. 5I) and female (Fig. 5J) fly populations appeared with completely impaired climbing activities, since none of the tested flies proved capable to climb over the specified cylinder mark. Similarly, another study has reported that among 16 genes encoding E2 enzymes, only the RNAi-mediated UbcD1 targeting resulted in neuroblast overgrowth 94 . protein deubiquitination deregulation in neuronal cells during Drosophila aging. Ubiquitination, the post-translational modification of proteins by ubiquitin, can be reversed by deubiquitinating enzymes (DUBs), a specific family of proteases that catalyze the removal of ubiquitin (or ubiquitin-like) molecules from target substrates, modulating protein stability and signaling 31,33 . DUBs are divided into 6 structurally distinct subfamilies based on the architecture of their catalytic domains and mechanisms of action 31,33,95,96 . In the human genome, approximately 100 DUBs are encoded, while in the fly there have been identified around 45-family members 31,95,97,98 . DUBs serve as key regulators of several intracellular processes, such as NFκB activation (USP11 and 15), c-Myc stability (USP28), p53 stability (USP2a, 7 and 10), DNA-damage response (USP1) and apoptosis (USP7 and 28) 27,31,95,96,99 . Aberrant expression patterns and mutant forms of DUBs have been implicated in pathogenesis of several human diseases, including cancer and neurological pathologies 31,95,96,98 , and, as such, DUBs have emerged to represent attractive drug targets for pharmacological intervention strategies 27,29,99 .
Ubiquitous suppression of most DUBs in Drosophila has been associated with severe deregulation of fly development, motility and longevity 98 . Hence, in an effort to in vivo examine the significance of deubiquitination process in neuronal tissues during Drosophila aging, we herein generated transgenic flies carrying downregulated expression of the dUBP64 protease (a deubiquitinating enzyme). Usp47, which is the human ortholog of the fly dUBP64 deubiquitinating enzyme, has been found to interact with the E3 ubiquitin ligase SCF 100 and to play a key role in the control of axonal growth during neuronal development 101 , while its depletion from different cell lines has been associated with decreased cell survival 100 . In Drosophila, dUBP64 functions as a modifier of position-effect variegation 102 and controller of cell-fate decision during eye development, by regulating the transcriptional repressor tramtrack 103 .
In contrast to male transgenic flies (Fig. 6A), female populations, at their 50% survival rate, were presented with a reduction of approximately 40 days in their viability, as compared to control populations (Fig. 6B). Moreover, both male and female dUBP64-targeted flies were characterized by similar to controls climbing activities (Fig. 7A,B). Similar fly-sex-specific differential longevity profiles have been also reported in response to pathogen infection and altered mitochondrial dynamics 104,105 . Furthermore, to investigate the effects of DUB overexpression in lifespan and climbing activity, transgenic flies overexpressing the -yeast-UBP2 protease (a deubiquitinating enzyme), specifically in neuronal tissues, were generated through utilization of the elav.L-GAL4 driver. Both male (Fig. 6C) and female (Fig. 6D) transgenic flies were presented with significantly reduced viabilities and climbing activities (Fig. 7C,D), as compared to control populations. It seems that UBP2 overexpression critically compromises Drosophila longevity and climbing activity, and that neuronal cells are unable to counterbalance, or neutralize, the proteotoxic stress being induced by UBP2 abundance. Ubiquitination pathway is intrinsically required for the regulation of synaptic growth and function in Drosophila, with the -yeast-UBP2 deubiquitinating enzyme overexpression specifically in nervous system leading to synaptic overgrowth and defect in neurotransmitter release 106 . Overexpression of UBP2 has been also associated with synaptic-elimination delay in postsynaptic muscles 107 . Axon and dendrite pruning are fundamental for development of proper circuitry in