BACKGROUND: In fly brains, the Drosophila Adar (adenosine deaminase acting on RNA) enzyme edits hundreds of transcripts to generate edited isoforms of encoded proteins. Nearly all editing events are absent or less efficient in larvae but increase at metamorphosis; the larger number and higher levels of editing suggest editing is most required when the brain is most complex. This idea is consistent with the fact that Adar mutations affect the adult brain most dramatically. However, it is unknown whether Drosophila Adar RNA editing events mediate some coherent physiological effect. To address this question, we performed a genetic screen for suppressors of Adar mutant defects. Adar5G1 null mutant flies are partially viable, severely locomotion defective, aberrantly accumulate axonal neurotransmitter pre-synaptic vesicles and associated proteins, and develop an age-dependent vacuolar brain neurodegeneration. RESULTS: A genetic screen revealed suppression of all Adar5G1 mutant phenotypes tested by reduced dosage of the Tor gene, which encodes a pro-growth kinase that increases translation and reduces autophagy in well-fed conditions. Suppression of Adar5G1 phenotypes by reduced Tor is due to increased autophagy; overexpression of Atg5, which increases canonical autophagy initiation, reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar mutant phenotypes tested. Endosomal microautophagy (eMI) is another Tor-inhibited autophagy pathway involved in synaptic homeostasis in Drosophila. Increased expression of the key eMI protein Hsc70-4 also reduces aberrant accumulation of synaptic vesicle proteins and suppresses all Adar5G1 mutant phenotypes tested. CONCLUSIONS: These findings link Drosophila Adar mutant synaptic and neurotransmission defects to more general cellular defects in autophagy; presumably, edited isoforms of CNS proteins are required for optimum synaptic response capabilities in the brain during the behaviorally complex adult life stage.

CEITEC Masaryk University Kamenice 735 5 A35 CZ 62 500 Brno Czech Republic

Centre for Integrative Physiology Euan MacDonald Centre for Motor Neurone Disease Research Hugh Robson Building University of Edinburgh George Square Edinburgh EH8 9XD UK

MRC Human Genetics Unit Institute of Genetics and Molecular Medicine at the University of Edinburgh Crewe Road Edinburgh EH4 2XU UK

National Centre for Biomolecular Research Faculty of Science Masaryk University Kamenice 5 625 00 Brno Czech Republic

See more in PubMed

Keegan LP, McGurk L, Palavicini JP, Brindle J, Paro S, Li X, Rosenthal JJ, O'Connell MA. Functional conservation in human and Drosophila of Metazoan ADAR2 involved in RNA editing: loss of ADAR1 in insects. Nucleic Acids Res. 2011;39(16):7249–7262. doi: 10.1093/nar/gkr423. PubMed DOI PMC

Keegan L, Khan A, Vukic D, O'Connell M. ADAR RNA editing below the backbone. Rna. 2017;23(9):1317–1328. doi: 10.1261/rna.060921.117. PubMed DOI PMC

Sommer B, Köhler M, Sprengel R, Seeburg PH. RNA editing in brain controls a determinant of ion flow in glutamate-gated channels. Cell. 1991;67:11–19. doi: 10.1016/0092-8674(91)90568-J. PubMed DOI

Higuchi M, Maas S, Single F, Hartner J, Rozov A, Burnashev N, Feldmeyer D, Sprengel R, Seeburg P. Point mutation in an AMPA receptor gene rescues lethality in mice deficient in the RNA-editing enzyme ADAR2. Nature. 2000;406:78–81. doi: 10.1038/35017558. PubMed DOI

Hogg M, Paro S, Keegan LP, O'Connell MA. RNA editing by mammalian ADARs. Adv Genet. 2011;73:87–120. doi: 10.1016/B978-0-12-380860-8.00003-3. PubMed DOI

Palladino MJ, Keegan LP, O'Connell MA, Reenan RA. A-to-I pre-mRNA editing in Drosophila is primarily involved in adult nervous system function and integrity. Cell. 2000;102(4):437–449. doi: 10.1016/S0092-8674(00)00049-0. PubMed DOI

Chen L, Rio DC, Haddad GG, Ma E. Regulatory role of dADAR in ROS metabolism in Drosophila CNS. Brain Res Mol Brain Res. 2004;131(1–2):93–100. doi: 10.1016/j.molbrainres.2004.08.013. PubMed DOI

Ma E, Gu XQ, Wu X, Xu T, Haddad GG. Mutation in pre-mRNA adenosine deaminase markedly attenuates neuronal tolerance to O2 deprivation in Drosophila melanogaster. J Clin Invest. 2001;107(6):685–693. doi: 10.1172/JCI11625. PubMed DOI PMC

Stapleton M, Carlson JW, Celniker SE. RNA editing in Drosophila melanogaster: new targets and functional consequences. Rna. 2006;12(11):1922–1932. doi: 10.1261/rna.254306. PubMed DOI PMC

Graveley BR, Brooks AN, Carlson JW, Duff MO, Landolin JM, Yang L, Artieri CG, van Baren MJ, Boley N, Booth BW, et al. The developmental transcriptome of Drosophila melanogaster. Nature. 2011;471(7339):473–479. doi: 10.1038/nature09715. PubMed DOI PMC

St Laurent G, Tackett M, Nechkin S, Shtokalo D, Antonets D, Savva Y, Maloney R, Kapranov P, Lawrence C, Reenan R. Genome-wide analysis of A-to-I RNA editing by single-molecule sequencing in Drosophila. Nat Struct Mol Biol. 2013;20:1333–1339. doi: 10.1038/nsmb.2675. PubMed DOI

Liscovitch-Brauer N, Alon S, Porath HT, Elstein B, Unger R, Ziv T, Admon A, Levanon EY, Rosenthal JJ, Eisenberg E. Trade-off between transcriptome plasticity and genome evolution in cephalopods. Cell. 2017;169(2):191–202. doi: 10.1016/j.cell.2017.03.025. PubMed DOI PMC

Sapiro AL, Shmueli A, Henry GL, Li Q, Shalit T, Yaron O, Paas Y, Li JB, Shohat-Ophir G. Illuminating spatial A-to-I RNA editing signatures within the Drosophila brain. Proc Natl Acad Sci. 2019;116(6):2318–2327. doi: 10.1073/pnas.1811768116. PubMed DOI PMC

Yablonovitch AL, Deng P, Jacobson D, Li JB. The evolution and adaptation of A-to-I RNA editing. PLoS Genet. 2017;13(11):e1007064. doi: 10.1371/journal.pgen.1007064. PubMed DOI PMC

Palladino MJ, Keegan LP, O'Connell MA, Reenan RA. dADAR, a Drosophila double-stranded RNA-specific adenosine deaminase is highly developmentally regulated and is itself a target for RNA editing. RNA. 2000;6:1004–1018. doi: 10.1017/S1355838200000248. PubMed DOI PMC

Hanrahan CJ, Palladino MJ, Bonneau LJ, Reenan RA. RNA editing of a Drosophila sodium channel gene. Ann N Y Acad Sci. 1998;868:51–66. doi: 10.1111/j.1749-6632.1999.tb11273.x. PubMed DOI

Keegan LP, Brindle J, Gallo A, Leroy A, Reenan RA, O'Connell MA. Tuning of RNA editing by ADAR is required in Drosophila. EMBO J. 2005;24(12):2183–2193. doi: 10.1038/sj.emboj.7600691. PubMed DOI PMC

Südhof TC. The presynaptic active zone. Neuron. 2012;75(1):11–25. doi: 10.1016/j.neuron.2012.06.012. PubMed DOI PMC

Kawasaki F, Zou B, Xu X, Ordway RW. Active zone localization of presynaptic calcium channels encoded by the cacophony locus of Drosophila. J Neurosci. 2004;24(1):282–285. doi: 10.1523/JNEUROSCI.3553-03.2004. PubMed DOI PMC

Diegelmann S, Nieratschker V, Werner U, Hoppe J, Zars T, Buchner E. The conserved protein kinase-A target motif in synapsin of Drosophila is effectively modified by pre-mRNA editing. BMC Neurosci. 2006;7:76. PubMed PMC

Hoopengardner B, Bhalla T, Staber C, Reenan R. Nervous system targets of RNA editing identified by comparative genomics. Science. 2003;301(5634):832–836. doi: 10.1126/science.1086763. PubMed DOI

Amoyel M, Anderson AM, Bach EA. JAK/STAT pathway dysregulation in tumors: a Drosophila perspective. Semin Cell Dev Biol. 2014;28:96–103. doi: 10.1016/j.semcdb.2014.03.023. PubMed DOI PMC

Bhogal B, Jepson JE, Savva YA, Pepper ASR, Reenan RA, Jongens TA. Modulation of dADAR-dependent RNA editing by the Drosophila fragile X mental retardation protein. Nat Neurosci. 2011;14(12):1517–1524. doi: 10.1038/nn.2950. PubMed DOI PMC

Maldonado C, Alicea D, Gonzalez M, Bykhovskaia M, Marie B. Adar is essential for optimal presynaptic function. Mol Cell Neurosci. 2013;52:173–180. doi: 10.1016/j.mcn.2012.10.009. PubMed DOI PMC

Robinson J, Paluch J, Dickman D, Joiner W. ADAR-mediated RNA editing suppresses sleep by acting as a brake on glutamatergic synaptic plasticity. Nat Commun. 2016;7:1–9. PubMed PMC

Hall MN. TOR and paradigm change: cell growth is controlled. Mol Biol Cell. 2016;27(18):2804–2806. doi: 10.1091/mbc.e15-05-0311. PubMed DOI PMC

Chang YY, Neufeld TP. Autophagy takes flight in Drosophila. FEBS Lett. 2010;584(7):1342–1349. doi: 10.1016/j.febslet.2010.01.006. PubMed DOI PMC

Ryder E, Ashburner M, Bautista-Llacer R, Drummond J, Webster J, Johnson G, Morley T, Chan YS, Blows F, Coulson D, et al. The DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency resource. Genetics. 2007;177(1):615–629. doi: 10.1534/genetics.107.076216. PubMed DOI PMC

Spradling AC, Stern D, Beaton A, Rhem EJ, Laverty T, Mozden N, Misra S, Rubin GM. The Berkeley Drosophila Genome Project gene disruption project: single P-element insertions mutating 25% of vital Drosophila genes. Genetics. 1999;153(1):135–177. PubMed PMC

Bellen HJ, Levis RW, Liao G, He Y, Carlson JW, Tsang G, Evans-Holm M, Hiesinger PR, Schulze KL, Rubin GM, et al. The BDGP gene disruption project: single transposon insertions associated with 40% of Drosophila genes. Genetics. 2004;167(2):761–781. doi: 10.1534/genetics.104.026427. PubMed DOI PMC

Anant S, Davidson NO. Molecular mechanisms of apolipoprotein B mRNA editing. Curr Opin Lipidol. 2001;12(2):159–165. doi: 10.1097/00041433-200104000-00009. PubMed DOI

Yasuyama K, Meinertzhagen IA, Schürmann FW. Synaptic organization of the mushroom body calyx in Drosophila melanogaster. J Comp Neurol. 2002;445(3):211–226. doi: 10.1002/cne.10155. PubMed DOI

Barnstedt O, Owald D, Felsenberg J, Brain R, Moszynski J-P, Talbot CB, Perrat PN, Waddell S. Memory-relevant mushroom body output synapses are cholinergic. Neuron. 2016;89(6):1237–1247. doi: 10.1016/j.neuron.2016.02.015. PubMed DOI PMC

Kitamoto T, Ikeda K, Salvaterra PM. Regulation of choline acetyltransferase/lacZ fusion gene expression in putative cholinergic neurons of Drosophila melanogaster. J Neurobiol. 1995;28(1):70–81. doi: 10.1002/neu.480280107. PubMed DOI

Li X, Overton IM, Baines RA, Keegan LP, O'Connell MA. The ADAR RNA editing enzyme controls neuronal excitability in Drosophila melanogaster. Nucleic Acids Res. 2014;42(2):1139–1151. doi: 10.1093/nar/gkt909. PubMed DOI PMC

Hay BA, Wolff T, Rubin GM. Expression of baculovirus P35 prevents cell death in Drosophila. Development. 1994;120(8):2121–2129. PubMed

Harris-Warrick RM. Ion channels and receptors: molecular targets for behavioral evolution. J Comp Physiol A. 2000;186(7–8):605–616. doi: 10.1007/s003590000133. PubMed DOI

Jacinto E, Hall MN. TOR signalling in bugs, brain and brawn. Nat Rev Mol Cell Biol. 2003;4:117. doi: 10.1038/nrm1018. PubMed DOI

Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S. Regulation of lifespan in Drosophila by modulation of genes in the TOR signaling pathway. Curr Biol. 2004;14(10):885–890. doi: 10.1016/j.cub.2004.03.059. PubMed DOI PMC

Barcelo H, Stewart MJ. Altering Drosophila S6 kinase activity is consistent with a role for S6 kinase in growth. Genesis. 2002;34(1–2):83–85. doi: 10.1002/gene.10132. PubMed DOI

Rusten TE, Lindmo K, Juhasz G, Sass M, Seglen PO, Brech A, Stenmark H. Programmed autophagy in the Drosophila fat body is induced by ecdysone through regulation of the PI3K pathway. Dev Cell. 2004;7(2):179–192. doi: 10.1016/j.devcel.2004.07.005. PubMed DOI

Menon S, Dibble CC, Talbott G, Hoxhaj G, Valvezan AJ, Takahashi H, Cantley LC, Manning BD. Spatial control of the TSC complex integrates insulin and nutrient regulation of mTORC1 at the lysosome. Cell. 2014;156(4):771–785. doi: 10.1016/j.cell.2013.11.049. PubMed DOI PMC

Cheng LY, Bailey AP, Leevers SJ, Ragan TJ, Driscoll PC, Gould AP. Anaplastic lymphoma kinase spares organ growth during nutrient restriction in Drosophila. Cell. 2011;146(3):435–447. doi: 10.1016/j.cell.2011.06.040. PubMed DOI

Nezis IP, Simonsen A, Sagona AP, Finley K, Gaumer S, Contamine D, Rusten TE, Stenmark H, Brech A. Ref(2)P, the Drosophila melanogaster homologue of mammalian p62, is required for the formation of protein aggregates in adult brain. J Cell Biol. 2008;180(6):1065–1071. doi: 10.1083/jcb.200711108. PubMed DOI PMC

Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C, et al. Autophagy suppresses tumorigenesis through elimination of p62. Cell. 2009;137(6):1062–1075. doi: 10.1016/j.cell.2009.03.048. PubMed DOI PMC

Uytterhoeven V, Lauwers E, Maes I, Miskiewicz K, Melo MN, Swerts J, Kuenen S, Wittocx R, Corthout N, Marrink S-J. Hsc70-4 deforms membranes to promote synaptic protein turnover by endosomal microautophagy. Neuron. 2015;88(4):735–748. doi: 10.1016/j.neuron.2015.10.012. PubMed DOI

Mukherjee A, Patel B, Koga H, Cuervo AM, Jenny A. Selective endosomal microautophagy is starvation-inducible in Drosophila. Autophagy. 2016;12(11):1984–1999. doi: 10.1080/15548627.2016.1208887. PubMed DOI PMC

Wang Y-C, Lauwers E, Verstreken P. Presynaptic protein homeostasis and neuronal function. Curr Opin Genet Dev. 2017;44:38–46. doi: 10.1016/j.gde.2017.01.015. PubMed DOI

Tekirdag KA, Cuervo AM. Chaperone-mediated autophagy and endosomal microautophagy: joint by a chaperone. J Biol Chem 2017:jbc. R117. 818237:5414-24. PubMed PMC

Truckenbrodt S, Viplav A, Jahne S, Vogts A, Denker A, Wildhagen H, Fornasiero EF, Rizzoli SO. Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission. EMBO J. 2018;37(15). PMID: 29950309. PubMed PMC

Denker A, Krohnert K, Buckers J, Neher E, Rizzoli SO. The reserve pool of synaptic vesicles acts as a buffer for proteins involved in synaptic vesicle recycling. Proc Natl Acad Sci U S A. 2011;108(41):17183–17188. doi: 10.1073/pnas.1112690108. PubMed DOI PMC

Denker A, Bethani I, Krohnert K, Korber C, Horstmann H, Wilhelm BG, Barysch SV, Kuner T, Neher E, Rizzoli SO. A small pool of vesicles maintains synaptic activity in vivo. Proc Natl Acad Sci U S A. 2011;108(41):17177–17182. doi: 10.1073/pnas.1112688108. PubMed DOI PMC

Futerman AH, van Meer G. The cell biology of lysosomal storage disorders. Nat Rev Mol Cell Biol. 2004;5(7):554–565. doi: 10.1038/nrm1423. PubMed DOI

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997;25(17):3389–3402. doi: 10.1093/nar/25.17.3389. PubMed DOI PMC

Kretzschmar D, Hasan G, Sharma S, Heisenberg M, Benzer S. The swiss cheese mutant causes glial hyperwrapping and brain degeneration in Drosophila. J Neurosci. 1997;17(19):7425–7432. doi: 10.1523/JNEUROSCI.17-19-07425.1997. PubMed DOI PMC

Tschape JA, Hammerschmied C, Muhlig-Versen M, Athenstaedt K, Daum G, Kretzschmar D. The neurodegeneration mutant lochrig interferes with cholesterol homeostasis and Appl processing. EMBO J. 2002;21(23):6367–6376. doi: 10.1093/emboj/cdf636. PubMed DOI PMC

Zaccheo O, Dinsdale D, Meacock PA, Glynn P. Neuropathy target esterase and its yeast homologue degrade phosphatidylcholine to glycerophosphocholine in living cells. J Biol Chem. 2004;279(23):24024–24033. doi: 10.1074/jbc.M400830200. PubMed DOI

Dermaut B, Norga KK, Kania A, Verstreken P, Pan H, Zhou Y, Callaerts P, Bellen HJ. Aberrant lysosomal carbohydrate storage accompanies endocytic defects and neurodegeneration in Drosophila benchwarmer. J Cell Biol. 2005;170(1):127–139. doi: 10.1083/jcb.200412001. PubMed DOI PMC

Becker LE, Yates AJ. Textbook of neuropathology. Baltimore: Williams and Williams; 1991.

Kehl SR, Soos BLA, Saha B, Choi SW, Herren AW, Johansen T, Mandell MA: TAK1 converts Sequestosome 1/p62 from an autophagy receptor to a signaling platform. EMBO Rep. 2019;20(9):e46238. PubMed PMC

Tian X, Gala U, Zhang Y, Shang W, Jaiswal SN, Di Ronza A, Jaiswal M, Yamamoto S, Sandoval H, Duraine L. A voltage-gated calcium channel regulates lysosomal fusion with endosomes and autophagosomes and is required for neuronal homeostasis. PLoS Biol. 2015;13(3):e1002103. doi: 10.1371/journal.pbio.1002103. PubMed DOI PMC

Mannion NM, Greenwood SM, Young R, Cox S, Brindle J, Read D, Nellaker C, Vesely C, Ponting CP, McLaughlin PJ, et al. The RNA-editing enzyme ADAR1 controls innate immune responses to RNA. Cell Rep. 2014;9(4):1482–1494. doi: 10.1016/j.celrep.2014.10.041. PubMed DOI PMC

Livingston JH, Lin JP, Dale RC, Gill D, Brogan P, Munnich A, Kurian MA, Gonzalez-Martinez V, De Goede CG, Falconer A, et al. A type I interferon signature identifies bilateral striatal necrosis due to mutations in ADAR1. J Med Genet. 2014;51(2):76–82. doi: 10.1136/jmedgenet-2013-102038. PubMed DOI

Rice GI, Kasher PR, Forte GM, Mannion NM, Greenwood SM, Szynkiewicz M, Dickerson JE, Bhaskar SS, Zampini M, Briggs TA. Mutations in ADAR1 cause Aicardi-Goutieres syndrome associated with a type I interferon signature. Nat Genet. 2012;44(11):1243–1248. doi: 10.1038/ng.2414. PubMed DOI PMC

Duran A, Amanchy R, Linares JF, Joshi J, Abu-Baker S, Porollo A, Hansen M, Moscat J, Diaz-Meco MT. p62 is a key regulator of nutrient sensing in the mTORC1 pathway. Mol Cell. 2011;44(1):134–146. doi: 10.1016/j.molcel.2011.06.038. PubMed DOI PMC

Nagy P, Kárpáti M, Varga Á, Pircs K, Venkei Z, Takáts S, Varga K, Érdi B, Hegedűs K, Juhász G. Atg17/FIP200 localizes to perilysosomal Ref (2) P aggregates and promotes autophagy by activation of Atg1 in Drosophila. Autophagy. 2014;10(3):453–467. doi: 10.4161/auto.27442. PubMed DOI PMC

Terajima H, Yoshitane H, Ozaki H, Suzuki Y, Shimba S, Kuroda S, Iwasaki W, Fukada Y. ADARB1 catalyzes circadian A-to-I editing and regulates RNA rhythm. Nat Genet. 2017;49(1):146–151. doi: 10.1038/ng.3731. PubMed DOI

Penn AC, Balik A, Greger IH. Reciprocal regulation of A-to-I RNA editing and the vertebrate nervous system. Front Neurosci. 2013;7:61. doi: 10.3389/fnins.2013.00061. PubMed DOI PMC

Gurung S, Evans AJ, Wilkinson KA, Henley J. ADAR2 mediated Q/R editing of GluK2 regulates homeostatic plasticity of kainate receptors. J Cell Sci. 2018;131(24). 10.1242/jcs.222273. PubMed PMC

Krestel HE, Shimshek DR, Jensen V, Nevian T, Kim J, Geng Y, Bast T, Depaulis A, Schonig K, Schwenk F. A genetic switch for epilepsy in adult mice. J Neurosci. 2004;24(46):10568–10578. doi: 10.1523/JNEUROSCI.4579-03.2004. PubMed DOI PMC

Jepson JE, Savva YA, Yokose C, Sugden AU, Sahin A, Reenan RA. Engineered alterations in RNA editing modulate complex behavior in Drosophila: regulatory diversity of adenosine deaminase acting on RNA (ADAR) targets. J Biol Chem. 2011;286(10):8325–8337. doi: 10.1074/jbc.M110.186817. PubMed DOI PMC

Adenosine Deaminase Acting on RNA (ADAR) Enzymes: A Journey from Weird to Wondrous

ADAR2 enzymes: efficient site-specific RNA editors with gene therapy aspirations

Adar RNA editing-dependent and -independent effects are required for brain and innate immune functions in Drosophila