RESEARCH ARTICLE

Implications of the Sap47 null mutation for synapsinphosphorylation, longevity, climbing proficiency and behaviouralplasticity in adult DrosophilaBeatriz Blanco-Redondo1,2,3,§§, Nidhi Nuwal2, Susanne Kneitz4, Tulip Nuwal2, Partho Halder2,*, Yiting Liu2,‡,Nadine Ehmann5,6,7, Nicole Scholz3,5, Annika Mayer1,§, Jorg Kleber8, Thilo Kahne9, Dominique Schmitt1,Madhumala K. Sadanandappa1,10,¶, Natalja Funk2,**, Viera Albertova1,2, Charlotte Helfrich-Forster2,Mani Ramaswami10,‡‡, Gaiti Hasan10, Robert J. Kittel5,6,7, Tobias Langenhan3,5, Bertram Gerber8,11,12

and Erich Buchner1,2,§§

ABSTRACTThe Sap47 gene of Drosophila melanogaster encodes a highlyabundant 47 kDa synaptic vesicle-associated protein. Sap47 nullmutants show defects in synaptic plasticity and larval olfactoryassociative learning but the molecular function of Sap47 at thesynapse is unknown. We demonstrate that Sap47 modulates thephosphorylation of another highly abundant conserved presynapticprotein, synapsin. Site-specific phosphorylation of Drosophila synapsinhas repeatedly been shown to be important for behavioural plasticity butit was not known where these phospho-synapsin isoforms are localizedin the brain. Here, we report the distribution of serine-6-phosphorylatedsynapsin in the adult brain and show that it is highly enriched in rings ofsynapses in the ellipsoid body and in large synapses near the lateraltriangle. The effects of knockout of Sap47 or synapsin on olfactoryassociative learning/memory support the hypothesis that both proteins

operate in the samemolecular pathway.We therefore asked if this mightalso be true for other aspects of their function.We show that knockout ofSap47 but not synapsin reduces lifespan, whereas knockout of Sap47and synapsin, either individuallyor together, affects climbing proficiency,as well as plasticity in circadian rhythms and sleep. Furthermore,electrophysiological assessment of synaptic properties at the larvalneuromuscular junction (NMJ) reveals increased spontaneous synapticvesicle fusion and reduced paired pulse facilitation in Sap47 andsynapsin single and double mutants. Our results imply that Sap47 andsynapsin cooperate non-uniformly in the control of synaptic properties indifferent behaviourally relevant neuronal networks of the fruitfly.

KEY WORDS: Synaptic proteins, Knockout, Behaviour, Plasticity,Age-related decline

INTRODUCTIONThe Sap47 gene was identified in Drosophila by using amonoclonal antibody from the Würzburg hybridoma library(Reichmuth et al., 1995; Hofbauer et al., 2009). Sap47 representsa founding member of the superfamily of proteins containing a BSDdomain, which is observed in BTF2-like transcription factors,Sap47 homologues, and DOS2-like proteins (Doerks et al., 2002).In third instar larvae and adults, Sap47 is concentrated in synapticneuropil and in synaptic boutons, where the protein is mainly foundin close proximity to synaptic vesicles (Reichmuth et al., 1995;Saumweber et al., 2011). However, Sap47 mRNA in embryos isdetected prior to synaptogenesis (Flybase; Gramates et al., 2017),indicating that Sap47 is not an exclusively synapse-specific protein.Drosophila Sap47 null mutants are viable and fertile (Funk et al.,2004) but third instar larvae show a ∼50% reduction in the ability tolearn and/or remember the association of an odorant with arewarding tastant (Saumweber et al., 2011; Kleber et al., 2015).Basic synaptic transmission is normal in Sap47 null mutants, butvoltage clamp recordings at larval neuromuscular junctions revealedenhanced synaptic depression during high frequency stimulation,indicating a defect in short-term synaptic plasticity (Saumweberet al., 2011).

The human orthologue of Sap47, termed SYAP1, was found to bedifferentially regulated by tamoxifen in breast cancer cells (Al-Dhaheriet al., 2006) and SYAP1 mRNA is detected in most human tissues(Chang et al., 2001). Syap1 interacts with the telomere componentPOT1 (Lee et al., 2011) and associates with proteasome subunits(Havugimana et al., 2012). Recently, Syap1 (also known as BSTA orBSD domain-containing signal transducer and Akt interactor) wasshown to play an essential role in adipocyte differentiation that requiredReceived 19 March 2019; Accepted 29 August 2019

1Institute of Clinical Neurobiology, University of Wurzburg, 97078 Wurzburg,Germany. 2Department of Neurobiology and Genetics, Biocenter of the Universityof Wurzburg, 97074 Wurzburg, Germany. 3Rudolf Schonheimer Institute ofBiochemistry, Division of General Biochemistry, Leipzig University, 04103 Leipzig,Germany. 4Department of Physiological Chemistry, Biocenter of the University ofWurzburg, 97074 Wurzburg, Germany. 5Department of Neurophysiology, Institute ofPhysiology, University of Wurzburg, 97070 Wurzburg, Germany. 6Department ofAnimal Physiology, Institute of Biology, Leipzig University, 04103 Leipzig, Germany.7Carl-Ludwig-Institute for Physiology, Leipzig University, 04103 Leipzig, Germany.8Leibniz Institute of Neurobiology, 39118 Magdeburg, Germany. 9Institute ofExperimental Internal Medicine, Otto von Guericke University, 39120 Magdeburg,Germany. 10National Centre for Biological Sciences, Tata Institute of FundamentalResearch, Bangalore, Karnataka 560065, India. 11Institute of Biology, University ofMagdeburg, 39120 Magdeburg, Germany. 12Center for Behavioral Brain Sciences,39106 Magdeburg, Germany.*Present address: Laboratory for Test Development, CSL Behring GmbH,35041 Marburg, Germany. ‡Present address: Clinical Development & OperationsMedical Writing, Pfizer (China) R&D Center, Shanghai, China. §Present address:Universitatsklinikum Bonn, Institut fur Neuropathologie, Sigmund-Freud-Str. 25,53127 Bonn, Germany. ¶Present address: Department of Molecular and SystemsBiology, Geisel School of Medicine at Dartmouth, Hanover, NH 03755, USA.**Present address: Hertie Institute for Clinical Brain Research andGerman Center forNeurodegenerative Diseases, University Clinic Tubingen, 72076 Tubingen,Germany. ‡‡Present address: TrinityCollege Institute of Neuroscience, Trinity CollegeDublin, Dublin 2, Ireland.

§§Authors for correspondence (Beatriz.Blanco-Redondo@medizin.uni-leipzig.de;E_Buchner@ukw.de)

B.B.-R., 0000-0003-0246-2893; N.N., 0000-0003-2728-6865; S.K., 0000-0001-9425-3698; P.H., 0000-0002-9503-5999; Y.L., 0000-0002-9839-396X; N.E., 0000-0002-6577-6913; N.S., 0000-0001-8087-8451; A.M., 0000-0001-7931-0316; D.S.,0000-0002-7888-5318; M.K.S., 0000-0001-6071-365X; V.A., 0000-0002-0163-0771; C.H.-F., 0000-0002-0859-9092; M.R., 0000-0001-7631-0468; G.H., 0000-0001-7194-383X; R.J.K., 0000-0002-9199-4826; T.L., 0000-0002-9061-3809; B.G.,0000-0003-3003-0051; E.B., 0000-0003-3031-8936

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the suppression of the FoxC2 transcription factor gene. It was observedthat Syap1 enhances phosphorylation of protein kinase B1 (PKB1/Akt1) at Ser473 in certain mammalian cell lines after growth factorstimulation. In addition, the BSD domain was demonstrated to beessential for the interaction of Syap1 andAkt1,which in turn appears todepend on mTORC2-mediated phosphorylation of Syap1 (Yao et al.,2013). In mice, Syap1 has been detected in all tested regions of thenervous system and also in non-neural tissues such as muscle or liver(Schmitt et al., 2016). In cultured primary motoneurons, Syap1 isconcentrated in the perinuclear region near the Golgi complex and isalso found in axons and growth cones. In these cells, however, Syap1knockdown or knockout does not reduce total Akt phosphorylation(Schmitt et al., 2016). In the postnatal mouse brain, Syap1 is widelydistributed in synaptic neuropil with high concentrations inglutamatergic synaptic regions, and is also detected in perinuclearstructures close to the Golgi of specific neuronal somata. Similarly toSap47 null mutant flies, Syap1 knockout mice are viable and fertile(Schmitt et al., 2016) but show distinct deficiencies inmotor behaviour(von Collenberg et al., 2019).Synapsins constitute a family of conserved vesicle-associated

synaptic phospho-proteins (Greengard et al., 1993). They are involvedin synaptic vesicle clustering and appear to regulate the reserve pool ofsynaptic vesicles in a phosphorylation-dependent manner. Vertebratesynapsins are substrates for various kinases, including PKA, CaMKI,CaMKII, CaMKIV, MAPK, Src and Cdk1/5, which regulate bindingof actin and synaptic vesicles and neurotransmitter release (reviewedby Cesca et al., 2010). In particular, impairment of synapsin functionmodifies synaptic plasticity as revealed by paired-pulse facilitation aswell as post-tetanic and long-term potentiation. Mutations in thesynapsin genes of humans are associated with epilepsy, aggressivebehaviour, learning problems and autism (Garcia et al., 2004; Fassioet al., 2011; Corradi et al., 2014). Indeed, SYN1 mutations areconsidered to be one cause of X-chromosome-linked intellectualdisability (Ropers and Hamel, 2005).The Drosophila synapsin gene (Syn) was identified as the first

invertebrate member of the synapsin protein family (Klagges et al.,1996). At larval neuromuscular junctions (NMJs) synapsin directlyinteracts with the endocytic scaffolding protein Dap160. Theresulting functional complex contains epidermal growth factorreceptor substrate 15 (Eps15) and is essential for the correctlocalization of synapsin and the re-clustering of synaptic vesicles inactive NMJs (Akbergenova and Bykhovskaia, 2007, 2010; Kohet al., 2007; Pechstein and Shupliakov, 2010; Denker and Rizzoli,2010; Winther et al., 2015). Syn null mutants show defects inbehaviour, including impaired olfactory habituation (Sadanandappaet al., 2013) and associative learning and memory (Godenschwegeet al., 2004; Michels et al., 2005, 2011; Knapek et al., 2010;Diegelmann et al., 2013; Kleber et al., 2015; Walkinshaw et al.,2015; Niewalda et al., 2015). These defects can be ‘rescued’ bytransgenic expression of wild-type synapsin but not by synapsinwith mutated phosphorylation sites. Thus the tissue distribution ofphosphorylated synapsin in the adult brain is of particular interest.Here, we use an antiserum specific for synapsin phosphorylated atSer6 to report this distribution for the first time.Considering that knockout of either synapsin or Sap47, or both, has

similar effects on associative learning and that the effects are notadditive in the double mutant (Saumweber et al., 2011; Kleber et al.,2015), wewondered if this applies to other phenotypes as well.Withinthis context, we describe previously uncharacterized behavioural traitsof adult Sap47 and Syn null mutants and investigate the interaction ofthe two synaptic proteins by molecular, electrophysiological andgenetic techniques. We present evidence that synapsin and Sap47

functionally cooperate differentially in different behavioural settingsbut apparently do not interact directly.

MATERIALS AND METHODSFly strainsTwo lines of Canton S wild type (CSNF and CSV, denoted as WTCS1and WTCS2) maintained separately for more than 10 years wereused. The Sap47156CS and Syn97CS mutants denoted as Sap−/− andSyn−/−, respectively, are P-element jump-out null mutant linesbrought into CSNF background by at least 12 generations of back-crossing and have been described in detail previously (Saumweberet al., 2011; Funk et al., 2004; Godenschwege et al., 2004; Michelset al., 2005, 2011; Sadanandappa et al., 2013). The Syn79 mutant(denoted as Syn−/−79) represents an independent synapsin null allele(Godenschwege et al., 2004). Sap47156w, Sap47201w, Sap47208w andSyn79w are non-Cantonized independent jump-out null mutants ofthe respective P-element lines (Funk et al., 2004; Godenschwegeet al., 2004). Note that the epitopes of the monoclonal antibodiesagainst synapsin (Synorf1) and Sap47 (nc46) used here are locatedin the N-terminal domains common to all known isoforms(Godenschwege et al., 2004; Funk et al., 2004; Saumweber et al.,2011) verifying that all isoforms are eliminated by the respectivemutations. The nervous system-specific rescue of the Sap47156

mutant was accomplished by using the F1 generation of elav-Gal4;;Sap47156 crossed to w;;UAS-Sap47-RF,Sap47156 as described bySaumweber et al. (2011). Sap47-RF represents the most stronglyexpressed nervous system specific splice variant of the Sap47 gene(FlyAtlas2; Leader et al., 2018). The Sap-Syn double mutants weregenerated as follows: from individual recombinant chromosomesobtained by mating F1 female offspring of a Sap47156CS×Syn97CS

cross to TM3/TM6 double balancer males, stocks were establishedand balanced flies were screened by PCR for deficiency of bothgenes. Four independent recombinants were isolated, which all werehomozygous lethal but viable as transheterozygotes, indicating thatall four recombinant chromosomes contained different third sitelethal mutations. The transheterozygote stocks are designatedSap-SynNS17, Sap-SynNS62, Sap-SynNF and Sap-SynV. From onerecombinant chromosome, Sap-SynV, the third site lethality wasremoved by outcrossing to WT CSV flies for six generations, usingsingle-fly PCR to identify recombinant chromosomes, therebygenerating the homozygous viable recombinants Sap-SynV1, Sap-SynV2, Sap-SynV3. The lack of transcripts was verified by RT-PCR(Fig. S1). No significant differences among the three double-mutantlines were detected in any of the tests employed in the present study.No significant change of Syn transcript in Sap47156 or of Saptranscript in Syn97 mutants were observed (Fig. S1). The mutationswere also verified by western blots using antibodies againstsynapsin and Sap47, which demonstrated the absence of allisoforms of both proteins (Fig. S2). The Syn(S6A,S533A) line,kindly provided by Birgit Michels (Michels et al., 2011), expressesa synapsin cDNA with point mutations replacing Ser6 and Ser533with Ala under the control of elav-Gal4 in the Syn null background(Michels et al., 2011). Ser6 mutation prevents the phosphorylationof Ser6, a site specifically recognized by anti-PSyn(S6).

Generation of the Latrophilin (Cirl) mutant [w1118; Cirlko attPCirl

loxP-w+-loxP;; (Cirlko w+)] and rescue [w1118; Cirlko

{w+mC=pTL370[Cirl]}attPCirl loxP;; (Cirlrescue w+)] strains used inthis study have been described previously (Scholz et al., 2015). Thew;UAS-Aβ42;+ transgenic line was obtained from the BloomingtonStock Center (no. 33769). w;gmr-Gal4;+, elav-Gal4;+;+, elav-Gal4;+;Sap47156, and the double balancer w;Sco/CyO;TM6,Tb,Hu/MKRS,Sb were kindly provided by Burkhard Poeck, Stephan Sigrist,

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Birgit Michels, and Thomas Raabe, respectively. Using the doublebalancer, homozygous w;UAS-Aβ42;Sap47156 and w;gmr-Gal4;Sap47156 recombinants were generated. w;UAS-Aβ42;Sap47156 wascrossed with eitherw;gmr-Gal4;+ or elav-Gal4;+;+ orw;gmr-Gal4;Sap47156 or elav-Gal4;+;Sap47156 driver lines to generate the fliesexpressing Aβ42 either in the retina (gmr-Gal4) or in the nervoussystem (elav-Gal4) in Sap47156/+ heterozygous or Sap47156

homozygous background, respectively.The Sap47rescue line (elav-Gal4/+;UAS-Sap47-RF/+;Sap47156/

Sap47156) was generated by crossing elav-Gal4;;Sap47156 withUAS-Sap47-RF;Sap47156 described previously (Saumweber et al.,2011). The Sap47 deficiency in the parental lines and Sap47expression in the rescue line were verified by western blotting.The lines expressing Gal4 in different sets of ring neurons of the

central complex, c819-Gal4, 189y-Gal4, c232-Gal4 (Renn et al.,1999), EB1-Gal4 (Spindler and Hartenstein, 2010) and ftz-ng-Gal4/TM3,Sb (Kuntz et al., 2012) were obtained from Burkhard Poeckand crossed with a line with 10xUAS-GFP constructs on the Xchromosome, which was kindly provided by Christian Wegener.

AntibodiesMonoclonal antibodies from the Würzburg hybridoma collection(Hofbauer et al., 2009) were used against the following proteins:Sap47 (nc46; 1:200 for WB, 1:50 for IHC), Syn [Synorf1 (3C11)1:100 for WB, 1:20 for IHC], bruchpilot (nc82; 1:100), CSP (ab49;1:200 for WB, 1:50 for IHC). These antibodies are available fromDSHB. Anti-PSyn(S6) rabbit serum against synapsin phosphorylatedat Ser6 has been described previously (Sadanandappa et al., 2013)and was used at 1:200 (WB) or 1:500 (IHC). The specificity ofbinding of the PSyn(S6) antiserum to synapsin phosphorylated atSer6 is demonstrated by the lack of signals in transgenic fliesexpressing synapsin with an S6A mutation in a synapsin nullbackground, although the anti-Syn antibody Synorf1 proves theexpression of the mutated synapsin [Fig. S2B, top, lane Syn(S6)].Specificity of nc46 and Synorf1 antibodies has been demonstratedpreviously (Saumweber et al., 2011; Funk et al., 2004; Klagges et al.,1996; Godenschwege et al., 2004; Michels et al., 2005, 2011) andwas verified here again by western blotting as no signals wereobtained with Syn or Sap47 antibodies for the respective mutants(Fig. S2A,B). Cy3-conjugated anti-HRP (Jackson ImmunoResearch)stains Drosophila neuronal membranes via a cross-reaction withDrosophila Na-K-ATPase and was used at 1:250. Secondaryantibodies, goat anti-rabbit peroxidase coupled (WB: 1:1000) andgoat anti-mouse peroxidase coupled (WB: 1:1000) and fluorophore-coupled antibodies (IHC 1:500) were provided by JacksonLaboratory, Farmington, CT, USA.

Western blotting and immunohistochemistryFly heads were homogenized in Laemmli sample buffer, proteinswere separated by standard SDS-PAGE, transferred to a nitrocellulosemembrane by wet blotting (Bio-Rad Laboratories, München,Germany), and detected after incubation with specific primary andHRP-coupled secondary antibodies by chemiluminescence (ECL,GE Healthcare Europe, Freiburg, Germany). For comparison ofnative versus de-phosphorylated synapsin, fly heads werehomogenized in buffer A (25 mmol l−1 Tris-HCl, 150 mmol l−1

NaCl, 10% glycerol, 0.1%NP40, pH 7.7), to only one of two aliquots(each containing 1 head equivalent) 40 U of shrimp alkalinephosphatase (Promega, Madison WI, USA) were added (AP+),both aliquots (AP+ and AP−) were incubated for 15 min at 37°C (toactivate the enzyme), the reaction was stopped with 2× Laemmlibuffer, and the samples were loaded on a standard SDS gel. For

separation of protein complexes under non-denaturing conditions, weused blue native polyacrylamide gel electrophoresis (Wittig andSchägger, 2008) with the NativePAGE buffer system (Invitrogen,Karlsruhe, Germany) following the manufacturer’s instructions.Single lanes were cut from the blue native gel and subjected toSDS PAGE using a singlewell 4–12%Bis–tris gel and the associatedbuffer system (NuPAGE, Invitrogen) according to the manufacturer’sinstructions. Proteins were transferred to a nitrocellulose membraneby wet blotting and detected by ECL as described above.

For immunohistochemistry of whole-mount fly brains, wefollowed the procedure described by Wu and Luo (2006).Preparations of fly heads for cryo-sectioning requires removal ofproboscis and air sacks below the brain, fixation for 3 h in fresh 4%buffered paraformaldehyde, freeze protection in 25% sucrose inRinger’s solution (128 mmol l−1 NaCl, 4.7 mmol l−1 KCl,1.7 mmol l−1 CaCl, 0.7 mmol l−1 Na2HPO4, 0.35 mol l−1

KH2PO4, pH 7.4) overnight, and sectioning at 30 µm thicknessusing a cryostat microtome. Staining for fluorescence microscopyhas been described previously (Halder et al., 2011; Blanco-Redondo et al., 2013). Image stacks were taken at 2 µm z-stepsusing an Olympus Fluoview FV1000 confocal microscopeequipped with an Olympus UPLSAPO 20× (air) objective.Whenever the WT image of a CLSM (confocal laser-scanningmicroscopy) scan was contrast enhanced (40–50%) to compensatefor staining variability due to different z-positions within the 30 µmsection the corresponding mutant image was contrast enhanced bythe same value. No signals are obtained at the same parametersettings when the first antibody is omitted. Immunohistochemistryof larval neuromuscular junctions was performed as previouslydescribed (Schmid and Sigrist, 2008).

2D electrophoresisFor 2D electrophoresis, iso-electric focusing by the ZOOMIPGRunner system was used (Invitrogen, Carlsbad, CA, USA).100 fly heads were separated from bodies and appendages byfreezing in liquid nitrogen and vigorous shaking using twosuccessive sieves. The heads were homogenized in 100 µl of thesample homogenizing mix (ZOOM 2D Protein Solubilizer 1) andprotease inhibitors. The homogenate was then centrifuged twice andthe post-nuclear supernatant was incubated with 1 µl N,N-dimethylacetamide (DMA; Sigma) for 15 min. Protein alkylationwas stopped with 2 µl of 2 mol l−1 dithiothreitol (DTT; Sigma).25 µl of this homogenate were added to the rehydration mixture asrecommended by the manufacturer. Thereafter, the sample wasloaded into the sample loading well (ZOOM IPG Runner Cassette).The ZOOM Strip (pH 3–10) was inserted into the cassette andloaded and sealed, the ZOOM IPG Runner Cassette was left at 18°Covernight. The next day, the cassette was placed in the ZOOM IPGCore and slid into the Mini-Cell Chamber of the IPG Runner andlocked. The proteins went through five voltage steps before beingplaced into the single-well 4–12% Bis-Tris NuPAGE 2D gel andbeing treated as a regular western blot as described above.

qRT-PCR analysisFor quantification of transcript levels in the mutants by real-timePCR total RNA from fly heads was isolated using the RNeasy minikit (QIAGEN, Hilden, Germany, following the manufacturer’sinstructions) and reverse transcribed. The Rotor-Gene Q SYBRGreen system (QIAGEN, Hilden, Germany) was applied to 100 ngof purified cDNA following the manufacturer’s instructions. Primersequences used were: Synapsin, for 5′-CTTAACGTTCATCGGC-CATT-3′ and rev 5′-AGGGGTTCGCTTCGTTACTA-3′; Sap47,

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for 5′-TAAAAGTTGGAGAGCCAGGA-3′ and rev 5′-GGTGG-CTTCGGATACTAATG-3′; Cirl, for 5′-GGATGATGCTCATGG-ATTG-3′ and rev 5′-AAAGCCCCGTAGTCAAGAG-3′.

ElectrophysiologyTwo-electrode voltage clamp (TEVC) recordings (Axoclamp 900 Aamplifier, Molecular Devices) were performed on male third instarDrosophila larvae, muscle 6 in segments A2 and A3 at roomtemperature using intracellular electrodes (resistance 10–20 MΩ),filled with 3 mol l−1 KCl, as described previously (Ljaschenkoet al., 2013). Composition of extracellular haemolymph-likesolution (HL-3; Stewart et al., 1994) was as follows (inmmol l−1): NaCl 70, KCl 5, MgCl2 20, NaHCO3 10, trehalose 5,sucrose 115, Hepes 5 and CaCl2 1, pH adjusted to 7.2. For analyses,only cells with an initial membrane resistance ≥4 MΩ andmembrane potential of at least −50 mV were accepted. Cells wereheld at −60 mV, except for minis (miniature excitatory postsynapticcurrents), where holding potential was adjusted to −80 mV (for90 s). To elicit action potential-evoked excitatory postsynapticcurrents (eEPSCs), brief (300 µs) pulses were applied via a suctionelectrode at typically 10–20 V. Paired-pulse recordings wereperformed with increasing inter-stimulus intervals of (in ms): 10,30, 100, 300 and 1000 with 10 s rest in between the recordings. Forlow frequency stimulation and paired-pulse recordings, 10 singletraces were averaged per cell and inter-pulse interval. The amplitudeof the second response in 10 ms inter-pulse recordings wasmeasured from the peak to the point of interception with theextrapolated first response. Recordings were sampled at 10 kHz andlow-pass filtered at 1 kHz. Analyses were carried out using Clampfit10.5 (Molecular Devices) and Sigmaplot 12.5 (Systat Software).Data are presented as means±s.e.m. and statistics employed the non-parametric rank-sum test (versus controls). For electrophysiologicalrecordings and analyses, all genotypes were blinded.

Lifespan analysisFor each genotype (CSNF, CSV, Syn97, Sap156, Sap-SynV3,Sap47rescue) five vials of 10 males each were kept at 25°C.Genotypes were encoded for fully blind evaluation. Twice a weekthe flies were transferred to fresh food vials, and the numbers ofdead flies were counted. The Kolmogorov–Smirnov test was used todetermine statistical significance of the differences betweengenotypes. This test is not based on standard errors of individualage points. All error bars but one per genotype have therefore beenomitted for clarity.

Climbing assayRapid induced negative geotaxis (Liu et al., 2015; Cao et al., 2017;Manjila and Hasan, 2018) was used as a simple test for sensory-motor performance. Experiments were done in the late afternoon.Adult male and female flies were assayed separately, but since noconsistent differences between the genders were noted, the datawere pooled. For each of the genotypes 6 vials of 10 flies each werekept at 25°C and transferred to fresh vials twice a week. Prior to theclimbing experiment, flies of the same agewere transferred to emptyvials (2.5 cm diameter; maximum climbing height, 7.5 cm) for30 min for acclimation. In a box holding the vials with the differentgenotypes, the flies were tapped to the bottom and allowed to climbupward for 4 s before a photograph of the assembly was taken. Afteranother 10 s, the procedure of tapping, waiting and image capturewas repeated, for a total of 10–12 times. The position of thegenotypes in the box was randomized and encoded for fully blindevaluation. Photos of the assembly were analysed using the

threshold and particle analysis features of ImageJ and a customwritten Basic subroutine to score the flies’ positions. For each age ofthe flies, the mean of the fly positions in each vial was scored andaveraged over the 10–12 repeats for each genotype. Normalizationof each experiment to the response of CS did not reduce standarddeviations of the mutant data, indicating that technical variationssuch as differences in tapping or environmental effects like time ofday, temperature, weather conditions, etc. did not systematicallyinfluence the results. The climbing success of the mutants relative toWTwas averaged over all ages. The Student’s t-test with Bonferronicorrection (six comparisons) was used to determine statisticalsignificance of the differences between the four genotypes.

Flight duration assayThis assay was performed as described in detail previously(Agrawal et al., 2013; Manjila and Hasan, 2018). Briefly, flieswere anesthetized for 10–15 min on ice and then attached with nailpolish between head and thorax to a thin wire. Flight was initiatedby a gentle air puff and the time to a spontaneous termination offlight was recorded. The Student’s t-test was used to determinestatistical significance of the differences between genotypes.

Circadian rhythms and sleepThe locomotor activity of single male flies was recorded using theTriKinetics DAM2 System (TriKinetics, Waltham, MA, USA) at20±0.5°C and 60±1% relative humidity, as described previously(Gmeiner et al., 2013). For the first 6 days, flies were recordedunder 12 h:12 h light:dark (LD) cycles and subsequently for 13subsequent days in constant darkness (DD). Illumination duringthe light phase was provided by white LEDs (Lumitronix LED-Rechnik, Jungingen, Germany) and set to 47.6 μW cm−2.Actograms of individual flies were plotted and the free-runningperiod and power was calculated by Sokolov–Bushell periodogramanalysis using the program ActogramJ (Schmid et al., 2013).

For testing the plasticity of the circadian clock, flies were rearedunder different photoperiods (LD 8:16, LD 12:12 and LD 16:8) andthen recorded for 14 days in DD. Sleep analysis was only performedduring LD 12:12 cycles. Sleep was defined as a period of inactivitylonger than 5 min (Hendricks et al., 2000; Shaw et al., 2000).Calculations of total sleep were performed for each hour of theday using a macro written in Microsoft Excel 2007 (courtesy ofDr Taishi Yoshii).

For 12 h sleep deprivation, the glass tubes with the flies weretaken out from TriKinetics system at lights-off and placed on aprogrammable rotator (Multi Bio RS-24, BioSan). The programmewas set to rotate at 18 rpm 4 times in one direction and then 4 timesin the opposite direction. Each directional shift was separated bythree 5 deg vibro-rotations. After sleep deprivation, flies werere-inserted into the Trikinetics system and recorded for a further 3days. All procedures were carried out under LD 12:12 at 20°C.

RESULTSKnockout of Sap47 alters the phosphorylation status ofsynapsinIn order to obtain information on the molecular function ofSap47 we attempted to identify its interaction partners. Afterimmunoprecipitation, we failed to detect any signals in silver-stained gels that were not also detected by our anti-Sap47 antibody(nc46). We also systematically compared western blots of WT andSap−/− head homogenates probed with antibodies against variousdifferent synaptic proteins. In this context, we discovered aqualitatively altered synapsin signal of the monoclonal antibody

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Synorf1 in all three independent Sap47 null mutants, Sap47156,Sap47201 and Sap47208, irrespective of their genetic background[Canton-S (CS) or w1118 (w)] (Fig. 1A). In Sap47 null mutants, anadditional synapsin-specific band at slightly higher apparentmolecular weight could be discerned (arrow in Fig. 1A, AP−).Interestingly, this additional signal could be eliminated by treatingthe head homogenate with alkaline phosphatase (Fig. 1A, AP+),

hinting at a hyper-phosphorylation of synapsin. However, massspectrometric analysis of synapsin peptides after liquidchromatography (LC-MS/MS) identified only a single phospho-serine in adult Sap−/− mutants that was not phosphorylated in WT(S574 in Fig. S3), at least within those regions of the protein forwhich the present methods had sufficient coverage. Indeed,coverage of synapsin phosphorylation by LC-MS/MS hasrepeatedly been reported to be partial (Kleber et al., 2015;Niewalda et al., 2015; Nuwal et al., 2011). We were therefore notdiscouraged from investigating Ser6 phosphorylation, which wasnot covered by the present LC-MS/MS approach. Using a custom-made rabbit antiserum [anti-PSyn(S6)] (Sadanandappa et al., 2013)against an N-terminal synapsin peptide containing phosphorylatedSer6, we confirmed by western blotting that Ser6 phosphorylationin Sap−/− mutants was reduced compared with WT levels(Fig. S2A,C). Comparison of western blot signals of anti-PSyn(S6) antiserum and the pan-Syn antibody Synorf1 revealedthat the read-through synapsin isoforms of 143 kDa (Klagges et al.,1996) were more efficiently phosphorylated at Ser6 compared withthe short isoforms of 70–80 kDa (Fig. S2D).

By 2D electrophoresis, approximately 20 synapsin isoforms of70–80 kDa were separated that are recognized by the monoclonalantibody Synorf1 (Fig. 1B). Of these, the isoforms of the lower rowtentatively labelled 17 to 21 in Fig. 1B (arrow in Fig. 1D) were notphosphorylated at Ser6 while isoforms of the middle and upper rowswere phosphorylated at this amino acid (Fig. 1C).

Repeated attempts to co-immunoprecipitate synapsin with theSap47 antibody or Sap47 with the Synorf1 antibody failed to showany sign of direct binding of the two proteins (not shown). Wetherefore tested if the two proteins migrate in a non-denaturing gel inthe same protein complex. By subjecting the protein complexes ofWT fly heads homogenized in non-denaturing buffer first to non-denaturing electrophoresis (horizontal dimension in Fig. 1E) andthen to SDS-PAGE (vertical dimension in Fig. 1E), we show thatsynapsin isoforms are found in protein complexes of >450 kDa,whereas Sap47 co-migrated with complexes of <450 kDa (Fig. 1E).Thus, it seems unlikely that the two proteins in the brain tissueinteract directly as components of the same macromolecularcomplex.

Knockout of Sap47 reduces lifespanSyap1, the mammalian homologue of Sap47, has been implicated inmodulating the target of rapamycin (TOR) pathway (Yao et al.,2013) which plays a central role in the regulation of lifespan (deCabo et al., 2014). We therefore wondered if mutation of the Sap47gene influencesDrosophila lifespan. Considering the similar effectsof knockout of Sap47 or synapsin on associative learning and theclear influence of the Sap47mutation on synapsin isoforms, we alsoasked whether the roles of the two genes for longevity are likewisesimilar and whether possible defects add up in double mutants.Similarly to the single-mutation lines, three viable Sap-Syn double-mutant lines generated by recombination (see Materials andMethods) showed no obvious phenotype. Quantitative comparisonwith WT, however, revealed clear deficiencies. The results of thequantification of survival of Sap−/− and Syn−/−, as well as Sap−/−,Syn−/− flies are shown in Fig. 2A. The Sap47 gene had a cleardetrimental influence on lifespan. Under our experimentalconditions, we found no significant difference in survival timesbetween the Syn−/− line and two Canton-S WT strains, or betweenSap−/− and Sap−/−, Syn−/− flies, but the latter two lines showedabout a 38% shorter life time (50% survival score) compared withthe WT (Fig. 2A). Pan-neural expression of the most abundant

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brain-specific Sap47 mRNA (RF splice variant, see Discussion)using the UAS-Gal4 system with elav-Gal4 as driver rescued theshort lifespan of the Sap−/− mutant (Fig. S4A).

Climbing proficiency is impaired in Sap−/−, Syn−/− and Sap−/−,Syn−/− fliesCompromised motor performance is a hallmark of manyneurodegenerative diseases. We wondered how the loss of twoproteins abundantlyexpressed inmost orall presynaptic terminals affectmotor performance with and without induction of neurodegeneration.Climbing, a simple test formotor performance,makes use of a negativegeotactic reflex that can be elicited by physical disturbance (seeMaterials and Methods). Mean climbing proficiency was reduced by27.6% in Syn−/− (P=0.011), by 49.9% in Sap−/− (P=0.0016) and by66.5% in Sap−/−, Syn−/− doublemutants (average of the 3Sap-Syn linesSap-SynNS62, Sap-SynV2, Sap-SynV3; P=0.000013) compared withWT-CS animals. With increasing age, climbing proficiencydeteriorated at roughly similar rates in all four genotypes (Fig. 2B).The climbingdefect of theSap−/−mutantwas rescuedby the pan-neuralexpression of the Sap47mRNA RF splice variant (Fig. S4B). We alsotested the impairment of motor function in Sap−/− mutants in a flightduration test (Agrawal et al., 2013; Manjila and Hasan, 2018).Sap−/− knockout flies terminated air-puff-induced flight 2.45 minearlier than WT control flies (Fig. S4C, P=0.00104).

Age-related decline in climbing proficiency can be accelerated byinduction of neurodegeneration via pan-neural (elav-Gal4)expression of human Aβ42 peptide (Liu et al., 2015; Fernendez-Funez et al., 2015; Ling et al., 2009; Ping et al., 2015; Rogers et al.,2012; Sofola et al., 2010; Wang et al., 2015; Costa et al., 2011). Weinvestigated whether the Sap−/− deficiency had any influence onthis effect (Fig. 2C). As controls, we used the driver gmr-Gal4 toobtain flies expressing Aβ42 only in the retina, which leads to a‘rough eye’ phenotype but does not impair brain function (Costaet al., 2011; Prüßing et al., 2013; Wang et al., 2015; Barucker et al.,2015). Similarly to the data shown in Fig. 2A, in these controls,homozygous Sap−/− deficiency significantly reduced climbingproficiency by 20% (P=0.0015) compared with heterozygousdeficiency. Surprisingly, when human Aβ42 was expressed pan-neurally using the elav-Gal4 driver, no such difference betweenhomozygous versus heterozygous Sap−/− deficiency was observed(P=0.132) (Fig. 2C) (see Discussion). Importantly, the rate of age-related decline in motor performance by Aβ42 expression was alsonot modified by the presence or absence of Sap47 (Fig. 2C),suggesting that the molecular mechanisms by which the Aβ42transgene and the Sap47 gene cause the age-related climbing defectare not independent. Aβ42 expression appears to block thedeleterious effect of Sap47 deficiency on climbing.

Plasticity in circadian rhythms and sleep is reduced inSyn−/−,Sap−/− and Sap-Syn double mutantsSap47 mRNA was found to be enriched in the ventral lateral clockneurons (LNvs) and showed different levels of accumulation duringthe day and night (Kula-Eversole et al., 2010), suggesting that it maybe linked with the circadian clock or sleep. To test the influence ofSap47 on rhythms and sleep, we monitored the locomotor activity ofSap−/−, Syn−/− or Syn-Sap double mutants under light–dark cyclesand constant darkness. Furthermore, we monitored the plasticity ofthe circadian period after rearing the flies under different day lengths(LD 8:16, LD 12:12, LD 16:8). A previous study had shown that fliesreared under short days (LD 8:16) exhibit a significantly longerperiod length under following constant darkness (DD) conditionscompared with flies reared under extended daylight (LD 16:8)

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(Tomioka et al., 1997). To test a possible effect of Sap47 and Syn onsleep plasticity, we recorded the sleep rebound of the mutants after a12-h night of sleep deprivation, which is known to cause significantlyincreased sleep during the subsequent day in WT flies (Hendrickset al., 2000; Shaw et al., 2000).We found that the basic activity/sleep patterns of WT and mutant

flies were very similar (Fig. 3A). Under LD conditions, all fliesshowed locomotor activity in the morning and evening that wasinterrupted by sleep during the middle of the day (the siesta) and atnight (a two-way ANOVA revealed no influence of the genotype onday time and night time sleep; F3,198=1.188; P=0.757). Sleep duringthe siesta appeared slightly more pronounced in the mutants than inthe WT (Fig. 3A,C,D), but this difference was not significant in aBonferroni post hoc test (P=1.000). Under constant darkness (DD),all flies showed significant circadian rhythms with a strain-specificperiod length (Fig. 3B). Rearing the flies under short and long dayssignificantly changed period length and power in the WT strains, butnot in the mutants (Fig. 3B). In WT flies, period was significantlyshorter (P<0.001) and power lower (P<0.001) in flies that were rearedunder long days as compared with those reared under short days.These differences were absent in the mutants (P=1.000), suggestingthat they lack the WT-like plasticity in circadian behaviour. Afterrescuing Sap47 in all neurons, the WT plasticity in period (P<0.001)and power (P=0.008) was restored (Fig. 3B).After sleep deprivation, Sap−/− and Syn−/−, as well as Sap-Syn

double mutants lacked the WT-like sleep rebound (Fig. 3C,D).WhereasWT flies slept significantly more during the entire followingday (P<0.001), the mutants did so only during the first 2 h of the day(arrows in Fig. 3C). When the amount of sleep in the mutants beforeand after sleep deprivation was compared over the entire followingday, no significant differences were found (P=1.000).

Postsynaptic currents are differentially affected in Syn−/−,Sap−/− and Sap-Syn double mutantsTo study the influence of Sap47 and synapsin on neurotransmission,two-electrode voltage clamp (TEVC) recordings were performedfrom larval neuromuscular junctions (NMJs; Fig. 4). Spontaneouspresynaptic neurotransmitter release can be assessed by measuringminiature postsynaptic currents (minis) elicited by individual vesiclefusions. Mini frequencies were subtly but significantly increased inboth single mutants and nearly tripled in the double mutant. Miniamplitudes were reduced in Sap−/− larvae (Sap47156), indicating aslight decrease in transmitter content of synaptic vesicles and/orpostsynaptic sensitivity compared with WT CS (CSNF) (Fig. 4A,B).Next, we investigated eEPSCs. While basal eEPSC amplitudes(0.2 Hz stimulation) were comparable in all genotypes (Fig. 4C), weobserved diminished paired-pulse facilitation in single and doublemutants upon closely spaced stimulation (Fig. 4D,E).Alerted by a possible genetic interaction of Syn and Cirl by

microarray and qPCR (Fig. S1) (Nuwal, 2010), we investigatedsynapsin levels in synaptic boutons ofCirl null mutants. Quantificationof anti-synapsin immunofluorescence in larval nerve musclepreparations of Cirl null mutants indicates a significant reduction ofsynapsin content in type Ib synaptic boutons, a defect that was rescuedto WT values upon re-constitution of the Cirl locus (Fig. 5).

Distribution of Sap47 and Ser6-phosphorylated synapsin inwild-type brainsThe vertebrate Sap47 orthologue Syap1 is localized close to the Golgicomplex of neuronal perikarya in addition to its concentration inneuropil (Schmitt et al., 2016).We therefore repeated immunostainingsof frozenDrosophila brain sections using a Sap47 antibody (nc46) and

observed that, in addition to the prominent localization of Sap47 insynaptic neuropil (Reichmuth et al., 1995; Funk et al., 2004), there isalso significant labelling of axonal fibre tracts and a subset of perikaryain the cellular cortex of the brain (Fig. 6A,C). In contrast, the activezone protein bruchpilot (BRP) (Hofbauer et al., 2009; Wagh et al.,2006; Kittel et al., 2006; Wichmann and Sigrist, 2010) is exclusivelyfound in synaptic neuropil (Fig. 6B).

Intrigued by the importance of synapsin phosphorylation forbehavioural plasticity, we investigated the distribution of totalsynapsin and synapsin phosphorylated at Ser6 in the adultDrosophila brain by performing immunohistochemicalexperiments. Fig. 7, Fig. S4D and Fig. S5A,B show whole-mountbrains and frozen horizontal head sections of white-eyed w;;Syn+/+

(w1118) and w;;Syn−/− null mutant flies stained with the monoclonalantibody Synorf1 (3C11) and the anti-PSyn(S6) antiserumdescribed above. All flies were in the w1118 background to avoidautofluorescence signals from eye pigments diffusing into thebrain during preparation and fixation. As reported previously(Godenschwege et al., 2004), and shown here by comparisonwith the synapsin knockout (Figs S4D and S5A,B), synapsindistribution in the WT is ubiquitous throughout the neuropil. ThePSyn(S6) antiserum, on the other hand, shows significant specificimmunoreactivity in the entire central brain neuropil (whole-mountbrain preparation in Fig. 7A,B, cryo-sections in Figs S4D and S5A,B),as well as very strong specific staining of various unidentifiedsynapses, including a ring of synapses in the ellipsoid body and setsof apparently large synapses at the rim of the lateral triangle (Fig. 7Band cryosection in C,D). Comparison of Fig. S5A,B left and middlecolumns indicates that the strong PSyn(S6) staining is indeed due toenhanced phosphorylation rather than stronger synapsin expression.We conclude that synapsin is partially phosphorylated at Ser6 inmost synapses of the central brain but strongly hyper-phosphorylated at this amino acid in a subset of central complexsynapses and several other structures that show enhanced PSyn(S6)staining. No qualitative difference in the distribution of synapsinand Ser6-phosphorylated synapsin between Sap−/− mutants andWTwas detected in six experiments by staining cryo-sections ofw;;Syn+/+, w;;Sap47208 and w;;Syn−/− flies on the same microscopeslides with Syn and PSyn(S6) antibodies. In the visual system,phosphorylation of synapsin at Ser6 is apparently low; however,detailed analysis is not feasible because, as revealed by stainingin the Syn−/− mutant, the PSyn(S6) antiserum crossreacts with anunknown antigen widely distributed in several synaptic layers ofthe visual system (Fig. S4D, middle column). This antigen couldpossibly also be responsible for the weak PSyn(S6) westernsignal in the Syn−/− mutant lane of Fig. S2D marked by anasterisk.

In a first attempt to identify the cells whose synapses containsynapsin hyper-phosphorylated at Ser6 in the synaptic rings of theellipsoid body, we used fiveGal4 lines to selectively express greenfluorescent protein (GFP) in specific subsets of so-called ‘ring’neurons. In Fig. 7C,D (EB1-Gal4) and Fig. S6, we compare theGal4-driven GFP expression in ellipsoid body ring neurons usingthe lines c819-Gal4 (R2/R4; Renn et al., 1999), 189y-Gal4 (R3;Renn et al., 1999; Kuntz et al., 2012), c232-Gal4 (R3/R4d; Rennet al., 1999; Thran et al., 2013), EB1-Gal4 (R2; Spindler andHartenstein, 2010) and ftz-ng-Gal4 (R4; Kuntz et al., 2012) withthe distribution of endogenous synapsin hyper-phosphorylated atSer6. None of these Gal4 lines showed GFP expressioncolocalizing with PSyn(S6) staining, as revealed by carefulinspection of individual optical sections with 40× oil optics andconfocal microscopy.

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DISCUSSIONThe two genes under study here encode several protein isoforms.Alternative splicing generates at least 10 Sap47 transcripts (Flybase;Gramates et al., 2017), while in western blots nine separate bandshave been tentatively identified (Funk et al., 2004) of which the47 kDa isoform is by far the most abundant. For synapsin, fourtranscripts (Flybase; Gramates et al., 2017) and five isoforms havebeen described, of which three in SDS gels run at 70, 74 and80 kDa, while two generated by UAG read-through run at 143 kDa

(Klagges et al., 1996). Information on the N-terminus ofDrosophilasynapsin has been ambiguous. The sequence XKRGFSSGDLidentified by Edman degradation of synapsin purified from adultheads has significant homology with the vertebrate synapsinN-terminal A domain (Godenschwege et al., 2004). However,sequence comparisons with various invertebrate synapsingenes demonstrate that 16 amino acid codons upstream ofthis sequence are highly conserved, suggesting an alternativetranslation start which results in the N-terminal sequence:LNFSSFKSSFTSNVNFLKRGFSSGDL. As a result of thisN-terminal ambiguity, amino acid numbering may differ by 16amino acids, e.g. Ser6 in focus here is alternatively referred toas Ser22 in some publications (Kleber et al., 2015; Niewaldaet al., 2015) and, for easy comparison, in the sequences ofFig. S3.

In any event, the present hypothesis on the role of synapsin inolfactory associative short-term learning assumes that the stimulationof protein kinase A (PKA) leads to phosphorylation of synapsin atone or multiple sites. This is thought to result in a modification ofneurotransmitter release (Heisenberg, 2003; Diegelmann et al., 2013;Menzel, 2014; Hige et al., 2015; Gerber and Aso, 2017; Saumweberet al., 2018). This modified transmitter release is assumed to be thebasis for learned behaviour. There are two candidate sites (RRXS) forPKA-dependent phosphorylation of synapsin, the conserved Ser6 in

Fig. 3. Circadian rhythms and sleep in WT flies and Sap−/−and Syn−/−

mutants. (A) Actograms for a representative WT fly (WTCS) and Sap−/−

mutant. Flies were first recorded for 6 days in a 12 h:12 h light:dark (LD) cycleand then for 13 days under constant darkness (DD). The LD programme isindicated as white and black bars on top of the actograms. (B) Period lengthand power of the free-running (DD) rhythm of flies that were reared under short(LD 8:16), normal (LD 12:12) and long (LD 16:8) photoperiods (20–32 flieswere recorded for each condition). Asterisks indicate the strains in which periodor power depended significantly on the photoperiod. *P<0.01, ANOVA followedby a Bonferroni post hoc test. (C) Sleep profiles for WTCS, Sap−/− (Sap47156),Syn−/− (Syn97) and Sap−/−,Syn−/− (Sap-SynV1) mutants before and after sleepdeprivation (SD) for the entire night. For each strain, 20–30 flies were recorded.Arrows indicate the short period of sleep rebound in themutants. (D) Number ofhours the flies slept during the day and the night before and after sleepdeprivations. A significant increase in sleep after sleep deprivation is indicatedby an asterisk.

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mutants. Representative traces (A) and analysis (B) of minis at larval NMJs. Mini frequency was increased in Sap−/− (P=0.014, n=10), Syn−/− (P=0.039, n=12),and Sap−/−, Syn−/− double mutants (Sap-SynNS62) (P≤0.001, n=13). The average mini amplitude was affected only in Sap−/− mutants (P=0.004, n=10).(C) Amplitudes of eEPSCs, recorded at 0.2 Hz, were comparable in all genotypes. (D,E) Example traces of paired-pulse recordings [30 ms inter-stimulus interval(ISI), stimulation artefact removed for clarity] (D) demonstrate less synaptic facilitation upon closely spaced stimulation in both single and double mutants (E)[30ms ISI: Sap−/− (P=0.015, n=10), Syn−/− (P=0.001, n=12), and Sap−/−, Syn−/− double mutants (P=0.003, n=13)]. Scale bars: 2 nA, 50 ms (A); 10 nA, 10 ms(D). *P≤0.05, **P≤0.01, ***P≤0.001 using the non-parametric rank sum test versus WT control.

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the N-terminal A domain and the non-conserved Ser533 betweendomains C and E. The PKA target site in domain A is removed inmost synapsins of larval and adult Drosophila by RNA editing,resulting in RXXS (Diegelmann et al., 2006). The ectopic expressionof such an edited cDNA in the mushroom body rescues the learningdefect of the null mutant (larvae: Michels et al., 2011; adults:Niewalda et al., 2015). This rescue is prevented if synapsin withmutated RRXS sites (S6A and S533A mutations) is expressed(Michels et al., 2011). Ser→Alamutation prevents phosphorylation atthese sites. Also, olfactory short-term habituation, a simple form ofbehavioural plasticity, critically depends on the expression ofsynapsin that can be phosphorylated at Ser6 and/or Ser533 inGABA-ergic local interneurons. In this experimental paradigm,activity of calcium and calmodulin-dependent kinase II (CaMKII) isrequired, which may also target these sites irrespective of editing(target consensus RXXS) (Sadanandappa et al., 2013). Whether Ser6or Ser533, or both, are essential for behavioural plasticity inDrosophila remains to be elucidated. It is also not known if PKA

can activate, directly or indirectly, CaMKII and in this wayphosphorylate Ser6 and/or Ser533 of synapsin irrespective of RNAediting. Thus additional experiments are required to clarify the role ofPKA and CaMKII and the detailed molecular modifications ofsynapsin in behavioural plasticity. For vertebrates, it has recently beenshown that in an aqueous environment synapsin can form a distinctliquid phase that rapidly disassembles upon phosphorylation byCaMKII (Milovanovic et al., 2018). It remains to be investigatedwhether this process is relevant for synapsin function in thebehavioural plasticity discussed here. No information is presentlyavailable on the function of the two large synapsin isoforms. Of note,these large isoforms are more efficiently phosphorylated at Ser6 thanthe small isoforms (Fig. S2D).

In qualitativewestern blots, in addition to the synapsin signals seenin the WT, a band at a slightly larger apparent molecular weight isreliably observed in three independent Sap47 null mutant alleles.These additional western signals were not present when thehomogenate is treated with alkaline phosphatase, indicating that the

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shifted signals may represent phosphorylated synapsin isoforms(Fig. 1A). Quantification of synapsin signals in non-saturated westernblots does not indicate an upregulation of total synapsin expression inSap−/−mutants. Mass spectrometric analysis of synapsins from adultWT heads showed that at least 32 amino acids of Drosophilasynapsins may be phosphorylated (Nuwal et al., 2011; Niewaldaet al., 2015). Comparison of synapsin phosphorylation in WT andSap−/−mutant larvae revealed a complex pattern of hyper- and hypo-phosphorylation in the mutant at 23 sites (Kleber et al., 2015). Here,we observe the phosphorylation of synapsin Ser574 in adult Sap−/−

mutants, which has not been detected in the WT (Fig. S3, amino acidnumbering according to upstream N-terminus). Interestingly,synapsin phosphorylation at Ser6 has been found only once in MSstudies (Niewalda et al., 2015), but synapsin phosphorylated at thissite is readily detected in western blots using the PSyn(S6) antiserum,which is specific for phosphorylated Ser6 of synapsin (Fig. S2); thissignal is reduced in Sap−/− mutants (Fig. S2A,C,D). Given that adirect binding of both proteins seems unlikely (Fig. 1E), Sap47 mightmodulate synapsin phosphorylation by regulating site-specifickinases or phosphatases to prevent hyper-phosphorylation at somesites while enhancing phosphorylation of Ser6. This hypothesiswould be compatible with the available data but requires furtherexperimental support.In order to test the possibility that Sap47 has an effect on lifespan

and to determine to what extent the apparent interdependence ofSap47 and synapsin influences viability, we compared survival timeof the two single mutants and the double knockout flies. Our resultssuggest that the decline in viability of Sap−/− mutants isindependent of synapsin (Fig. 2A). This is in stark contrast toassociative learning, which depends equally on both proteins.

In mammals, Syap1 is strongly expressed in various tissues suchas muscles and liver (Schmitt et al., 2016). RNA-seq data fromFlyAtlas2 (Leader et al., 2018) show that in Drosophila five Sap47mRNA splice variants (RE, RF, RG, RI, FJ) are specificallyexpressed in the adult nervous system, whereas one transcript (RA)is found in all other analysed tissues, albeit at approximately 10-foldlower abundance. At the protein level, Sap47 in Drosophila has sofar been described only in the nervous system. Repeated earlierattempts to detect differences in Sap47 staining between WT andSap47156 null mutants outside the nervous system have failed. Thiscould have been caused by difficulties identifying weakimmunohistochemical signals on top of background due tounspecific antibody binding or might point to inefficient mRNAtranslation outside the nervous system. We report here thattransgenic pan-neural expression of the most abundant brain-specific Sap47 splice variant (RF) in the null mutant rescues theviability and climbing deficiencies of the mutant (Fig. S4A,B). Thisdemonstrates that the expression of Sap47 outside the nervoussystem is not required for normal lifespan and climbing ability. Thefunctional relevance of expression of the Sap47 RA splice variantmay thus be subtle and needs to be investigated in a separate study,which could then also clarify how to interpret the striking increase inlifespan of the rescued flies compared with the WT (Fig. S4A).

Age-related decline of motor performance is a standard test inDrosophila models of neurodegenerative disease (Pendleton et al.,2002; Barone and Bohmann, 2013; Long et al., 2014). We wanted todetermine how knockout of synapsin or Sap47, or both, affectsamount and rate of age-related decline of climbing proficiency inrapid induced negative geotaxis. Our results suggest that synapsin andSap47 control climbing through independent pathways such that their

A Sap47

R

BRP

Na-K-ATPase Merge

B

C

La

Me

Lo

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SAP47

Fig. 6. Distribution of Sap47 in the adult Drosophila brain. (A) Immunohistochemical staining of horizontal frozen brain sections with nc46 (anti-Sap47) verifyhigh concentrations of Sap47 in synaptic neuropil but also reveal significant Sap47 levels in axons of the inner optic chiasm and the connections betweenoptic lobes and lateral protocerebrum (asterisks) as well as in subsets of neuronal perikarya (arrows). R, retina; La, lamina; Me, medulla; Lo, lobula; LP, lobulaplate. (B) For comparison, staining with the active zone marker anti-bruchpilot (BRP) is strictly confined to synaptic neuropil. (C) The section of the lateral medullacortex at higher magnification illustrates the selective Sap47 localization in few perikarya (left), counterstained with anti-Na-K-ATPase (middle), mergedin right image. Dorsal is up. Scale bars: 20 µm (A,B), 10 µm (C).

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defects are compounded in the double mutant (Fig. 2B; see alsoFig. S4B for a neuronal rescue of the Sap47 phenotype in climbing).Of note, only the amount, not the age-related rate of decline ofclimbing proficiency was different in the three genotypes, suggestingthat the mutations affect the efficiency of climbing but not the age-related decline in motor function. This interpretation is supported bythe observation that the accelerated age-related decline in climbinginduced by transgenic pan-neural expression of human Aβ42 peptideis not further enhanced by homozygous Sap47 deficiency (Fig. 2C).The suppression of the climbing deficiency of Sap−/−mutants in fliesexpressing Aβ42 (compare violet with red curves in Fig. 2C) couldindicate an interesting functional interaction of Sap47 and Aβ42.In experiments comparing genome-wide gene expression in a

subset of adult head clock neurons – the ventral lateral neurons(LNvs) identified by expression of pigment dispersing factor (PDF) –with expression in randomly selected adult head neurons,

Sap47 mRNA was found to be enriched at lights on (ZT0) but notat lights off (ZT12) during a 24 h light–dark cycle (Kula-Eversoleet al., 2010). Synapsin concentration in the adult brain, on the otherhand, was shown to increase during sleep deprivation (Gilestroet al., 2009). We therefore wondered if knockout of Sap47 and/orsynapsin modified circadian activity and/or sleep behaviour. Wefound that the typical locomotor activity pattern with a morning andan evening peak interrupted by a siesta and a pronounced night-timesleep was also displayed by the mutants (Fig. 3A,C). However, afterrearing flies under LD 8:16 conditions (short days, long nights) onlyWT flies showed the typical extended period and increasedamplitude of activity cycles under DD conditions (Fig. 3B),indicating that Sap47 and synapsin may be required for plasticadaptations of the free-running activity cycle. Similarly, sleeprebound requiring a ‘memory’ of sleep deprivation is reduced in themutants (Fig. 3C,D). These observations of impaired behavioural

AL

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A MergeAnti-SynAnti-PSyn(S6) Fig. 7. Distribution of synapsin and Ser6-phosphorylated synapsin in the adult Drosophila brain.(A,B) Z-projections of frontal confocal optical sections of awhole-mount preparation of a Drosophila brain stained withanti-Syn (magenta) and anti-PSyn(S6) (green) reveal thepresence of both total and Ser6-phosphorylated synapsinthroughout the synaptic neuropil of the central brain. (A) 20×objective; scale bar: 100 µm. (B) Subset of optical sections40× objective; scale bar: 50 µm. (C) Maximum intensityprojection of the confocal stack of an oblique frontal 30 µmfrozen section stained with anti-Syn (magenta) and anti-PSyn(S6) (green) reveals high concentrations of Ser6-phosphorylated synapsin in a subset of synapses near thelateral triangle, the lateral edge of the fan-shaped body (FB)and, most prominently, in rings of synapses of the ellipsoidbody (EB). AL, antennal lobe; LPR, lateral protocerebrum.(D) The same section shown in C with GFP expressiondriven by EB1-Gal4 (green), illustrating the lack of co-expression of GFP and PSyn(S6) (red). Anti-Syn isrepresented here in blue. Dorsal/posterior is up. Scale bar:20 µm (C,D).

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plasticity are in line with earlier reports on defects in olfactory short-term habituation of Syn−/− mutants and in olfactory associativeconditioning in Sap−/− and Syn−/− mutants referred to above.Previous work on Sap47 and synapsin single mutants showed

normal basal but impaired evoked, high-frequency synaptictransmission (Godenschwege et al., 2004; Saumweber et al., 2011;Akbergenova and Bykhovskaia, 2010). Our electrophysiologicalrecordings of mutant NMJs show that evoked transmitter release isindeed altered upon closely spaced paired stimulation. At shortintervals, paired-pulse facilitation is reduced non-additively, i.e. to asimilar extent in both single and double mutants (Fig. 4E); given thatpaired-pulse facilitation is almost absent in the single mutants,baseline effects need to be considered for this measure of evokedrelease. However, the almost threefold increase in mini frequency inSap-Syn double mutants (Fig. 4B) could result from an additive effectof the individual mutations. Note that a similar additivity, whichwould be inconsistent with an obligatory cooperative function ofsynapsin and Sap47, is observed in climbing deficiency. This raisesthe question as to whether there could be a functional link betweenincreased mini frequencies, which are indicative of increasedspontaneous release, and climbing defects.The reduced synapsin levels in larval neuro-muscular boutons

of Cirl mutants (Fig. 5) are difficult to interpret. Cirl codesfor an adhesion-type G-protein-coupled receptor known forits interaction with α-Latrotoxin, which causes massiveneurotransmitter release from Drosophila NMJs (Umbach et al.,1998). Drosophila Latrophilin/Cirl has recently been shown tomodulate ionotropic receptor currents in mechanosensory neurons(Scholz et al., 2017) but is likely to have additional functions in theadult brain as its depletion by pan-neuronal knockdown induceshyperactivity and reduced sleep (van der Voet et al., 2016).Additional experiments are required to explain the presentobservations of reduced Cirl mRNA in synapsin mutants(‘rescued’ by the Sap−/− mutation, Fig. S1) and the effect of Cirlknockout on synapsin levels.The distribution of synapsin phosphorylated at Ser6 is highly

intriguing (Fig. 4D and Fig. S5A,B). It now needs to be investigatedto which cells the synapses that contain the extremely highconcentrations of synapsin phosphorylated at Ser6 belong(Fig. 7C,D and Fig. S5A,B). Initial attempts to answer thisquestion for the central complex using transgenic flies expressingGFP in subsets of ring neurons (Fig. S6) were inconclusive.

ConclusionsAlthough the current study provides the most detailed account ofSap47 expression and function to date, the need for further research isobvious. The present work may serve as guide for such continuedefforts. In particular, it shows that Sap47 is found not only at synapticboutons of larval motoneurons and in most neuropil regions of larvaland adult nervous system, but it is also detected in axons andselectively in subsets of neuronal cell bodies. Lack of Sap47 leads topremature death, climbing defects, impaired behavioural plasticity,reductions in paired-pulse facilitation and causes modifiedphosphorylation of synapsin. In particular, the latter data suggest anintegratedmode of action of these two highly abundant evolutionarilyconserved neuronal proteins.

AcknowledgementsStocks obtained from the Bloomington Drosophila Stock Center (NIHP40OD018537) were used in this study.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: B.B.-R., C.H.-F., G.H., R.J.K., T.L., B.G., E.B.; Methodology:B.B.-R., N.N., S.K., T.N., P.H., Y.L., N.E., N.S., A.M., J.K., T.K., D.S., M.K.S., N.F.,V.A., C.H.-F., M.R., G.H., R.J.K., T.L., B.G., E.B.; Software: S.K., T.N., T.K., E.B.;Validation: B.B.-R., N.N., S.K., T.N., P.H., Y.L., N.E., N.S., A.M., J.K., T.K., M.K.S.,N.F., V.A., C.H.-F., M.R., G.H., R.J.K., T.L., B.G., E.B.; Formal analysis: B.B.-R.,N.N., S.K., T.K., C.H.-F., R.J.K., E.B.; Investigation: B.B.-R., N.N., S.K., T.N., P.H.,Y.L., N.E., N.S., A.M., J.K., T.K., D.S., M.K.S., N.F., V.A., E.B.; Resources: S.K.,T.K., C.H.-F., R.J.K., T.L., B.G., E.B.; Data curation: B.B.-R., N.N., S.K., P.H., Y.L.,N.E., N.S., A.M., J.K., T.K., N.F., C.H.-F., R.J.K., T.L., B.G., E.B.; Writing - originaldraft: E.B.; Writing - review & editing: B.B.-R., T.N., N.E., N.S., D.S., C.H.-F., R.J.K.,B.G., E.B.; Visualization: B.B.-R., N.N., P.H., Y.L., N.E., N.S., A.M., J.K., T.K., N.F.,C.H.-F., R.J.K., T.L., B.G., E.B.; Supervision: S.K., T.K., C.H.-F., M.R., G.H., R.J.K.,B.G., E.B.; Project administration: C.H.-F., T.L., E.B.; Funding acquisition: R.J.-K.,T.L., B.G., E.B.

FundingThe study was supported by grants from the Deutsche Forschungsgemeinschaft(http://www.dfg.de/) (SFB554, SFB581, GK1156 to E.B.; FOR 2149/P01 and P03,SFB 1047/A05, TRR 166/C03, LA2861/7-1 to T.L.; FOR 2149/P03, SFB 1047/A05,TRR 166/B04, KI1460/4-1 to R.J.K.; FO207/14-2 to C.H.-F.; and CRC 779 andGE1091/4-1 to B.G.). N.E. was supported by a grant from the German ExcellenceInitiative to the Graduate School of Life Sciences, University Wurzburg, GSC106/3(PostDoc Plus program). The visits of B.B.-R. and A.M. to NCBS/TIFR Bangalore,India and of M.K.S. to the University of Wurzburg were supported by short-termfellowships from the Deutscher Akademischer Austauschdienst (DAAD).

Supplementary informationSupplementary information available online athttp://jeb.biologists.org/lookup/doi/10.1242/jeb.203505.supplemental

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